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Studies on the Availability of Nitrogen from Various Ammoniated Products for Rumen Bacteria and Dairy Cattle
STUDIES
ON
THE
AVAILABILITY
OF
NITROGEN
FROM
VARIOUS AMMONIATED PRODUCTS FOR RUMEN BACTE:R, I A A N D D A I R Y C A T T L E R. 1~. D A V I S , ~ ]~. I t . W A S S E R M A N , ~ J. K . L O O S L I , AND C. t / . G R I P P I N
De~art~tcnt of A n i ~ a l Husbandry, Cornell University, Ithaca, N. Y.
The circumstances essential for the efficient utilization of urea nitrogen b y the r u m i n a n t have been reviewed by Reid (13). One serious problem concerning the utilization of urea and ammonia is their r a p i d disappearance f r o m the rumen. Removal of either of these soluble nitrogenous substances f r o m the sphere of r u m e n bacterial action makes them useless as sources of protein for the host animal. I n an a t t e m p t to solve this problem, m a n y industrial products and feeds have been i m p r e g n a t e d with ammonia u n d e r v a r y i n g conditions of temp e r a t u r e and pressure to induce chemicM combinations that might release utilizable nitrogen more in proportion to the rate at which energy f r o m complex carbohydrates becomes available. Thus, the energy-dependent protein synthetic reactions would have conditions more suitable for the conversion of nonprotein nitrogen to protein. Tests of several of these products have been r e p o r t e d by different authors. M a c G r u d e r et al. (10) reported on the feeding of an ammoniated building byproduct. E x p e r i m e n t s with ammoniated citrus pulp, beet pulp, molasses, and other feeds have been reported by various workers (4, 7, 11, 12, 15). I t has been generally concluded t h a t nitrogen f r o m these a m m o n i a t e d compounds is available to r u m e n bacteria. However, some of these experiments have been conducted u n d e r conditions which do not allow a critical assessment of the availability of nitrogen f r o m ammoniated materials. I n some studies, the aminohinted products furnished either a relatively small p r o p o r t i o n of the total nitrogen of the ration or the nitrogen in the ration was fed at or above the a n i m a l ' s requirement. W h e n F e r g u s o n and Reeves (4) took these factors into consideration, they found t h a t the nitrogen in ammoniated beet p u l p was relatively unavailable in comparison with urea. I n order to obtain more data on the availability of nitrogen f r o m ammoniated products, a series of in vitro studies with r u m e n bacteria and a series of balance studies with d a i r y steers were conducted. IN VITRO EXPERIMENTS
Procedures. A modification of the in vitro technique of H u h t a n e n and Gall (5) was employed in assessing the availability of nitrogen in ammoniated feed products. The f e r m e n t a t i o n was confined to a semipermeable sac suspended in a l~eceived for publication December 24, 1954. Present address: Dairy Department, University of Maryland, College Park. Present address: University of Tennessee - - Atomic Energy Commission Agricultural Research Program, Oak Ridge. 677
678
•. F. DAVIS ET AL
buffer-salt solution in order to decrease by dialysis the concentration of deleterious f e r m e n t a t i o n by-products (9). The criteria of nitrogen availability were the a m o u n t Of cellulose digestion a n d / o r protein synthesis which occurred in the presence of limiting quantities of s u p p l e m e n t a r y nitrogen in the t r e a t e d products. I n addition, these manifestations of r u m e n bacterial activity were compared with the effect of equivalent levels of ammonia-nitrogen. Urea was not employed, since high levels of urease activity are present in the r u m e n (17) and p r e l i m i n a r y work had shown t h a t the r a p i d breakdown of urea adversely affected the p H within the artificial rumen. The essential differences in the present procedure as comp a r e d to that of the aforementioned investigators are noted as follows : (a) The artificial rumens were incubated within a desiccator made anaerobic b y displacing air with CO.o ; (b) phenol red was employed as an internal p H indicator when periodic neutralizations with I N . K O H were made during the course of f e r m e n t a t i o n ; (c) the incubation period was usually 24 hours a n d never exceeded 30 hours; (d) the fiber substrate consisted of an alfalfa leaf meal-cellulose (Solka-Floc) m i x t u r e (1:4) ; (e) dried distillers molasses solubles were added, where noted, as a source of factors shown to be s t i m u l a t o r y for cellulose digestion (16). The buffer-salt solution shown in Table 1 was based on the solution used b y B u r r o u g h s et al. (2) in their artificial r u m e n studies. TABLE 1
Composition of the buffer-salt solution employed in the in vitro st,~dy
K~.HPO~ KH_~PO4 NaHCO~ KC1 NaC1 CaCI_~
(%) 0.46 0.46 0.25 0.56 0.47 0,04
MgSO~ • 7H~O Na_~SO4 FeCI_. l~nC12 Methionine
(%) 0.12 0.07 0.04 0.012 0.07
pH--7.2
The ammoniated test product, containing 4.4 mg. nitrogen, 500 rag. fiber mixture, and 100 mg. dried distillers molasses solubles, was placed within the dialyzing membrane. The dialyzing m e m b r a n e and contents, a f t e r insertion into a small bottle, were sterilized by autoclaving at 15 p.s.i, for 10 minutes. Buffersalt solution (50 ml.) was added to the bottle to serve as a source of inorganic nutrients, as a buffer, and as a recipient for substances diffusing through the semipermeable membrane. Ten ml. of this buffer-salt solution was placed within the sac. The active inoeulum consisted of 5 ml. of a washed h e a v y suspension of r u m e n microorganisms f r o m a fistulated steer fed a ration of late-cut grass hay, molasses, urea, and minerals. A f t e r the period of incubation, the residual fiber was separated f r o m the tureen microorganisms b y differential centrifugation. The cellulose in the fiber was determined b y a modification of the method of C r a m p t o n a n d M a y n a r d (3). The bacterial protein was estimated by hydrolyzing the protein with concentrated HzSO~, and the subsequent determination of ammonia-nitrogen b y the nesslerization technique of LePage (8).
AVAILABILITY
OF NITROGEN
FROM
AMMONIATED
PRODUCTS
679
The ammoniated products, obtained from three manufacturers, had been treated with anhydrous ammonia at various temperatures and pressures to induce the chemical combinations. In certain cases the exact ammoniation procedure was not known, but inspection of the product indicated the severity of the treatment. The products are more thoroughly described in the presentation of the balance studies. In conjunction with these and other artificial rumen studies, the microbial population was examined by standard microscopic techniques and by the growth dilution method of Huhtanen et al. (6).
Results. Examination of the microbial population indicated that the centrifugation procedure did not significantly alter the numbers and types of bacteria present. However, certain changes were obvious in the fermentation liquor after incubation. The protozoal population, which was vigorous in the inoculum, was less apparent after incubation. The cultural technique revealed that a fast growing facultative streptococcus was usually 100 times more numerous, whereas the microscopic examination of gram stains showed the bacterial population to be less diverse after incubation. However, the bacteria present in the greatest number culturally post-incubation were similar to those considered the most numerous in the pre-incubation sample, and the bacteria most frequently observed microscopically were morphologically similar to a known cellulose digester previously isolated from the rumen. All of the above findings indicate that the population had changed during incubation under artificial conditions. However, significant similarities existed between the in vitro and in vivo microorganisms. This cursory bacteriological examination indicated that certain important reservations should accompany any in vitro data when extrapolating to the live animal. The results of the comparative nitrogen availability in the ammoniated products as shown by in vitro methods are presented in Table 2. In the first two series both cellulose digestion and protein synthesis were determined, but only cellulose digestion was estimated in the third and fourth series. Belasco (1) indicated that cellulose digestion was a valid indication o f nitrogen utilization or availability in vitro. Statistical evaluation of the data in Table 2 revealed that the correlation coefficient between the two measurements (cellulose digestion vs. protein synthesis) was 0.920 (degrees of freedom = 8), which is significant at the 0.01 probability level. The data suggest that the procedures employed in ammoniation significantly reduce the availability of nitrogen in all of the products tested except one. This exception, Series II and IV, ammoniated f urfural residue 3A, was ammoniated at atmospheric pressure followed by acid neutralization. This would indicate that the nitrogen in that particular sample was present as the ammonium ion per se or readily convertible to the ammonium ion in vitro. The other products in this fourth series were treated by high temperature, high pressure, saccharification, or combinations of these treatments. The ammoniation procedure which the citrus pulp and dried distillers molasses solubles received was unknown. It is apparent that the methods employed to introduce nitrogen into these products
R. F, DAVIS ET AL
680
TABLE 2
Effect of ammoniated by-products on eelhdose digestion and protein synthesis by ru~en organisms in vitro Supplement •
Cellulose digestion b
(%)
Protein synthesis b
(mg.)
Series I ( A m m o n i a t e d citrus p u l p ) ¢ N o n a m m o n i a t e d citrus p u l p 5.4 N o n a m m o n i a t e d citrus p u l p plus NH~C1 16.9 A m m o n i a t e d citrus p u l p ( S a m p l e ~:19) 11.2 A m m o n i a t e d citrus p u l p ( S a m p l e ;~20) 9.1
6.0 35.0 13.5 9.0
Series I I ( A m m o n i a t e d f u r f u r a l residue) c Control 10.1 NH4C1 23.6 A m m o n i a t e d f u r f u r a l residue ~3A 24.8 A m m o u i a t e d f u r f u r a ] residue ' ' A ' ' 16.6 A m m o n i a t e d f u r f u r a l residue ' ' B ' ' 15.5
4.4 34.4 38.1 18.1 24.4
Series I I I ( A m m o n i a t e d dried distillers molasses solubles) d Nonammoniated DDMS 11.9 N o n a m m o n i a t e d D D M S plus NH~C1 32.0 A m m o n i a t e d D D M S ~tl 10.7 A m m o n i a t e d D D M S ~3 14.5 A m m o n i a t e d I ) D M S ~$4 8.4 A m m o n i a t e d D D M S ~5 11.9 A m m o n i a t e d D D M S ~6 8.0 Control NH~C1 Ammoniated Ammoniated Ammoniated Ammoniated Ammoniated Ammoniated
Series I V ( A m m o n i a t e d f u r f u r a l residue) ~ 11.0 21.9 f u r f u r a l residue 1A 9.0 f u r f u r a l residue 2A 10.1 f u r f u r a l residue 3A 20.6 f u r f u r a l residue 4A 13.2 f u r f u r a l residue 5A 13,8 f u r f u r a l residue 6A 14.6
• The s u p p l e m e n t a l a m m o n i a t e d b y - p r o d u c t s or NIt4C1 c o n t r i b u t e d 4.4 rag. N to each fermentation apparatus. b E a c h value r e p r e s e n t s the mean of duplicate samples f r o m two experiments. B a s a l : a l f a l f a leaf meal - - cellulose m i x t u r e (0.5 g.) p l u s dried distillers molasses solubles (0.1 g.). d B a s a l : a l f a l f a leaf meal - - cellulose m i x t u r e (0.5 g . ) .
were sufficiently drastic (with the exception noted) to depress its availability to rumen bacteria as compared to ammonium chloride. Variations also existed among these products in the availability of their nitrogen. Some products showed an increased cellulose digestion and/or protein syntheuis above the negative control whereas others provided no available nitrogen. The possibility of inhibitory substances present in any of these products was not eliminated during the course of this investigation. BALANCE AND DIGESTION STUDIES
Procedure. Three balance studies, all conducted in a similar manner, were used to determine the availability of nitrogen from several ditTerent sources of ammoniated products. Four dairy steers, ranging in weight from 400 to 550 lb., were used in each experiment. The steers in each replicate were closely matched.
681
A V A I L A B I L I T Y OF N I T R O G E N FRO~a/i A M M O N I A T E D P R O D U C T S
Experimental periods consisted of a minimum of I week preliminary feeding and 1 week during which total collection of feces and urine Was made. The design of the experiment was a 4 × 4 Latin square. Animals were fed their preliminary rations in individual p e n s ; collections were made in crates especially designed for the separation and quantitative collection of urine and feces. Aliquot samples of all urine and feces were taken daily and composited. These were stored in closed containers under toluene and refrigerated until the end of the collection period, at which time samples were taken for proximate analysis. Feces samples were dried at approximately 60 ° C. for a minimum of 48 hours and allowed to come to equilibrium with the moisture in the atmosphere for a minimum of 48 hours before being g r o u n d in a Wiley mill and placed in sealed containers for analysis. All chemical analyses were conducted by accepted procedures. Trial 1 was conducted with three different ammoniated residues from the m a n u f a c t u r e of furfural. Original materials used in this process include corn cobs, oat hulls, cottonseed hulls, and other fibrous substances. Residue A had been hydrolyzed for 1/2 hour a f t e r sulfuric acid extraction to produce furfural, residue B had been hydrolyzed for 11/2 hours, and residue C for 31/~ hours. The experiment was designed to determine whether the different treatments affected TABLE 3 t~ations used in studying the value of three ammoniated furfural residues as sources of nitrogen for ruminants
Ingredient
Ration A
Ration B
Ration C
Ration D
(%) -23.0 --35.0 15.0 25.0 1.0 1.0
(%) --23.0 -35.0 15.0 25.0 1.0 1.0
(%)
Product A Product B Product C Soybean oil meal Corn meal Molasses Hay Bone meal Salt
(%) 23.0 ----35.0 15.0 25.0 1.0 1.0 100.0
100.0
100.0
100.0
--23.0 35.0 15.0 25.0 1.0 1.0
the availability of nitrogen from these products in comparison with the availability of nitrogen from soybean oil meal. The residue products were finely divided PoWders which were very light and dusty. The composition of the rations fed is shown in Table 3. The rations were prepared by mixing the corn meal, molasses, minerals, and ammoniated product or soybean meal in a small mechanical feed mixer. The hay, which was late-cut, low-protein timothy, was chopped to 1- to 2-in. lengths and mixed with the rest of the ration at the time of feeding. All animals received the same amount of feed d u r i n g each period. I n addition to the minerals listed, each animal received 50 g. of monosodium phosphate (NaH2PO~) daily after the first period. These rations were fed at a level which more than met the nitrogen requirement of the animals in the study.
R. F. DAVIS ET AL
682
TABLE 4 Daily ration ingredients used in studying the value of ammoniated molasses and ammoniated furfural residues as sources of nitrogen for ruminants :Ration Soybean oil m e a l
Ingredient ~
Hay Corn m e a l Molasses Ammoniated molasses S o y b e a n oil m e a l F u r a m e a l m i x l~o. 10 F u r a m e a l m i x No. 11 Total
NH~ molasses
Furameal No. 10
1%rameal No. 11
(g.)
(g.)
(g.)
(g.)
2268 898 1315 0 500 0 0
2268 1452 0 1315 0 0 O
2268 90 1088 0 0 1588 O
2268 O 1043 0 0 O 1906
4981
5035
5034
5217
a Also f e d w e r e : salt, free c h o i c e ; dicalcium p h o s p h a t e , 50 g / d a y ; cobalt, 0.3 r a g / d a y ; v i t a m i n A s u p p l e m e n t , twice weekly ( N o p c a y ' ' 10 ' ' s u p p l i e d b y N o p c o Chemical Co., H a r r i s o n , N. J . ) .
I n Trial 2 ammoniated molasses and soybean meal were employed, in addition to two products f r o m the residues of f u r f u r a l production. Residue No. 1 was a f u r f u r a l b y - p r o d u c t ammoniated at pressures greater than atmosphere. Residue No. 2 was the same material ammoniated at atmospheric pressures. The ammoniated molasses t r e a t m e n t was not known. The objectives of this s t u d y were the same as in Trial 1, that is, to determine the relative availability of nitrogen f r o m these sources as compared to soybean oil meal. F u r f u r a l residues were fed in a pelleted m i x t u r e containing a p p r o x i m a t e l y 53% of the residue, 13% molasses, 32% corn meal, and 2% minerals. E'qual amounts of h a y and molasses were included in all rations. The daily rations consumed are r e p o r t e d in Table 4. The nitrogen content of all rations was a d j u s t e d to the lower level of Morrison's recommendations. Since the n u t r i e n t content of the residues was unknown, no a t t e m p t was made to adjust T D N intake. However, the T D N content of the control ration fell within the r a n g e of Morrison's recommendations. TABLE 5 Daily ration ingredients used in studying the value of ammoniated DDMS and urea as sources of nitrogen for ruminants Rations I
Control
II NH, DDMS
III
IV
DDMS
Alfalfa
(g.)
(g.)
(g.)
(g.)
Timothy hay Alfalfa hay Corn m e a l DDMS NH4 D D M S Urea
2722 O 1769 O O 45
2722 O 1315 O 499 9
2722 O 1733 36 0 45
2268 454 1783 0 O 31
Total
4536
4536
4536
4536
AVAILABILITY OF NITROGEN Ft~OM AMMONIATED PRODUCTS
~
TABLE 6
Chemical co~aposition of the feed ingredients used in three balance studges involving ammoniatcd products
Water
(%)
Crude Nitrogen a protein
(%)
(~)
Ether extract
Crude fiber
Ash
W a t e r , aeid~ a n d b a s e soluble m a t t e r ~
(%)
(%)
(~)
(~)
1.9 2.4 2.2 2.0 3.4 3.4 --
45.6 51.3 54.1 33.0 2.1 6.7
3.7 3.8 3.9 3.6 1.4 5.7 7.6
22.0 16.5 16.5 47.0 72.7 28.8 56.6
1.9 2.0 --2.0 4.6 1.4
17.2 18.0 38.6 2.2 6.8
6.1 6.5 6.5 6.6 3.4 1.7 5.4
43.9 46.1 44.4 61.0 40.7 69.6 30.5
2.9 1.7 3.0 3.9 2.1 1.9
2.0 1.9 2.0 2.2 35.2 25.5
1.6 11.3 5.9 1.4 3.0 5.5
67.1 59.1 61.8 67.0 46.6 38.5
Trial I Residue A Residue B Residue C Hay Corn meal S o y b e a n oil meal Molasses
20.8 19.4 17.0 9.6 12.0 9.5 32.3
Residue m i x t u r e 1 Residue m i x t u r e 2 A m m o n i a t e d molasses Molasses Hay Corn meal Soybean oil meal
12.2 11.2 32.8 28.9 8.8 12.9 11.6
Control mix NH4 D D M S mix D D M S mix Mix fed w i t h a l f a l f a Timothy hay Alfalfa hay
10.5 10.9 11.1 11.6 7.6 10.2
6.0 6.6 6.3
--
-
-
-
4.8 8.4 45.9 3.5 Trial II
2.6
18.7 16.2 16.3 3.5 6.5 9.0 44.3 Trial I I I 15.9 15.1 16.2 13.9 5.5 18.4
a N i t r o g e n is r e p o r t e d as such in the a m m o n i a t e d products, since it is d o u b t f u l if there is more t h a n a very small a m o u n t of p r o t e i n material. b R e p o r t e d in this m a n n e r since the t e r m nitrogen-free e x t r a c t p r o b a b l y does n o t a p p l y to the a m m o n i a t e d products. This column includes nitrogen-free extract f o r the n a t u r a l products.
Trial 3 was a similar study, in which nitrogen from ammoniated dried distillers molasses solubles (DDMS) was compared with nitrogen from urea i n rations which contained the nonammoniated DDMS and a small amount of alfalfa. Previous studies indicated that DDMS had a stimulatory effect on the digestion of cellulose and the utilization of nonprotein nitrogen in vitro. These materials were included in this study in order to determine whether this effect could be demonstrated with animals. The rations fed in this experiment are reported in Table 5. Chemical compositions of all the feed ingredients are included in Table 6. Results. Digestion coefficients obtained in all three trials are reported i n Table 7. It will be noted that, compared with the soybean oil meal (control) ration, the rations which included the three different furfural residues exhibited a greatly lowered digestibility of dry matter. The digestibility of nitrogen was severely decreased when the residue products were the only source of supplemental nitrogen. Crude fiber also was digested at a much lower rate from the samples representing the furfural residue. This may be explained by the fact that this furfural residue had been already severely treated with sulfuric acid,
68~
R.F.
D A V I S E T AL
TABLE 7 Average digestion coefficients obtained from rations fed in three balance studies involving ammoniated products
Nitrogen
Ether extract
Crude fiber
Water, acid, and base soluble material ~
(%)
(%)
(%)
(%)
62 60 62 75
Trial I 51 52 47 66
69 73 72 77
30 31 27 54
74 74 75 80
Residue I Residue I I Ammoniated molasses Soybean oil meal
60 b 61 b 64 67
Trial I I 42 b 43 b 45 b 56
65 70 69
41 b 43 47 49
73 74 75 78
Corn and urea Corn and ammoniated DDMS Corn, urea, and DDMS Corn, urea, and alfalfa
63 60 64 68
Trial I I I 64 45 65 66
69 71 76 80
47 48 50 53
71 68 71 76
Dry matter (%)
:Residue A Residue B Residue C Soybean oil meal
Ration characteristics
66
Calculated in the same manner as nitrogen free extract but given this title due to the possible presence of nitrogen compounds. b Differences from control statistically significant at P =- < 0.05. a n d the r e m a i n i n g fiber p r o b a b l y was n o t s u b j e c t to a t t a c k b y b a c t e r i a l e n z y m e s i n the r u m i n a n t ' s digestive tract. The d i g e s t i b i l i t y of m a t e r i a l s soluble i n water, acids a n d bases, which w o u l d n o r m a l l y be called n i t r o g e n - f r e e extract, was also significantly reduced. S i m i l a r t r e n d s were n o t e d i n T r i a l 2. The d i g e s t i b i l i t y of d r y m a t t e r n i t r o g e n a n d of c r u d e fiber was r e d u c e d w h e n the f u r f u r a l residues were i n c l u d e d i n the r a t i o n . I n this t r i a l a m m o n i a t e d molasses p r o v i d e d a n i t r o g e n d i g e s t i b i l i t y o n l y s l i g h t l y b e t t e r thaa~ t h a t o b t a i n e d w i t h the a m m o n i a t e d f u r f u r a l p r o d u c t s . The d i g e s t i b i l i t y of n i t r o g e n f r o m a m m o n i a t e d D D M S i n T r i a l 3 was striki n g l y lower t h a n the coefficients o b t a i n e d for u r e a n i t r o g e n . T h e d i g e s t i b i l i t y of d r y m a t t e r f r o m the a m m o n i a t e d D D M S r a t i o n was also somewhat poorer t h a n f r o m other r a t i o n s . The i n c l u s i o n of 1 lb. of a l f a l f a h a y i n the r a t i o n i n c r e a s e d the d i g e s t i b i l i t y of all the n u t r i e n t s studied. N i t r o g e n b a l a n c e d a t a are i n c l u d e d i n T a b l e 8. I t m a y be seen t h a t the a m o u n t of n i t r o g e n a p p a r e n t l y absorbed reflects the r e d u c e d d i g e s t i b i l i t y of n i t r o g e n f r o m a m m o n i a t e d p r o d u c t s i n all t r i a l s . The values f r o m T r i a l 1 are s o m e w h a t h i g h e r t h a n those in the two other t r i a l s , because n i t r o g e n was overfed i n this s t u d y , whereas it was fed at the m i n i m u m r e q l l i r e m e n t i n T r i a l s 2 a n d 3. I n T r i a l 1 the absorbed n i t r o g e n f r o m the a m m o n i a t e d p r o d u c t s was n o t as well r e t a i n e d as t h a t f r o m s o y b e a n oil meal. I n T r i a l 2 r e s i d u e No. 2 a n d a m m o n i a t e d molasses p r o v i d e d absorbed n i t r o g e n i n a f o r m which was r e t a i n e d to the same e x t e n t as n i t r o g e n f r o m s o y b e a n oil m e a l , N i t r o g e n absorbed f r o n l r e s i d u e No. 1 was r e t a i n e d to a lesser degree. A b s o r b e d n i t r o g e n f r o m a m m o n i a t e d D D M S was
AVAILABILITY OF NITROGEN FROM AMMONIATED :PRODUCTS
685
TABLE 8
Average nitrogen balance data obtained from three balance studies involving ammoniated products Nitrogen Ration characteristics
Consumed
(g.)
P e r cent of
Apparently a b s o r b e d Retained
(g.)
A b s o r b e d hr retained
C o n s u m e d hr retained
(g.)
(%)
(%)
24 29 22 44
34 39 36 43
18 20 16 28
]3 17 16 20
38 49 46 45
17 22 21 25
20 12 26 23
43 39 56 45
28 18 37 30
Trial I Residue A Residue B Residue C Soybean oil meal
133 144 133 154
68 75 63 102 Trial I I
Residue I Residue I I A m m o n i a t e d molasses Soybean oil meal
74 78 79 79
Corn and urea Corn and a m m o n i a t e d D D M S Corn, urea, a n d D D M S Corn, urea, a n d a l f a l f a
71 68 71 77
29 33 35 44 Trial I I I 46 31 46 51
retained poorly in comparison to that from other sources (Trial 3). The inclusion of DDMS in the ration a p p a r e n t l y markedly improved the retention of absorbed nitrogen. The apparent improvement in the retention of digested nitrogen when DDMS was included in the ration is interesting in the light of in vitro experiments that have indicated increased cellulose digestion and utilization of nonprotein nitrogen by rumen bacteria when it was included in the media (16). DISCUSSION
The use and availability of nitrogen from ammoniated industrial by-products and ammoniated feeds by ruminants still require considerable investigation before a thorough knowledge will be obtained. It has been theorized that nitrogen from these sources might be as readily available to the microorganisms existing in the rumen as that from urea or natural protein. Several studies have indicated good or acceptable growth and use of nitrogen from these sources. A critical evaluation of some of these studies indicates that relatively low levels of nitrogen were supplied from the ammoniated products and that nitrogen from other sources was f r e q u e n t l y fed at levels above the minimum r e q u i r e m e n t s of the animals. Under these conditions one cannot obtain a critical evaluation of the utilization of nitrogen from any source. The studies here reported indicate that ammoniated industrial by-products and some ammoniated feeds do provide a source of nitrogen which can be utilized to a certain degree by r u m i n a n t microorganisms. However, this nitrogen does not appear to be available to the same degree as nitrogen from natural protein and is not as well utilized as nitrogen from urea. Similar results have been reported by Ferguson and Reeves (~/).
686
R . F . DAVIS ET AL
Since the chemical combinations t h a t occur when nitrogen is incorporated in feeds and industrial by-products are not well understood, it is not amiss to speculate t h a t m a n y of these compounds m a y be of a n a t u r e t h a t is not subject to attack b y enzymes n o r m a l l y occurring in the rumen. Unless this nitrogen is convertible to a utilizable f o r m by dissolution or enzyme hydrolysis, one would not expect it to be available for use b y the microorganisms. A p p a r e n t l y , f u r t h e r research is necessary to find an ideal source of nonprotein nitrogen for ruminants. A m m o n i a t e d products m a n u f a c t u r e d u n d e r more subtle or better controlled conditions could result in an ideal source of a nonprotein nitrogen for microorganisms. This could be a p r o d u c t which would be attacked b y some normal enzyme system occurring in the r u m e n and f r o m which the nitrogen was liberated at a p p r o x i m a t e l y the same rate as the energy necessary for the synthesis of protein. Another factor which enters into the feasibility of using a m m o n i a t e d industrial by-products as a source of nitrogen for r u m i n a n t s is the fact t h a t m a n y of them, as shown in this paper, contain relatively low levels of energy or available nutrients. I t appears economically unfeasible to t r a n s p o r t large amounts of relatively inert materials long distances for feeds. A source of nonprotein nitrogen which is concentrated or else contains appreciable amounts of energy would a p p e a r to be much more practical. The possibility of toxic reactions m u s t be considered in evaluating ammoniated products as a source of nitrogen. Recent reports b y l~ichardson et al. (14) have indicated t h a t animals fed fairly low levels of ammoniated molasses have shown nervous symptoms. A p p a r e n t l y , other workers have made similar observations. Until more is known about the chemical combinations a p p e a r i n g f r o m products a m m o n i a t e d u n d e r relatively severe conditions, caution should be used in the feeding of these materials. SUMMARY
A series of studies on the in vitro and in vivo utilization of nitrogen from a m m o n i a t e d industrial by-products and ammoniated feed stuffs has been reported. The results of both types of studies indicate t h a t nitrogen f r o m ammoniated products is not available to r m n e n bacteria or to the host to the same degree as is nitrogen f r o m u r e a or f r o m n a t u r a l protein sources. ACKNOWLEDGMENTS These studies were supported in part by The Quaker Oats Company, Chicago, and Publicker Industries, Inc., Philadelphia. REFERENCES (1) BEbASCO, I. J. New Nitrogen Feed Compounds for Ruminants - - a Laboratory Evaluation. J. Animal Sci., 13: 601. 1954. (2) BURROUGHS,W., FRANK, N. A., GERLAUGH, P., AND BETHKE, !t. M. Preliminary Observations upon the Factors Influencing Cellulose Digestion by Rumen Microorganisms. J. Nutrition, 40: 9. 1950.
AVAILABILITY OF NITROGEN FROM AMMONIATED PRODUCTS
687
(3) CRAMPTON,E. W., AND MAYNARD, l'J. A. The Relation of Cellulose and Lignin Content to the Nutritive Value of Animal Feeds. J. Nutrition, 15: 383. 1938. (4) F~RGUSON, W. S., AND REEVES, O. Nutritive Value of Ammoniated Sugar Beet Pulp. J. Agr. Sci., 33: 95. 1943. (5) HU~TANEN, C. N., AND GALL, L. S. The Miniature Artificial Rumen and Its Uses. J . Animal Sci., 11: 766. 1952. (6) C. HUHTANEN, N., ROGERS, M. R., AND GALL, .~. S. Improved Technique for Isolating and Purifying Rumen Bacteria. J. Bacteriol., 64: 17. 1952. (7) KNODT, C. B., WILLIAMS, J . B., AND BRUMBAUGH, J . Ammoniated Cane Molasses and Similar Products in the Rations of Dairy Calves. J. Dairy Sci., 34: 1042. 1951, (8) LEPAGE, G. A. Manometric Techniques and Tissue Metabolism. Burgess Publishing Co., Minneapolis, Minn. 1951. (9) LOUW, J. G., WILLIAMS, H. H., AND MAYNARD, L. A. A New Method for the Study in vitro of Rumen Digestion. Science, 110: 1. 1949. (10) MAcGI~IYDE4¢,N. D., KNODT, C. B., AND WILLIAI~S, P. S. The Value of Ammoniated Industrial By-Products in the Rations of Dairy Heifers and Cows. J. Dairy Sei., 34: 1055. 1951. (11) McCALL, R., AND GRAHA:k~, W. R., JR. The Value of Several Ammoniated Products as Feed for Beef Cattle. J. Animal Sci., 12: 798. 1953. (12) MILLAR, H. C. Ammoniated Sugar Beet Pulp as a New Nitrogenous Feed for Ruminants. J. Dairy Sci., 27: 255. 1944. (13) REID, J. T. Urea as a Protein Replacement for Ruminants: A Review. J. Dairy Sci., 36: 955. 1953. (14) RIChArDSON, D., SMITH, E. F., BAKE~, F. H., Cox, R. F., AND MCREYNOLDS, K. L. The Value of Ammoniated Molasses in Beef Cattle Wintering Rations 1953-1954. Kan. Agr. Expt. Sta., Circ. 308. 1954. (15) TILL~AN, A. D., AND KIDWELL, J. F. The Value of Ammoniated Condensed Distillers Molasses Solubles as a Feed for Beef Cattle. J. Anima~ Sci., 10: 934. 1951. (16) WASSEa~AN, R. H., DAVIS, R. F., AND LOOSLI, J. K. Unpublished data. Cornell University, Ithaca, N. ¥ . 1953. (17) WEGNER, M. I., BGO~'~, A. W., BOHSTEDT, G., AND HART, E. B. The in vitro Conversion of Inorganic Nitrogen to Protein by Microorganisms from the Cow's Rnmen. J. Dairy Sci., 23: 112. 1940.
ON
THE
AVAILABILITY
OF
NITROGEN
FROM
VARIOUS AMMONIATED PRODUCTS FOR RUMEN BACTE:R, I A A N D D A I R Y C A T T L E R. 1~. D A V I S , ~ ]~. I t . W A S S E R M A N , ~ J. K . L O O S L I , AND C. t / . G R I P P I N
De~art~tcnt of A n i ~ a l Husbandry, Cornell University, Ithaca, N. Y.
The circumstances essential for the efficient utilization of urea nitrogen b y the r u m i n a n t have been reviewed by Reid (13). One serious problem concerning the utilization of urea and ammonia is their r a p i d disappearance f r o m the rumen. Removal of either of these soluble nitrogenous substances f r o m the sphere of r u m e n bacterial action makes them useless as sources of protein for the host animal. I n an a t t e m p t to solve this problem, m a n y industrial products and feeds have been i m p r e g n a t e d with ammonia u n d e r v a r y i n g conditions of temp e r a t u r e and pressure to induce chemicM combinations that might release utilizable nitrogen more in proportion to the rate at which energy f r o m complex carbohydrates becomes available. Thus, the energy-dependent protein synthetic reactions would have conditions more suitable for the conversion of nonprotein nitrogen to protein. Tests of several of these products have been r e p o r t e d by different authors. M a c G r u d e r et al. (10) reported on the feeding of an ammoniated building byproduct. E x p e r i m e n t s with ammoniated citrus pulp, beet pulp, molasses, and other feeds have been reported by various workers (4, 7, 11, 12, 15). I t has been generally concluded t h a t nitrogen f r o m these a m m o n i a t e d compounds is available to r u m e n bacteria. However, some of these experiments have been conducted u n d e r conditions which do not allow a critical assessment of the availability of nitrogen f r o m ammoniated materials. I n some studies, the aminohinted products furnished either a relatively small p r o p o r t i o n of the total nitrogen of the ration or the nitrogen in the ration was fed at or above the a n i m a l ' s requirement. W h e n F e r g u s o n and Reeves (4) took these factors into consideration, they found t h a t the nitrogen in ammoniated beet p u l p was relatively unavailable in comparison with urea. I n order to obtain more data on the availability of nitrogen f r o m ammoniated products, a series of in vitro studies with r u m e n bacteria and a series of balance studies with d a i r y steers were conducted. IN VITRO EXPERIMENTS
Procedures. A modification of the in vitro technique of H u h t a n e n and Gall (5) was employed in assessing the availability of nitrogen in ammoniated feed products. The f e r m e n t a t i o n was confined to a semipermeable sac suspended in a l~eceived for publication December 24, 1954. Present address: Dairy Department, University of Maryland, College Park. Present address: University of Tennessee - - Atomic Energy Commission Agricultural Research Program, Oak Ridge. 677
678
•. F. DAVIS ET AL
buffer-salt solution in order to decrease by dialysis the concentration of deleterious f e r m e n t a t i o n by-products (9). The criteria of nitrogen availability were the a m o u n t Of cellulose digestion a n d / o r protein synthesis which occurred in the presence of limiting quantities of s u p p l e m e n t a r y nitrogen in the t r e a t e d products. I n addition, these manifestations of r u m e n bacterial activity were compared with the effect of equivalent levels of ammonia-nitrogen. Urea was not employed, since high levels of urease activity are present in the r u m e n (17) and p r e l i m i n a r y work had shown t h a t the r a p i d breakdown of urea adversely affected the p H within the artificial rumen. The essential differences in the present procedure as comp a r e d to that of the aforementioned investigators are noted as follows : (a) The artificial rumens were incubated within a desiccator made anaerobic b y displacing air with CO.o ; (b) phenol red was employed as an internal p H indicator when periodic neutralizations with I N . K O H were made during the course of f e r m e n t a t i o n ; (c) the incubation period was usually 24 hours a n d never exceeded 30 hours; (d) the fiber substrate consisted of an alfalfa leaf meal-cellulose (Solka-Floc) m i x t u r e (1:4) ; (e) dried distillers molasses solubles were added, where noted, as a source of factors shown to be s t i m u l a t o r y for cellulose digestion (16). The buffer-salt solution shown in Table 1 was based on the solution used b y B u r r o u g h s et al. (2) in their artificial r u m e n studies. TABLE 1
Composition of the buffer-salt solution employed in the in vitro st,~dy
K~.HPO~ KH_~PO4 NaHCO~ KC1 NaC1 CaCI_~
(%) 0.46 0.46 0.25 0.56 0.47 0,04
MgSO~ • 7H~O Na_~SO4 FeCI_. l~nC12 Methionine
(%) 0.12 0.07 0.04 0.012 0.07
pH--7.2
The ammoniated test product, containing 4.4 mg. nitrogen, 500 rag. fiber mixture, and 100 mg. dried distillers molasses solubles, was placed within the dialyzing membrane. The dialyzing m e m b r a n e and contents, a f t e r insertion into a small bottle, were sterilized by autoclaving at 15 p.s.i, for 10 minutes. Buffersalt solution (50 ml.) was added to the bottle to serve as a source of inorganic nutrients, as a buffer, and as a recipient for substances diffusing through the semipermeable membrane. Ten ml. of this buffer-salt solution was placed within the sac. The active inoeulum consisted of 5 ml. of a washed h e a v y suspension of r u m e n microorganisms f r o m a fistulated steer fed a ration of late-cut grass hay, molasses, urea, and minerals. A f t e r the period of incubation, the residual fiber was separated f r o m the tureen microorganisms b y differential centrifugation. The cellulose in the fiber was determined b y a modification of the method of C r a m p t o n a n d M a y n a r d (3). The bacterial protein was estimated by hydrolyzing the protein with concentrated HzSO~, and the subsequent determination of ammonia-nitrogen b y the nesslerization technique of LePage (8).
AVAILABILITY
OF NITROGEN
FROM
AMMONIATED
PRODUCTS
679
The ammoniated products, obtained from three manufacturers, had been treated with anhydrous ammonia at various temperatures and pressures to induce the chemical combinations. In certain cases the exact ammoniation procedure was not known, but inspection of the product indicated the severity of the treatment. The products are more thoroughly described in the presentation of the balance studies. In conjunction with these and other artificial rumen studies, the microbial population was examined by standard microscopic techniques and by the growth dilution method of Huhtanen et al. (6).
Results. Examination of the microbial population indicated that the centrifugation procedure did not significantly alter the numbers and types of bacteria present. However, certain changes were obvious in the fermentation liquor after incubation. The protozoal population, which was vigorous in the inoculum, was less apparent after incubation. The cultural technique revealed that a fast growing facultative streptococcus was usually 100 times more numerous, whereas the microscopic examination of gram stains showed the bacterial population to be less diverse after incubation. However, the bacteria present in the greatest number culturally post-incubation were similar to those considered the most numerous in the pre-incubation sample, and the bacteria most frequently observed microscopically were morphologically similar to a known cellulose digester previously isolated from the rumen. All of the above findings indicate that the population had changed during incubation under artificial conditions. However, significant similarities existed between the in vitro and in vivo microorganisms. This cursory bacteriological examination indicated that certain important reservations should accompany any in vitro data when extrapolating to the live animal. The results of the comparative nitrogen availability in the ammoniated products as shown by in vitro methods are presented in Table 2. In the first two series both cellulose digestion and protein synthesis were determined, but only cellulose digestion was estimated in the third and fourth series. Belasco (1) indicated that cellulose digestion was a valid indication o f nitrogen utilization or availability in vitro. Statistical evaluation of the data in Table 2 revealed that the correlation coefficient between the two measurements (cellulose digestion vs. protein synthesis) was 0.920 (degrees of freedom = 8), which is significant at the 0.01 probability level. The data suggest that the procedures employed in ammoniation significantly reduce the availability of nitrogen in all of the products tested except one. This exception, Series II and IV, ammoniated f urfural residue 3A, was ammoniated at atmospheric pressure followed by acid neutralization. This would indicate that the nitrogen in that particular sample was present as the ammonium ion per se or readily convertible to the ammonium ion in vitro. The other products in this fourth series were treated by high temperature, high pressure, saccharification, or combinations of these treatments. The ammoniation procedure which the citrus pulp and dried distillers molasses solubles received was unknown. It is apparent that the methods employed to introduce nitrogen into these products
R. F, DAVIS ET AL
680
TABLE 2
Effect of ammoniated by-products on eelhdose digestion and protein synthesis by ru~en organisms in vitro Supplement •
Cellulose digestion b
(%)
Protein synthesis b
(mg.)
Series I ( A m m o n i a t e d citrus p u l p ) ¢ N o n a m m o n i a t e d citrus p u l p 5.4 N o n a m m o n i a t e d citrus p u l p plus NH~C1 16.9 A m m o n i a t e d citrus p u l p ( S a m p l e ~:19) 11.2 A m m o n i a t e d citrus p u l p ( S a m p l e ;~20) 9.1
6.0 35.0 13.5 9.0
Series I I ( A m m o n i a t e d f u r f u r a l residue) c Control 10.1 NH4C1 23.6 A m m o n i a t e d f u r f u r a l residue ~3A 24.8 A m m o u i a t e d f u r f u r a ] residue ' ' A ' ' 16.6 A m m o n i a t e d f u r f u r a l residue ' ' B ' ' 15.5
4.4 34.4 38.1 18.1 24.4
Series I I I ( A m m o n i a t e d dried distillers molasses solubles) d Nonammoniated DDMS 11.9 N o n a m m o n i a t e d D D M S plus NH~C1 32.0 A m m o n i a t e d D D M S ~tl 10.7 A m m o n i a t e d D D M S ~3 14.5 A m m o n i a t e d I ) D M S ~$4 8.4 A m m o n i a t e d D D M S ~5 11.9 A m m o n i a t e d D D M S ~6 8.0 Control NH~C1 Ammoniated Ammoniated Ammoniated Ammoniated Ammoniated Ammoniated
Series I V ( A m m o n i a t e d f u r f u r a l residue) ~ 11.0 21.9 f u r f u r a l residue 1A 9.0 f u r f u r a l residue 2A 10.1 f u r f u r a l residue 3A 20.6 f u r f u r a l residue 4A 13.2 f u r f u r a l residue 5A 13,8 f u r f u r a l residue 6A 14.6
• The s u p p l e m e n t a l a m m o n i a t e d b y - p r o d u c t s or NIt4C1 c o n t r i b u t e d 4.4 rag. N to each fermentation apparatus. b E a c h value r e p r e s e n t s the mean of duplicate samples f r o m two experiments. B a s a l : a l f a l f a leaf meal - - cellulose m i x t u r e (0.5 g.) p l u s dried distillers molasses solubles (0.1 g.). d B a s a l : a l f a l f a leaf meal - - cellulose m i x t u r e (0.5 g . ) .
were sufficiently drastic (with the exception noted) to depress its availability to rumen bacteria as compared to ammonium chloride. Variations also existed among these products in the availability of their nitrogen. Some products showed an increased cellulose digestion and/or protein syntheuis above the negative control whereas others provided no available nitrogen. The possibility of inhibitory substances present in any of these products was not eliminated during the course of this investigation. BALANCE AND DIGESTION STUDIES
Procedure. Three balance studies, all conducted in a similar manner, were used to determine the availability of nitrogen from several ditTerent sources of ammoniated products. Four dairy steers, ranging in weight from 400 to 550 lb., were used in each experiment. The steers in each replicate were closely matched.
681
A V A I L A B I L I T Y OF N I T R O G E N FRO~a/i A M M O N I A T E D P R O D U C T S
Experimental periods consisted of a minimum of I week preliminary feeding and 1 week during which total collection of feces and urine Was made. The design of the experiment was a 4 × 4 Latin square. Animals were fed their preliminary rations in individual p e n s ; collections were made in crates especially designed for the separation and quantitative collection of urine and feces. Aliquot samples of all urine and feces were taken daily and composited. These were stored in closed containers under toluene and refrigerated until the end of the collection period, at which time samples were taken for proximate analysis. Feces samples were dried at approximately 60 ° C. for a minimum of 48 hours and allowed to come to equilibrium with the moisture in the atmosphere for a minimum of 48 hours before being g r o u n d in a Wiley mill and placed in sealed containers for analysis. All chemical analyses were conducted by accepted procedures. Trial 1 was conducted with three different ammoniated residues from the m a n u f a c t u r e of furfural. Original materials used in this process include corn cobs, oat hulls, cottonseed hulls, and other fibrous substances. Residue A had been hydrolyzed for 1/2 hour a f t e r sulfuric acid extraction to produce furfural, residue B had been hydrolyzed for 11/2 hours, and residue C for 31/~ hours. The experiment was designed to determine whether the different treatments affected TABLE 3 t~ations used in studying the value of three ammoniated furfural residues as sources of nitrogen for ruminants
Ingredient
Ration A
Ration B
Ration C
Ration D
(%) -23.0 --35.0 15.0 25.0 1.0 1.0
(%) --23.0 -35.0 15.0 25.0 1.0 1.0
(%)
Product A Product B Product C Soybean oil meal Corn meal Molasses Hay Bone meal Salt
(%) 23.0 ----35.0 15.0 25.0 1.0 1.0 100.0
100.0
100.0
100.0
--23.0 35.0 15.0 25.0 1.0 1.0
the availability of nitrogen from these products in comparison with the availability of nitrogen from soybean oil meal. The residue products were finely divided PoWders which were very light and dusty. The composition of the rations fed is shown in Table 3. The rations were prepared by mixing the corn meal, molasses, minerals, and ammoniated product or soybean meal in a small mechanical feed mixer. The hay, which was late-cut, low-protein timothy, was chopped to 1- to 2-in. lengths and mixed with the rest of the ration at the time of feeding. All animals received the same amount of feed d u r i n g each period. I n addition to the minerals listed, each animal received 50 g. of monosodium phosphate (NaH2PO~) daily after the first period. These rations were fed at a level which more than met the nitrogen requirement of the animals in the study.
R. F. DAVIS ET AL
682
TABLE 4 Daily ration ingredients used in studying the value of ammoniated molasses and ammoniated furfural residues as sources of nitrogen for ruminants :Ration Soybean oil m e a l
Ingredient ~
Hay Corn m e a l Molasses Ammoniated molasses S o y b e a n oil m e a l F u r a m e a l m i x l~o. 10 F u r a m e a l m i x No. 11 Total
NH~ molasses
Furameal No. 10
1%rameal No. 11
(g.)
(g.)
(g.)
(g.)
2268 898 1315 0 500 0 0
2268 1452 0 1315 0 0 O
2268 90 1088 0 0 1588 O
2268 O 1043 0 0 O 1906
4981
5035
5034
5217
a Also f e d w e r e : salt, free c h o i c e ; dicalcium p h o s p h a t e , 50 g / d a y ; cobalt, 0.3 r a g / d a y ; v i t a m i n A s u p p l e m e n t , twice weekly ( N o p c a y ' ' 10 ' ' s u p p l i e d b y N o p c o Chemical Co., H a r r i s o n , N. J . ) .
I n Trial 2 ammoniated molasses and soybean meal were employed, in addition to two products f r o m the residues of f u r f u r a l production. Residue No. 1 was a f u r f u r a l b y - p r o d u c t ammoniated at pressures greater than atmosphere. Residue No. 2 was the same material ammoniated at atmospheric pressures. The ammoniated molasses t r e a t m e n t was not known. The objectives of this s t u d y were the same as in Trial 1, that is, to determine the relative availability of nitrogen f r o m these sources as compared to soybean oil meal. F u r f u r a l residues were fed in a pelleted m i x t u r e containing a p p r o x i m a t e l y 53% of the residue, 13% molasses, 32% corn meal, and 2% minerals. E'qual amounts of h a y and molasses were included in all rations. The daily rations consumed are r e p o r t e d in Table 4. The nitrogen content of all rations was a d j u s t e d to the lower level of Morrison's recommendations. Since the n u t r i e n t content of the residues was unknown, no a t t e m p t was made to adjust T D N intake. However, the T D N content of the control ration fell within the r a n g e of Morrison's recommendations. TABLE 5 Daily ration ingredients used in studying the value of ammoniated DDMS and urea as sources of nitrogen for ruminants Rations I
Control
II NH, DDMS
III
IV
DDMS
Alfalfa
(g.)
(g.)
(g.)
(g.)
Timothy hay Alfalfa hay Corn m e a l DDMS NH4 D D M S Urea
2722 O 1769 O O 45
2722 O 1315 O 499 9
2722 O 1733 36 0 45
2268 454 1783 0 O 31
Total
4536
4536
4536
4536
AVAILABILITY OF NITROGEN Ft~OM AMMONIATED PRODUCTS
~
TABLE 6
Chemical co~aposition of the feed ingredients used in three balance studges involving ammoniatcd products
Water
(%)
Crude Nitrogen a protein
(%)
(~)
Ether extract
Crude fiber
Ash
W a t e r , aeid~ a n d b a s e soluble m a t t e r ~
(%)
(%)
(~)
(~)
1.9 2.4 2.2 2.0 3.4 3.4 --
45.6 51.3 54.1 33.0 2.1 6.7
3.7 3.8 3.9 3.6 1.4 5.7 7.6
22.0 16.5 16.5 47.0 72.7 28.8 56.6
1.9 2.0 --2.0 4.6 1.4
17.2 18.0 38.6 2.2 6.8
6.1 6.5 6.5 6.6 3.4 1.7 5.4
43.9 46.1 44.4 61.0 40.7 69.6 30.5
2.9 1.7 3.0 3.9 2.1 1.9
2.0 1.9 2.0 2.2 35.2 25.5
1.6 11.3 5.9 1.4 3.0 5.5
67.1 59.1 61.8 67.0 46.6 38.5
Trial I Residue A Residue B Residue C Hay Corn meal S o y b e a n oil meal Molasses
20.8 19.4 17.0 9.6 12.0 9.5 32.3
Residue m i x t u r e 1 Residue m i x t u r e 2 A m m o n i a t e d molasses Molasses Hay Corn meal Soybean oil meal
12.2 11.2 32.8 28.9 8.8 12.9 11.6
Control mix NH4 D D M S mix D D M S mix Mix fed w i t h a l f a l f a Timothy hay Alfalfa hay
10.5 10.9 11.1 11.6 7.6 10.2
6.0 6.6 6.3
--
-
-
-
4.8 8.4 45.9 3.5 Trial II
2.6
18.7 16.2 16.3 3.5 6.5 9.0 44.3 Trial I I I 15.9 15.1 16.2 13.9 5.5 18.4
a N i t r o g e n is r e p o r t e d as such in the a m m o n i a t e d products, since it is d o u b t f u l if there is more t h a n a very small a m o u n t of p r o t e i n material. b R e p o r t e d in this m a n n e r since the t e r m nitrogen-free e x t r a c t p r o b a b l y does n o t a p p l y to the a m m o n i a t e d products. This column includes nitrogen-free extract f o r the n a t u r a l products.
Trial 3 was a similar study, in which nitrogen from ammoniated dried distillers molasses solubles (DDMS) was compared with nitrogen from urea i n rations which contained the nonammoniated DDMS and a small amount of alfalfa. Previous studies indicated that DDMS had a stimulatory effect on the digestion of cellulose and the utilization of nonprotein nitrogen in vitro. These materials were included in this study in order to determine whether this effect could be demonstrated with animals. The rations fed in this experiment are reported in Table 5. Chemical compositions of all the feed ingredients are included in Table 6. Results. Digestion coefficients obtained in all three trials are reported i n Table 7. It will be noted that, compared with the soybean oil meal (control) ration, the rations which included the three different furfural residues exhibited a greatly lowered digestibility of dry matter. The digestibility of nitrogen was severely decreased when the residue products were the only source of supplemental nitrogen. Crude fiber also was digested at a much lower rate from the samples representing the furfural residue. This may be explained by the fact that this furfural residue had been already severely treated with sulfuric acid,
68~
R.F.
D A V I S E T AL
TABLE 7 Average digestion coefficients obtained from rations fed in three balance studies involving ammoniated products
Nitrogen
Ether extract
Crude fiber
Water, acid, and base soluble material ~
(%)
(%)
(%)
(%)
62 60 62 75
Trial I 51 52 47 66
69 73 72 77
30 31 27 54
74 74 75 80
Residue I Residue I I Ammoniated molasses Soybean oil meal
60 b 61 b 64 67
Trial I I 42 b 43 b 45 b 56
65 70 69
41 b 43 47 49
73 74 75 78
Corn and urea Corn and ammoniated DDMS Corn, urea, and DDMS Corn, urea, and alfalfa
63 60 64 68
Trial I I I 64 45 65 66
69 71 76 80
47 48 50 53
71 68 71 76
Dry matter (%)
:Residue A Residue B Residue C Soybean oil meal
Ration characteristics
66
Calculated in the same manner as nitrogen free extract but given this title due to the possible presence of nitrogen compounds. b Differences from control statistically significant at P =- < 0.05. a n d the r e m a i n i n g fiber p r o b a b l y was n o t s u b j e c t to a t t a c k b y b a c t e r i a l e n z y m e s i n the r u m i n a n t ' s digestive tract. The d i g e s t i b i l i t y of m a t e r i a l s soluble i n water, acids a n d bases, which w o u l d n o r m a l l y be called n i t r o g e n - f r e e extract, was also significantly reduced. S i m i l a r t r e n d s were n o t e d i n T r i a l 2. The d i g e s t i b i l i t y of d r y m a t t e r n i t r o g e n a n d of c r u d e fiber was r e d u c e d w h e n the f u r f u r a l residues were i n c l u d e d i n the r a t i o n . I n this t r i a l a m m o n i a t e d molasses p r o v i d e d a n i t r o g e n d i g e s t i b i l i t y o n l y s l i g h t l y b e t t e r thaa~ t h a t o b t a i n e d w i t h the a m m o n i a t e d f u r f u r a l p r o d u c t s . The d i g e s t i b i l i t y of n i t r o g e n f r o m a m m o n i a t e d D D M S i n T r i a l 3 was striki n g l y lower t h a n the coefficients o b t a i n e d for u r e a n i t r o g e n . T h e d i g e s t i b i l i t y of d r y m a t t e r f r o m the a m m o n i a t e d D D M S r a t i o n was also somewhat poorer t h a n f r o m other r a t i o n s . The i n c l u s i o n of 1 lb. of a l f a l f a h a y i n the r a t i o n i n c r e a s e d the d i g e s t i b i l i t y of all the n u t r i e n t s studied. N i t r o g e n b a l a n c e d a t a are i n c l u d e d i n T a b l e 8. I t m a y be seen t h a t the a m o u n t of n i t r o g e n a p p a r e n t l y absorbed reflects the r e d u c e d d i g e s t i b i l i t y of n i t r o g e n f r o m a m m o n i a t e d p r o d u c t s i n all t r i a l s . The values f r o m T r i a l 1 are s o m e w h a t h i g h e r t h a n those in the two other t r i a l s , because n i t r o g e n was overfed i n this s t u d y , whereas it was fed at the m i n i m u m r e q l l i r e m e n t i n T r i a l s 2 a n d 3. I n T r i a l 1 the absorbed n i t r o g e n f r o m the a m m o n i a t e d p r o d u c t s was n o t as well r e t a i n e d as t h a t f r o m s o y b e a n oil meal. I n T r i a l 2 r e s i d u e No. 2 a n d a m m o n i a t e d molasses p r o v i d e d absorbed n i t r o g e n i n a f o r m which was r e t a i n e d to the same e x t e n t as n i t r o g e n f r o m s o y b e a n oil m e a l , N i t r o g e n absorbed f r o n l r e s i d u e No. 1 was r e t a i n e d to a lesser degree. A b s o r b e d n i t r o g e n f r o m a m m o n i a t e d D D M S was
AVAILABILITY OF NITROGEN FROM AMMONIATED :PRODUCTS
685
TABLE 8
Average nitrogen balance data obtained from three balance studies involving ammoniated products Nitrogen Ration characteristics
Consumed
(g.)
P e r cent of
Apparently a b s o r b e d Retained
(g.)
A b s o r b e d hr retained
C o n s u m e d hr retained
(g.)
(%)
(%)
24 29 22 44
34 39 36 43
18 20 16 28
]3 17 16 20
38 49 46 45
17 22 21 25
20 12 26 23
43 39 56 45
28 18 37 30
Trial I Residue A Residue B Residue C Soybean oil meal
133 144 133 154
68 75 63 102 Trial I I
Residue I Residue I I A m m o n i a t e d molasses Soybean oil meal
74 78 79 79
Corn and urea Corn and a m m o n i a t e d D D M S Corn, urea, a n d D D M S Corn, urea, a n d a l f a l f a
71 68 71 77
29 33 35 44 Trial I I I 46 31 46 51
retained poorly in comparison to that from other sources (Trial 3). The inclusion of DDMS in the ration a p p a r e n t l y markedly improved the retention of absorbed nitrogen. The apparent improvement in the retention of digested nitrogen when DDMS was included in the ration is interesting in the light of in vitro experiments that have indicated increased cellulose digestion and utilization of nonprotein nitrogen by rumen bacteria when it was included in the media (16). DISCUSSION
The use and availability of nitrogen from ammoniated industrial by-products and ammoniated feeds by ruminants still require considerable investigation before a thorough knowledge will be obtained. It has been theorized that nitrogen from these sources might be as readily available to the microorganisms existing in the rumen as that from urea or natural protein. Several studies have indicated good or acceptable growth and use of nitrogen from these sources. A critical evaluation of some of these studies indicates that relatively low levels of nitrogen were supplied from the ammoniated products and that nitrogen from other sources was f r e q u e n t l y fed at levels above the minimum r e q u i r e m e n t s of the animals. Under these conditions one cannot obtain a critical evaluation of the utilization of nitrogen from any source. The studies here reported indicate that ammoniated industrial by-products and some ammoniated feeds do provide a source of nitrogen which can be utilized to a certain degree by r u m i n a n t microorganisms. However, this nitrogen does not appear to be available to the same degree as nitrogen from natural protein and is not as well utilized as nitrogen from urea. Similar results have been reported by Ferguson and Reeves (~/).
686
R . F . DAVIS ET AL
Since the chemical combinations t h a t occur when nitrogen is incorporated in feeds and industrial by-products are not well understood, it is not amiss to speculate t h a t m a n y of these compounds m a y be of a n a t u r e t h a t is not subject to attack b y enzymes n o r m a l l y occurring in the rumen. Unless this nitrogen is convertible to a utilizable f o r m by dissolution or enzyme hydrolysis, one would not expect it to be available for use b y the microorganisms. A p p a r e n t l y , f u r t h e r research is necessary to find an ideal source of nonprotein nitrogen for ruminants. A m m o n i a t e d products m a n u f a c t u r e d u n d e r more subtle or better controlled conditions could result in an ideal source of a nonprotein nitrogen for microorganisms. This could be a p r o d u c t which would be attacked b y some normal enzyme system occurring in the r u m e n and f r o m which the nitrogen was liberated at a p p r o x i m a t e l y the same rate as the energy necessary for the synthesis of protein. Another factor which enters into the feasibility of using a m m o n i a t e d industrial by-products as a source of nitrogen for r u m i n a n t s is the fact t h a t m a n y of them, as shown in this paper, contain relatively low levels of energy or available nutrients. I t appears economically unfeasible to t r a n s p o r t large amounts of relatively inert materials long distances for feeds. A source of nonprotein nitrogen which is concentrated or else contains appreciable amounts of energy would a p p e a r to be much more practical. The possibility of toxic reactions m u s t be considered in evaluating ammoniated products as a source of nitrogen. Recent reports b y l~ichardson et al. (14) have indicated t h a t animals fed fairly low levels of ammoniated molasses have shown nervous symptoms. A p p a r e n t l y , other workers have made similar observations. Until more is known about the chemical combinations a p p e a r i n g f r o m products a m m o n i a t e d u n d e r relatively severe conditions, caution should be used in the feeding of these materials. SUMMARY
A series of studies on the in vitro and in vivo utilization of nitrogen from a m m o n i a t e d industrial by-products and ammoniated feed stuffs has been reported. The results of both types of studies indicate t h a t nitrogen f r o m ammoniated products is not available to r m n e n bacteria or to the host to the same degree as is nitrogen f r o m u r e a or f r o m n a t u r a l protein sources. ACKNOWLEDGMENTS These studies were supported in part by The Quaker Oats Company, Chicago, and Publicker Industries, Inc., Philadelphia. REFERENCES (1) BEbASCO, I. J. New Nitrogen Feed Compounds for Ruminants - - a Laboratory Evaluation. J. Animal Sci., 13: 601. 1954. (2) BURROUGHS,W., FRANK, N. A., GERLAUGH, P., AND BETHKE, !t. M. Preliminary Observations upon the Factors Influencing Cellulose Digestion by Rumen Microorganisms. J. Nutrition, 40: 9. 1950.
AVAILABILITY OF NITROGEN FROM AMMONIATED PRODUCTS
687
(3) CRAMPTON,E. W., AND MAYNARD, l'J. A. The Relation of Cellulose and Lignin Content to the Nutritive Value of Animal Feeds. J. Nutrition, 15: 383. 1938. (4) F~RGUSON, W. S., AND REEVES, O. Nutritive Value of Ammoniated Sugar Beet Pulp. J. Agr. Sci., 33: 95. 1943. (5) HU~TANEN, C. N., AND GALL, L. S. The Miniature Artificial Rumen and Its Uses. J . Animal Sci., 11: 766. 1952. (6) C. HUHTANEN, N., ROGERS, M. R., AND GALL, .~. S. Improved Technique for Isolating and Purifying Rumen Bacteria. J. Bacteriol., 64: 17. 1952. (7) KNODT, C. B., WILLIAMS, J . B., AND BRUMBAUGH, J . Ammoniated Cane Molasses and Similar Products in the Rations of Dairy Calves. J. Dairy Sci., 34: 1042. 1951, (8) LEPAGE, G. A. Manometric Techniques and Tissue Metabolism. Burgess Publishing Co., Minneapolis, Minn. 1951. (9) LOUW, J. G., WILLIAMS, H. H., AND MAYNARD, L. A. A New Method for the Study in vitro of Rumen Digestion. Science, 110: 1. 1949. (10) MAcGI~IYDE4¢,N. D., KNODT, C. B., AND WILLIAI~S, P. S. The Value of Ammoniated Industrial By-Products in the Rations of Dairy Heifers and Cows. J. Dairy Sei., 34: 1055. 1951. (11) McCALL, R., AND GRAHA:k~, W. R., JR. The Value of Several Ammoniated Products as Feed for Beef Cattle. J. Animal Sci., 12: 798. 1953. (12) MILLAR, H. C. Ammoniated Sugar Beet Pulp as a New Nitrogenous Feed for Ruminants. J. Dairy Sci., 27: 255. 1944. (13) REID, J. T. Urea as a Protein Replacement for Ruminants: A Review. J. Dairy Sci., 36: 955. 1953. (14) RIChArDSON, D., SMITH, E. F., BAKE~, F. H., Cox, R. F., AND MCREYNOLDS, K. L. The Value of Ammoniated Molasses in Beef Cattle Wintering Rations 1953-1954. Kan. Agr. Expt. Sta., Circ. 308. 1954. (15) TILL~AN, A. D., AND KIDWELL, J. F. The Value of Ammoniated Condensed Distillers Molasses Solubles as a Feed for Beef Cattle. J. Anima~ Sci., 10: 934. 1951. (16) WASSEa~AN, R. H., DAVIS, R. F., AND LOOSLI, J. K. Unpublished data. Cornell University, Ithaca, N. ¥ . 1953. (17) WEGNER, M. I., BGO~'~, A. W., BOHSTEDT, G., AND HART, E. B. The in vitro Conversion of Inorganic Nitrogen to Protein by Microorganisms from the Cow's Rnmen. J. Dairy Sci., 23: 112. 1940.