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Influence of dietary protein on urea levels in blood and milk of buffalo cows
Livestock Production Science 55 (1998) 135–143
Influence of dietary protein on urea levels in blood and milk of buffalo cows a, a a b a G. Campanile *, C. De Filippo , R. Di Palo , W. Taccone , L. Zicarelli a
Dipartimento di Scienze Zootecniche, Via F. Delpino 1, 80137 Napoli, Italy b Laboratorio Analisi Centrale ASL, 2 Via Degli Imbimbo, Avellino, Italy Received 7 February 1997; accepted 28 January 1998
Abstract The aim of the study described herein, was to verify the influence of changing protein levels in buffalo cow diets on quantity–quality of milk yield, blood (BU) and milk (MU) urea, metabolic profile and milk freezing point (MFP). The experiment was carried out on eight buffaloes divided into two trials (trial 1 and trial 2), each trial divided into three periods (P1, P2 and P3). The levels of CP/ DM in the three periods were 9%, 12% and 9%, respectively. Buffaloes of trials 1 and 2 were different for days in milk (DIM 5 164 vs. 132, respectively) and for milk yield (7 kg vs. 10 kg, respectively). Protein requirements were consistently met in trial 1 buffaloes, while the subjects of trial 2 were protein deficient in periods P1 and P3. The increase of CP/ DM (P2) increased milk protein level and quantity, and MU in trial 2. The CP/ DM increase caused, moreover, a decrease, and therefore an improvement in the MFP, and an increase in BU values in both trials. Multiple regression analyses showed a strong link between MU and BU (R 2 5 0.769; P , 0.01) and between MU and BU as regards protein / energy ratio. The MFP, moreover, varied according to MU values (R 2 5 0.685; P , 0.01). The increase in protein concentration in subjects whose protein requirements had already been met, (trial 1) brought about an excess of protein which probably triggered a more intense gluconeogenesis confirmed by the higher glycemia levels (P , 0.01) in P2. In trial 2, on the other hand, the protein requirements met in P2 resulted in greater milk yield, which, not being backed up by the energy levels in the diet, caused a relative energy deficiency in the animals. This hypothesis is confirmed by the increase in b-hydroxybutyrate and lipoproteins as well as the lower body condition score levels in P2 vs. P1 and P3. The existing connection between protein / energy ratio and MU could indicate that in buffalo, as in cattle, MU can be used as a valid parameter in order to highlight the existence of an alteration in the protein / energy ratio of the diet. The normalization of the MFP upon the increase of the CP/ DM ratio confirms that in a diet rich in fermentable energy, it is necessary to increase the dietary protein concentration and meet requirements, in order to avoid abnormalities of the physical characteristics of the milk. 1998 Elsevier Science B.V. Keywords: Buffalo; Dietary protein; Metabolic profile; Milk urea
1. Introduction *Corresponding author. Tel.: 1 39 81 291663; fax: 1 39 81 292981; e-mail: [email protected]
The urea nitrogen concentration in plasma and milk in cattle is influenced by the amount of crude
0301-6226 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 98 )00123-7
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protein in the diet (Carlsson and Pehrson, 1994; Gonda and Lindberg, 1994; Baker et al., 1995), as well as by degradable intake protein (DIP) and undegradable intake protein (UIP). Moreover, it is clearly correlated with dietary protein supply relative to requirements (Baker et al., 1995). Roseler et al. (1993) also showed that urea concentrations in blood and milk are influenced by changes in DIP and UIP and influenced by increasing energy intake. The relation between the protein fractions of the diet and the protein / energy (P/ E) ratio in cattle have previously been described (Oltner and Wiktorsson, 1983; Oltner et al., 1985). Blood urea concentration decreased in lactating dairy cows, when an optimal level of ruminally fermentable carbohydrate was supplied to enhance the capture of DIP into microbial protein (Hoover and Miller, 1990). Excess degradable protein responsible for increases in blood and milk urea levels are shown to be poorly used in the production of milk proteins. In fact, Baker et al. (1995) noted an increase in ‘‘true’’ proteins when the DIP and UIP were consistent with production requirements. Azotemia is found to be closely correlated with milk urea concentration (Oltner and Wiktorsson, 1983; Gustaffson and Palmquist, 1993; Roseler et al., 1993; Baker et al., 1995). This parameter can be used, since it facilitates monitoring for sampling feeding adequacy. This analysis cannot replace conventional feed analysis, but can be utilized as a valuable complement to it (Oltner and Wiktorsson, 1983). The change in milk urea content in response to an altered dietary composition is very rapid in cattle (Oltner et al., 1985). Metabolic profile tests (Zicarelli et al., 1982, 1986; Bertoni et al., 1993a; Kobeisy and Ibrahim, 1993; Bertoni et al., 1994; Campanile et al., 1994, 1995) have been carried out on buffalo involving plasma (but not milk) urea concentrations, however studies into protein requirements during lactation in buffalo are fewer than in cattle. Protein concentrations used in lactating buffalo diets can be equal to or go below 12% on DM, since these concentrations have little influence on the quantity–quality of milk yield (Verna et al., 1992; Tripaldi, 1994; Verna et al., 1994). Sivaiah and Mudgal (1978) suggested the administration of 166.34 g to 126.03 g (Kurar and
Mudgal, 1980) of digestible crude protein / 100 g of milk produced protein, while according to Rai and Aggarwal (1991), the concentration of crude protein on dry matter should be between 11% and 14%. Tweatia and Bhatia (1996) state that the ideal protein content in the diet stimulates ruminal microflora. Moreover, Singh and Gupta (1984) detected a higher quantity of volatile fatty acids (VFAs) and ATP in buffalo cows fed a greater quantity of nitrogen with ammoniated straw. The present studies were aimed at verifying whether in buffaloes in midlactation, varying protein levels in the diet (in subjects whose protein requirements were or were not met) could influence the quantity–quality of milk yield, blood (BU) and milk urea (MU) nitrogen levels, metabolic profile and milk freezing point (MFP).
2. Materials and methods
2.1. Experimental animals and feed Trials were carried out in duplicate (trials 1 and 2) on eight buffaloes, each trial divided into three experimental periods (P1, P2, P3). In the first period P1, duration 40 days, subjects were fed a diet containing 9% crude protein / dry matter (CP/ DM). In the second period P2, duration 21 days, CP/ DM was increased to 12%. In the third period P3, duration 21 days, CP/ DM was reduced again to the level of P1. In trial 1, eight buffaloes 164 days in milk (DIM) were used, yielding an average of 7 kg / day of milk. In these subjects protein requirements were met. In trial 2, eight buffaloes 132 days DIM were used, yielding an average of 10 kg / day of milk. In these latter subjects, however, protein requirements in P1 1 P3 were not sufficiently met. It is important to note that the lactation period in buffalo is 270 days. Subjects were kept in cement paddocks and were fed once daily at 09:00 h for 10% orts. Feed consisted of total mixed ration based on corn silage, barley meal, soybean meal, sunflower meal, beet pulp, ryegrass hay and vitamin and mineral supplements. The different protein concentration in the diets was guaranteed by varying the quantity of soybean meal.
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Feed intake and orts for each trial were measured daily. Orts were sampled weekly on a random day. Analyses of individual foodstuffs and orts were carried out as per ASPA Commissione Valutazione Degli Alimenti (1980) methods and energy values calculated according to INRA (1988) equations. To calculate protein fractions (DIP, UIP, PDIN, PDIE), values obtained from fistulated buffaloes were used (ASPA Commissione Proteine nella Nutrizione e nell’Alimentazione dei Poligastrici, 1994; Di Lella et al., 1995; Infascelli, pers. comm.).
2.2. Milk sampling and analytical methods At the end of each trial period, milk samples from each buffalo were collected during morning and afternoon milking in order to evaluate milk composition (fat and protein), using the Foss Electric Milko-Scan 139 (calibrated with appropriate buffalo standard) and the MFP (using Genotec 030 osmometre). During the whole experimental period the daily milk yield was recorded. Milk urea (MU) was measured by the urease method-kits (Boheringer).
2.3. Standard milk and calculation of differences between intake and requirements Buffalo standard milk (ECM 5 740 kcal) was calculated using the formula for buffalo cows (Di Palo, 1992; Di Palo and Cheli, 1995): ([hfat (g / kg) 2 40 1 protein (g / kg) 2 31j0.01155] 1 1)milk yield. Estimation of the differences (D) between nutritive values intake and relative requirements is reported as follows: DM intake 5 91 g 3 kg metabolic weight 1 0.27 kg 3 kg ECM (Campanile et al., 1996); DCP 5 g CP intake 2 (80 g CP 3 100 kg live weight 1 2.7 g CP 3 g milk protein yield); DUFL 5 UFL (1700 kcal NEL) intake 2 [(1.4 1 0.6 3 100 kg live weight) 3 1.1 1 0.44 UFL 3 kg ECM)] (INRA, 1988); DPDI 5 PDI intake 2 (3.25 g PDI 3 kg of metabolic weight 1 1.55 3 g milk protein yield) (INRA, 1988).
2.4. Blood samples and analytical methods At the end of each experimental period blood samples were collected at 08.00 h, before feeding, from the jugular vein in vacutainer tubes. The tubes
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were centrifuged at 3000 rpm for 15 min. The recovered serum was stored at 2 188C until analyzed for metabolic profile. Immediately following sampling, the glycemia was measured on plasma by means of the colorimetric method. During each experimental period the following blood parameters were measured on serum using the enzymatic colorimetric method: urea (BU) (urease method), creatinine, total cholesterol, HDL cholesterol, triglycerides. Colorimetric methods were used to measure the total proteins; the serum protein fractions were evaluated by the electrophoresis technique. Sodium, potassium, chlorine were measured by means of a potentiometer using ionoselective electrodes. Following the results of the first trial, it was decided to analyse the b-hydroxybutyrate (BHBA), non esterified fatty acids (NEFAs), various serum enzymes such as ALT, AST, GGT, CPK, LDH (kinetic at 378C) using the enzymatic colorimetric method, and insulin by automated fluoro-immunometric method on AIA 1200; TOSO.
2.5. Body condition score Body condition scores were assigned using a scale of 1 to 9 (Wagner et al., 1988) (modified to the buffalo) at the end of each trial period.
2.6. Statistical analysis Correlation analyses (SPSS / PC 1 User Guide, 1986) were performed between blood and milk parameters of each trial and days in milk. No linear trend with time was observed so the differences between mean values of P1, P2 and P3 of each trial were tested using the SPSS / PC 1 software package Student’s t-test with pairwise procedure (1986). Linear regression analyses were performed for MFP and MU as dependent variable using BU as independent variable. Multiple linear regression were performed with SPSS / PC 1 stepwise procedure for: (i) MFP as dependent variable and five explicative variables: milk osmolarity, fat, protein, lactose and MU; (ii) MFP, MU and BU as dependent variables in function of dietary characteristics, intake of single diet compounds and difference between intake and requirements.
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Table 1 Characteristics of dry matter intake Trial
1
period
P1 1 P3
P2
P1 1 P3
P2
DMI (kg / d) UFL / DM
14.4 0.84
14.7 0.84
15.5 0.83
15.8 0.83
(%)
(%)
(%)
(%)
9.9 5.6 7.7 7.1 2.8 71.7 2.53 24.3 39.1 42.3 25.7 0.253
12.1 7.1 8.4 8.5 3.6 70.2 2.63 20.3 36.8 42.0 25.1 0.33
8.7 5.5 6.9 6.3 2.4 72.4 3.32 22.8 34.1 47.1 24.3 0.26
12.3 8.4 8.4 8.5 3.8 69.1 2.96 21.6 33.1 44.9 23.2 0.37
CP PDIN PDIE DIP UIP DIP/ CP Ether extract Starch NSC NDF ADF CP/ NSC
in the two trials. The DIP/ UIP rates did not change upon modification of the diet. In both trials milk production requirements for energy were met due to the energy density (UFL / DM) of the diet (Table 2). In particular, in trial 1, the buffaloes in full recovery phase ( . 160 DIM), were fed 8.7% more energy than required, notwithstanding a lower dry matter intake. In trial 2 a greater DMI was found, due to lower DIM and higher productive level. In this trial the theoretical energy requirements were met, except in P2. Protein requirements were met in trial 1, while in trial 2, during P1 and P3, a noticeable deficiency of about 350 g of crude proteins was evident. The deficiency would be estimated as only 54 g according to the specifications of Sivaiah and Mudgal (1978). If the data is analyzed according to the French (INRA, 1988) system (PDI), then the diet of trial 1 would also be considered deficient in PDI of approximately 103.5 g in P1 and P3, while in trial 2 the protein deficiency would be higher (2242 g). In both trials the availability of NSC enabled the synthesis of the nitrogen of the DIP into microbial protein (Table 1). Greater milk yield (Fig. 1) was measured in P1 and P2 vs. P3 in both trials 1 (P , 0.05) and 2 (P , 0.01) and was attributed to the DIM. If we consider ECM, these differences are not significant even if both trial groups verified greater
2
3. Results and discussion Table 1 shows the chemical composition of the dry matter intake of the two trials during the various periods. The different compositions of the dry matter intake in trial 2, in relation to the CP/ DM, starch / DM and CF / DM is mainly linked to the different type of orts in the two trials and partly to the quantity of corn grain present in the corn silage used
Table 2 Milk and milk protein yields, and differences between crude protein and energy intakes with their respective requirements Trial
1
2
period
P1
P2
P3
P1
P2
P3
ECM (kg) Milk protein / d (g) DMI (kg) CP (g) DCP 1 (g) DCP 2 (g) UFL intake DUFL DPDI
12.7 351 14.5 1435 17 1 214 12.2 1 1.05 2 127
13.4 346 14.7 1779 1 365 1 567 12.3 1 0.95 1 89
12.3 316 14.4 1426 1 93 1 268 12.1 1 1.19 2 80
16.9 463 15.6 1357 2 373 2 65 12.95 10 2 254
17.5 516 15.8 1943 1 70 1 426 13.11 2 0.107 1 63
15.9 441 15.4 1340 2 331 2 43 12.78 1 0.25 2 231
DCP 1 (g) 5 CP intake 2 CP requirement assessed by the authors. DCP 2 (g) 5 CP intake 2 CP requirement assessed by Sivaiah and Mudgal (1978). DUFL 5 UFL intake 2 UFL requirement. DPDI 5 PDI intake 2 PDI requirement.
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requirements suggested by the authors themselves and those of France (INRA, 1988), as opposed to those from India (Sivaiah and Mudgal, 1978). The MFP (Fig. 3) was found to be significantly higher (P , 0.01) in both trials when the CP/ DM of the diet was lower (P1 and P3 vs. P2); and in both trials the MFP values in P3 seem to be higher than in P1. The change in the diet (P2 vs. P1) showed a slight increase (not significant however) in the urea levels of the blood (Fig. 4), which went from 5.16 mmol / l Fig. 1. Mean milk and ECM yields in the three periods (P1, P2 and P3) separately for trials 1 and 2. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
milk yield during P2, since a decrease in milk yield is usually associated with an increase in milk lipid percentage due to an increase of the DIM (Fig. 2). With regards to trial 1, no change in the percentage or quantity of milk protein was recorded. In trial 2, the increase of CP/ DM in the diet from 9% to 12% led to an increase (P , 0.01) of protein levels and of protein quantity produced in the milk in P2 vs. P1 and P3 (Fig. 2). Only during P2 were protein requirements met, guaranteeing an adequate availability of nitrogen for milk protein biosynthesis. These results are more easily explained using the
Fig. 2. Mean milk fat and protein concentration in the three periods (P1, P2 and P3) separately for trials 1 and 2. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
Fig. 3. Mean milk freezing point in the three periods (P1, P2 and P3) separately for trials 1 and 2. (A, B on bars: P , 0.01).
Fig. 4. Mean blood urea levels separately for trials 1 and 2 and milk urea levels in trial 2 in the three periods (P1, P2 and P3). (A, B on bars: P , 0.01).
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(trial 1) and 6.23 mmol / l (trial 2) in P1 to 6.01 mmol / l and 6.47 mmol / l, respectively for the two trial groups in P2. In P3 the decrease of CP/ DM to the same levels as in P1 caused a noticeable decrease of the urea levels (P , 0.01) in both trials. In earlier treatments (P1 and P2) went to 3.36 mmol / l (trial 1) and 3.85 mmol / l (trial 2). A similar trend was also found in trial 1 with regard to MU (Fig. 4). The length of the period during which a diet with low protein concentration was fed influenced urea levels in blood and milk. In fact, the urea levels in blood and milk reached higher levels when a low-protein diet was given for a fairly long period of time, whereas levels were lower immediately following a sharp reduction in the protein contribution of the diet. Following sharp reduction in the protein content of the diet, a decline in circulating urea was verified. This occurred (as shown in our experiment) when a fermentable energy supply was present. We propose that an increase of insulin levels reduced or blocked the normal amino acid breakdown, and therefore, reduced the urea levels in circulation. An increase in protein concentration in the diet meeting requirements of the trial 2 buffaloes resulted in: an increase in milk yield (interrupting the physiological decrease due to DIM), stabilization of the MFP (P , 0.01) and an increase in milk protein percentage and urea nitrogen levels (MU). The major differences between trial 2 vs. trial 1 were the more marked increase of the CP-level and in particular, of the PDIN of the DMI in P2 vs. P1 and P3.
the diet (the protein / energy ratio), the MU level rises upon increasing the CP/ NSC ratio and decreasing the UIP/ NSC ratio. Thus: MU (mmol / l) 5 2 5.37 1 41.60 CP/ NSC 2 53.58 UIP/ NSC; R 2 5 0.906. Upon elaboration of the results of both trials the following weak relationships are observed: (a) BU (mmol / l) 5 13.58 1 16.23 CP/ NSC 2 0.04 g starch intake 2 1.03 UFL; R 2 5 0.574; (b) MFP 5 2 0.530 2 0.926 UIP/ NSC 1 0.00003 g starch intake; R 2 5 0.383; (c) MFP 5 2 0.473 1 0.01 BU (mmol / l); R 2 5 0.257. It should be emphasized that no association between MFP and serum osmolarity was found, as had been previously found in cattle (Peterson and Freeman, 1966). We also wish to point out that the increase of CP/ DM resulted in significantly higher blood concentrations of both K and Cl in these trials (Fig. 5). These studies show that BU and MU levels are positively influenced by the CP/ NSC ratio in the buffalo. In fact, the greater availability of rumen fermentable energy, can permit a better use of ammonia at ruminal level for microbial protein synthesis. Therefore, a smaller quantity of blood urea would be present (Journet et al., 1975).
3.2. Metabolic profile The metabolic profile examination showed that in P2 BHBA and total cholesterol blood levels (Fig. 6) increased in trial 2 (in trial 1 BHBA was not
3.1. Regression analyses The relationship between the MFP and the components of the milk in trial 2 is expressed by the following equation: MFP 5 2 0.413 2 0.487 MU (mmol / l) 2 0.0182 milk protein (%) 2 0.00095 milk (kg); R 2 5 0.685. With regards to this study whenever milk urea was measured, BU was positively related to MU over the three experimental periods. The relationship between MU and BU is expressed by the following equation, which is similar to results found by other authors (Roseler et al., 1993; Gonda and Lindberg, 1994): BU (mmol / l) 5 1.675 1 0.817 MU (mmol / l); R 2 5 0.769. When one changes the components of the DM in
Fig. 5. Mean serum levels of K, Na and Cl in the three periods (P1, P2 and P3) separately for trials 1 and 2. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
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Fig. 6. Mean blood glucose (Gluc) and total cholesterol (Chol) levels in the two trials and mean blood NEFA and BHBA levels in trial 2 in the three periods. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
measured) while blood glucose decreased. The greater quantity of protein fed in the diet, increased protein synthesis, which requires greater energy availability. Energy requirements were probably not met, because the energy level in the diet was not varied (Table 2). This could have caused a ‘‘relative’’ energy deficiency which could explain the increase of BHBA derived from lipid catabolism, evidenced by the transient increase in blood lipoprotein levels (Fig. 6). Moreover, the imbalance between energy used for milk production and energy intake is calculated at an estimated deficiency of 0.107 UFL per day. As a results of energy imbalance BCS decreased (although not significantly) 0.25 points in P2 and increased 0.64 points in the following period (P3), when excess energy of 0.25 UFL per day was estimated (Table 2). NEFA blood levels did not significantly increase (Fig. 6). Elevated insulin blood levels ( . 6 U / ml) characteristic of midlactation, did not permit the optimum use of this metabolic pathway. As previously proposed, in trial 1 where only the protein requirements were met in P1 and P3, the further increase of protein intake in P2 triggered a more intense gluconeogenesis evidenced by the glycemia increase (P , 0.01) and the stability of plasma lipoprotein (Fig. 6). In the second trial the increase of CP/ DM in the diet resulted in greater milk yield and an improvement in milk quality and also gave rise to increased
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Fig. 7. Mean serum levels of ALT, AST, CK and GGT in the three periods (P1, P2 and P3) in trial 2. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
serum enzymatic activity (Fig. 7) which was necessary to deal with the sudden increase in biosynthetic activity.
4. Conclusion In the Italian-bred Mediterranean buffalo cow, diets of low ratio protein / energy or with low protein content DM are common since high quantities (20– 34 kg) of corn silage are used. This sometimes causes an increase in the MFP often responsible for contractual problems within the dairy industry. In most cases an increase in diet protein concentration can normalize the MFP. Similar results have been found in Jersey cows (Peterson and Freeman, 1966). In those cases, higher MFP values were found in subjects fed low protein level diets while the same diets did not alter the MFP in Holstein cows (Peterson and Freeman, 1966). Those results and ours suggest that there may be one or more minor factors which affect the MFP. It should be remembered that Jersey and buffalo cows yield milk with a higher dry residue than the Holstein. In buffalo cows, as well as in dairy cows, dietary protein characteristics and P/ E ratio influence urea levels in blood and milk. This ratio (P/ E) must be further evaluated considering that the buffalo cow adapts itself to the lack of protein easier than the dairy cow (Bertoni et al., 1993b). It is important to remember that low values of BU and MU were
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reported only when we passed from high to low protein content diets in both trials. Smith (1969) reported that deficiency of nitrogen in ruminants decreases the efficiency of urea clearance by the kidney, increasing ruminal return and decreasing hematic levels. In agreement with Oltner and Wiktorsson (1983), we show that in buffalo cows the urea determination in milk can be used as a valid method of estimating the P/ E ratio but it cannot replace conventional feed analysis. From these results we also conclude that the buffalo cow diet should ensure a sufficient protein percentage, not only to meet requirements, but also taking into consideration the energy level, above all in diets rich in fermentable energy. Moreover if the task was only to meet requirements, then low protein concentrations would suffice (9% of DM) but these often cause alterations of the MFP.
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G. Campanile et al. / Livestock Production Science 55 (1998) 135 – 143 Rai, S.N., Aggarwal, S.K., 1991. Effect of substitution of green fodder with ammoniated straw on nutrient utilization and milk production in Murrah buffaloes. Buffalo J. 1, 51–61. Roseler, D.K., Ferguson, J.D., Sniffen, C.J., Herrema, J., 1993. Dietary protein degradability effect on plasma and milk urea nitrogen and milk nonprotein nitrogen in Holstein cows. J. Dairy Sci. 76, 525–534. Singh, S.P., Gupta, B.N., 1984. Effect of ammonia treatment of wheat straw on fibre digestion and volatile fatty acids production rates in the rumen of male Murrah buffaloes. Indian J. Anim. Nutr. 1, 27–30. Sivaiah, K., Mudgal, V.D., 1978. Effect of feeding different levels of protein and energy on feed utilization for growth and milk production in buffaloes (Bos bubalis). Annual Report, National Dairy Research Institute. Karnal, India, pp. 145–146. Smith, R.H., 1969. Reviews of the progress of dairy science. J. Dairy Res. 36, 313–331. SPSS / PC 1 User Guide. McGraw-Hill, NY, 1986. Tripaldi, C., 1994. Effetto di alcune diete sulle caratteristiche qualitative del latte di bufala. Agric. Ricerca 153, 79–86. Tweatia, B.S., Bhatia, S.K., 1996. Comparative studies in rumen ammonia anabolising enzymes, microbial and mineral profile between buffalo and cattle fed fibrous diet. Buffalo J. 12, 169–179.
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Verna, M., Bartocci, S., Amici, A., Agostini, M., 1992. Effect of diets with different energetic concentration on the qualitative and quantitative yield in lactating buffaloes. Proc. of the Inter. Symp. ‘‘Prospect of buffalo production in the Mediterranean and the Middle East’’, Cairo, 9–12 November, pp. 258–261. Verna, M., Bartocci, S., Amici, A., Agostini, M., 1994. Effetto di diete diverse sulle prestazioni produttive di bufale in lattazione. Agric. Ricerca 153, 73–78. Wagner, J.J., Lusby, J., Oltjen, J.W., Rakestraw, J., Wettermann, R.P., Walters, L.E., 1988. Carcass composition in mature Hereford cows: estimation and effect on daily metabolizable energy requirement during winter. J. Anim. Sci. 66, 603–612. Zicarelli, L., Intrieri, F., de Franciscis, G., 1982. Variazioni stagionali ed aziendali del profilo metabolico nei bufali. Riv. Zoot. Nutr. Anim. 8, 321–336. Zicarelli, L., Avallone, L., Salvatore, M., Pizzuti, G.P., 1986. Comportamento di alcune costanti ematochimiche nella bufala in gravidanza e lattazione. Atti XL Conv. Naz. S.I.S.Vet., pp. 569–572.
Influence of dietary protein on urea levels in blood and milk of buffalo cows a, a a b a G. Campanile *, C. De Filippo , R. Di Palo , W. Taccone , L. Zicarelli a
Dipartimento di Scienze Zootecniche, Via F. Delpino 1, 80137 Napoli, Italy b Laboratorio Analisi Centrale ASL, 2 Via Degli Imbimbo, Avellino, Italy Received 7 February 1997; accepted 28 January 1998
Abstract The aim of the study described herein, was to verify the influence of changing protein levels in buffalo cow diets on quantity–quality of milk yield, blood (BU) and milk (MU) urea, metabolic profile and milk freezing point (MFP). The experiment was carried out on eight buffaloes divided into two trials (trial 1 and trial 2), each trial divided into three periods (P1, P2 and P3). The levels of CP/ DM in the three periods were 9%, 12% and 9%, respectively. Buffaloes of trials 1 and 2 were different for days in milk (DIM 5 164 vs. 132, respectively) and for milk yield (7 kg vs. 10 kg, respectively). Protein requirements were consistently met in trial 1 buffaloes, while the subjects of trial 2 were protein deficient in periods P1 and P3. The increase of CP/ DM (P2) increased milk protein level and quantity, and MU in trial 2. The CP/ DM increase caused, moreover, a decrease, and therefore an improvement in the MFP, and an increase in BU values in both trials. Multiple regression analyses showed a strong link between MU and BU (R 2 5 0.769; P , 0.01) and between MU and BU as regards protein / energy ratio. The MFP, moreover, varied according to MU values (R 2 5 0.685; P , 0.01). The increase in protein concentration in subjects whose protein requirements had already been met, (trial 1) brought about an excess of protein which probably triggered a more intense gluconeogenesis confirmed by the higher glycemia levels (P , 0.01) in P2. In trial 2, on the other hand, the protein requirements met in P2 resulted in greater milk yield, which, not being backed up by the energy levels in the diet, caused a relative energy deficiency in the animals. This hypothesis is confirmed by the increase in b-hydroxybutyrate and lipoproteins as well as the lower body condition score levels in P2 vs. P1 and P3. The existing connection between protein / energy ratio and MU could indicate that in buffalo, as in cattle, MU can be used as a valid parameter in order to highlight the existence of an alteration in the protein / energy ratio of the diet. The normalization of the MFP upon the increase of the CP/ DM ratio confirms that in a diet rich in fermentable energy, it is necessary to increase the dietary protein concentration and meet requirements, in order to avoid abnormalities of the physical characteristics of the milk. 1998 Elsevier Science B.V. Keywords: Buffalo; Dietary protein; Metabolic profile; Milk urea
1. Introduction *Corresponding author. Tel.: 1 39 81 291663; fax: 1 39 81 292981; e-mail: [email protected]
The urea nitrogen concentration in plasma and milk in cattle is influenced by the amount of crude
0301-6226 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 98 )00123-7
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protein in the diet (Carlsson and Pehrson, 1994; Gonda and Lindberg, 1994; Baker et al., 1995), as well as by degradable intake protein (DIP) and undegradable intake protein (UIP). Moreover, it is clearly correlated with dietary protein supply relative to requirements (Baker et al., 1995). Roseler et al. (1993) also showed that urea concentrations in blood and milk are influenced by changes in DIP and UIP and influenced by increasing energy intake. The relation between the protein fractions of the diet and the protein / energy (P/ E) ratio in cattle have previously been described (Oltner and Wiktorsson, 1983; Oltner et al., 1985). Blood urea concentration decreased in lactating dairy cows, when an optimal level of ruminally fermentable carbohydrate was supplied to enhance the capture of DIP into microbial protein (Hoover and Miller, 1990). Excess degradable protein responsible for increases in blood and milk urea levels are shown to be poorly used in the production of milk proteins. In fact, Baker et al. (1995) noted an increase in ‘‘true’’ proteins when the DIP and UIP were consistent with production requirements. Azotemia is found to be closely correlated with milk urea concentration (Oltner and Wiktorsson, 1983; Gustaffson and Palmquist, 1993; Roseler et al., 1993; Baker et al., 1995). This parameter can be used, since it facilitates monitoring for sampling feeding adequacy. This analysis cannot replace conventional feed analysis, but can be utilized as a valuable complement to it (Oltner and Wiktorsson, 1983). The change in milk urea content in response to an altered dietary composition is very rapid in cattle (Oltner et al., 1985). Metabolic profile tests (Zicarelli et al., 1982, 1986; Bertoni et al., 1993a; Kobeisy and Ibrahim, 1993; Bertoni et al., 1994; Campanile et al., 1994, 1995) have been carried out on buffalo involving plasma (but not milk) urea concentrations, however studies into protein requirements during lactation in buffalo are fewer than in cattle. Protein concentrations used in lactating buffalo diets can be equal to or go below 12% on DM, since these concentrations have little influence on the quantity–quality of milk yield (Verna et al., 1992; Tripaldi, 1994; Verna et al., 1994). Sivaiah and Mudgal (1978) suggested the administration of 166.34 g to 126.03 g (Kurar and
Mudgal, 1980) of digestible crude protein / 100 g of milk produced protein, while according to Rai and Aggarwal (1991), the concentration of crude protein on dry matter should be between 11% and 14%. Tweatia and Bhatia (1996) state that the ideal protein content in the diet stimulates ruminal microflora. Moreover, Singh and Gupta (1984) detected a higher quantity of volatile fatty acids (VFAs) and ATP in buffalo cows fed a greater quantity of nitrogen with ammoniated straw. The present studies were aimed at verifying whether in buffaloes in midlactation, varying protein levels in the diet (in subjects whose protein requirements were or were not met) could influence the quantity–quality of milk yield, blood (BU) and milk urea (MU) nitrogen levels, metabolic profile and milk freezing point (MFP).
2. Materials and methods
2.1. Experimental animals and feed Trials were carried out in duplicate (trials 1 and 2) on eight buffaloes, each trial divided into three experimental periods (P1, P2, P3). In the first period P1, duration 40 days, subjects were fed a diet containing 9% crude protein / dry matter (CP/ DM). In the second period P2, duration 21 days, CP/ DM was increased to 12%. In the third period P3, duration 21 days, CP/ DM was reduced again to the level of P1. In trial 1, eight buffaloes 164 days in milk (DIM) were used, yielding an average of 7 kg / day of milk. In these subjects protein requirements were met. In trial 2, eight buffaloes 132 days DIM were used, yielding an average of 10 kg / day of milk. In these latter subjects, however, protein requirements in P1 1 P3 were not sufficiently met. It is important to note that the lactation period in buffalo is 270 days. Subjects were kept in cement paddocks and were fed once daily at 09:00 h for 10% orts. Feed consisted of total mixed ration based on corn silage, barley meal, soybean meal, sunflower meal, beet pulp, ryegrass hay and vitamin and mineral supplements. The different protein concentration in the diets was guaranteed by varying the quantity of soybean meal.
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Feed intake and orts for each trial were measured daily. Orts were sampled weekly on a random day. Analyses of individual foodstuffs and orts were carried out as per ASPA Commissione Valutazione Degli Alimenti (1980) methods and energy values calculated according to INRA (1988) equations. To calculate protein fractions (DIP, UIP, PDIN, PDIE), values obtained from fistulated buffaloes were used (ASPA Commissione Proteine nella Nutrizione e nell’Alimentazione dei Poligastrici, 1994; Di Lella et al., 1995; Infascelli, pers. comm.).
2.2. Milk sampling and analytical methods At the end of each trial period, milk samples from each buffalo were collected during morning and afternoon milking in order to evaluate milk composition (fat and protein), using the Foss Electric Milko-Scan 139 (calibrated with appropriate buffalo standard) and the MFP (using Genotec 030 osmometre). During the whole experimental period the daily milk yield was recorded. Milk urea (MU) was measured by the urease method-kits (Boheringer).
2.3. Standard milk and calculation of differences between intake and requirements Buffalo standard milk (ECM 5 740 kcal) was calculated using the formula for buffalo cows (Di Palo, 1992; Di Palo and Cheli, 1995): ([hfat (g / kg) 2 40 1 protein (g / kg) 2 31j0.01155] 1 1)milk yield. Estimation of the differences (D) between nutritive values intake and relative requirements is reported as follows: DM intake 5 91 g 3 kg metabolic weight 1 0.27 kg 3 kg ECM (Campanile et al., 1996); DCP 5 g CP intake 2 (80 g CP 3 100 kg live weight 1 2.7 g CP 3 g milk protein yield); DUFL 5 UFL (1700 kcal NEL) intake 2 [(1.4 1 0.6 3 100 kg live weight) 3 1.1 1 0.44 UFL 3 kg ECM)] (INRA, 1988); DPDI 5 PDI intake 2 (3.25 g PDI 3 kg of metabolic weight 1 1.55 3 g milk protein yield) (INRA, 1988).
2.4. Blood samples and analytical methods At the end of each experimental period blood samples were collected at 08.00 h, before feeding, from the jugular vein in vacutainer tubes. The tubes
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were centrifuged at 3000 rpm for 15 min. The recovered serum was stored at 2 188C until analyzed for metabolic profile. Immediately following sampling, the glycemia was measured on plasma by means of the colorimetric method. During each experimental period the following blood parameters were measured on serum using the enzymatic colorimetric method: urea (BU) (urease method), creatinine, total cholesterol, HDL cholesterol, triglycerides. Colorimetric methods were used to measure the total proteins; the serum protein fractions were evaluated by the electrophoresis technique. Sodium, potassium, chlorine were measured by means of a potentiometer using ionoselective electrodes. Following the results of the first trial, it was decided to analyse the b-hydroxybutyrate (BHBA), non esterified fatty acids (NEFAs), various serum enzymes such as ALT, AST, GGT, CPK, LDH (kinetic at 378C) using the enzymatic colorimetric method, and insulin by automated fluoro-immunometric method on AIA 1200; TOSO.
2.5. Body condition score Body condition scores were assigned using a scale of 1 to 9 (Wagner et al., 1988) (modified to the buffalo) at the end of each trial period.
2.6. Statistical analysis Correlation analyses (SPSS / PC 1 User Guide, 1986) were performed between blood and milk parameters of each trial and days in milk. No linear trend with time was observed so the differences between mean values of P1, P2 and P3 of each trial were tested using the SPSS / PC 1 software package Student’s t-test with pairwise procedure (1986). Linear regression analyses were performed for MFP and MU as dependent variable using BU as independent variable. Multiple linear regression were performed with SPSS / PC 1 stepwise procedure for: (i) MFP as dependent variable and five explicative variables: milk osmolarity, fat, protein, lactose and MU; (ii) MFP, MU and BU as dependent variables in function of dietary characteristics, intake of single diet compounds and difference between intake and requirements.
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Table 1 Characteristics of dry matter intake Trial
1
period
P1 1 P3
P2
P1 1 P3
P2
DMI (kg / d) UFL / DM
14.4 0.84
14.7 0.84
15.5 0.83
15.8 0.83
(%)
(%)
(%)
(%)
9.9 5.6 7.7 7.1 2.8 71.7 2.53 24.3 39.1 42.3 25.7 0.253
12.1 7.1 8.4 8.5 3.6 70.2 2.63 20.3 36.8 42.0 25.1 0.33
8.7 5.5 6.9 6.3 2.4 72.4 3.32 22.8 34.1 47.1 24.3 0.26
12.3 8.4 8.4 8.5 3.8 69.1 2.96 21.6 33.1 44.9 23.2 0.37
CP PDIN PDIE DIP UIP DIP/ CP Ether extract Starch NSC NDF ADF CP/ NSC
in the two trials. The DIP/ UIP rates did not change upon modification of the diet. In both trials milk production requirements for energy were met due to the energy density (UFL / DM) of the diet (Table 2). In particular, in trial 1, the buffaloes in full recovery phase ( . 160 DIM), were fed 8.7% more energy than required, notwithstanding a lower dry matter intake. In trial 2 a greater DMI was found, due to lower DIM and higher productive level. In this trial the theoretical energy requirements were met, except in P2. Protein requirements were met in trial 1, while in trial 2, during P1 and P3, a noticeable deficiency of about 350 g of crude proteins was evident. The deficiency would be estimated as only 54 g according to the specifications of Sivaiah and Mudgal (1978). If the data is analyzed according to the French (INRA, 1988) system (PDI), then the diet of trial 1 would also be considered deficient in PDI of approximately 103.5 g in P1 and P3, while in trial 2 the protein deficiency would be higher (2242 g). In both trials the availability of NSC enabled the synthesis of the nitrogen of the DIP into microbial protein (Table 1). Greater milk yield (Fig. 1) was measured in P1 and P2 vs. P3 in both trials 1 (P , 0.05) and 2 (P , 0.01) and was attributed to the DIM. If we consider ECM, these differences are not significant even if both trial groups verified greater
2
3. Results and discussion Table 1 shows the chemical composition of the dry matter intake of the two trials during the various periods. The different compositions of the dry matter intake in trial 2, in relation to the CP/ DM, starch / DM and CF / DM is mainly linked to the different type of orts in the two trials and partly to the quantity of corn grain present in the corn silage used
Table 2 Milk and milk protein yields, and differences between crude protein and energy intakes with their respective requirements Trial
1
2
period
P1
P2
P3
P1
P2
P3
ECM (kg) Milk protein / d (g) DMI (kg) CP (g) DCP 1 (g) DCP 2 (g) UFL intake DUFL DPDI
12.7 351 14.5 1435 17 1 214 12.2 1 1.05 2 127
13.4 346 14.7 1779 1 365 1 567 12.3 1 0.95 1 89
12.3 316 14.4 1426 1 93 1 268 12.1 1 1.19 2 80
16.9 463 15.6 1357 2 373 2 65 12.95 10 2 254
17.5 516 15.8 1943 1 70 1 426 13.11 2 0.107 1 63
15.9 441 15.4 1340 2 331 2 43 12.78 1 0.25 2 231
DCP 1 (g) 5 CP intake 2 CP requirement assessed by the authors. DCP 2 (g) 5 CP intake 2 CP requirement assessed by Sivaiah and Mudgal (1978). DUFL 5 UFL intake 2 UFL requirement. DPDI 5 PDI intake 2 PDI requirement.
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requirements suggested by the authors themselves and those of France (INRA, 1988), as opposed to those from India (Sivaiah and Mudgal, 1978). The MFP (Fig. 3) was found to be significantly higher (P , 0.01) in both trials when the CP/ DM of the diet was lower (P1 and P3 vs. P2); and in both trials the MFP values in P3 seem to be higher than in P1. The change in the diet (P2 vs. P1) showed a slight increase (not significant however) in the urea levels of the blood (Fig. 4), which went from 5.16 mmol / l Fig. 1. Mean milk and ECM yields in the three periods (P1, P2 and P3) separately for trials 1 and 2. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
milk yield during P2, since a decrease in milk yield is usually associated with an increase in milk lipid percentage due to an increase of the DIM (Fig. 2). With regards to trial 1, no change in the percentage or quantity of milk protein was recorded. In trial 2, the increase of CP/ DM in the diet from 9% to 12% led to an increase (P , 0.01) of protein levels and of protein quantity produced in the milk in P2 vs. P1 and P3 (Fig. 2). Only during P2 were protein requirements met, guaranteeing an adequate availability of nitrogen for milk protein biosynthesis. These results are more easily explained using the
Fig. 2. Mean milk fat and protein concentration in the three periods (P1, P2 and P3) separately for trials 1 and 2. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
Fig. 3. Mean milk freezing point in the three periods (P1, P2 and P3) separately for trials 1 and 2. (A, B on bars: P , 0.01).
Fig. 4. Mean blood urea levels separately for trials 1 and 2 and milk urea levels in trial 2 in the three periods (P1, P2 and P3). (A, B on bars: P , 0.01).
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(trial 1) and 6.23 mmol / l (trial 2) in P1 to 6.01 mmol / l and 6.47 mmol / l, respectively for the two trial groups in P2. In P3 the decrease of CP/ DM to the same levels as in P1 caused a noticeable decrease of the urea levels (P , 0.01) in both trials. In earlier treatments (P1 and P2) went to 3.36 mmol / l (trial 1) and 3.85 mmol / l (trial 2). A similar trend was also found in trial 1 with regard to MU (Fig. 4). The length of the period during which a diet with low protein concentration was fed influenced urea levels in blood and milk. In fact, the urea levels in blood and milk reached higher levels when a low-protein diet was given for a fairly long period of time, whereas levels were lower immediately following a sharp reduction in the protein contribution of the diet. Following sharp reduction in the protein content of the diet, a decline in circulating urea was verified. This occurred (as shown in our experiment) when a fermentable energy supply was present. We propose that an increase of insulin levels reduced or blocked the normal amino acid breakdown, and therefore, reduced the urea levels in circulation. An increase in protein concentration in the diet meeting requirements of the trial 2 buffaloes resulted in: an increase in milk yield (interrupting the physiological decrease due to DIM), stabilization of the MFP (P , 0.01) and an increase in milk protein percentage and urea nitrogen levels (MU). The major differences between trial 2 vs. trial 1 were the more marked increase of the CP-level and in particular, of the PDIN of the DMI in P2 vs. P1 and P3.
the diet (the protein / energy ratio), the MU level rises upon increasing the CP/ NSC ratio and decreasing the UIP/ NSC ratio. Thus: MU (mmol / l) 5 2 5.37 1 41.60 CP/ NSC 2 53.58 UIP/ NSC; R 2 5 0.906. Upon elaboration of the results of both trials the following weak relationships are observed: (a) BU (mmol / l) 5 13.58 1 16.23 CP/ NSC 2 0.04 g starch intake 2 1.03 UFL; R 2 5 0.574; (b) MFP 5 2 0.530 2 0.926 UIP/ NSC 1 0.00003 g starch intake; R 2 5 0.383; (c) MFP 5 2 0.473 1 0.01 BU (mmol / l); R 2 5 0.257. It should be emphasized that no association between MFP and serum osmolarity was found, as had been previously found in cattle (Peterson and Freeman, 1966). We also wish to point out that the increase of CP/ DM resulted in significantly higher blood concentrations of both K and Cl in these trials (Fig. 5). These studies show that BU and MU levels are positively influenced by the CP/ NSC ratio in the buffalo. In fact, the greater availability of rumen fermentable energy, can permit a better use of ammonia at ruminal level for microbial protein synthesis. Therefore, a smaller quantity of blood urea would be present (Journet et al., 1975).
3.2. Metabolic profile The metabolic profile examination showed that in P2 BHBA and total cholesterol blood levels (Fig. 6) increased in trial 2 (in trial 1 BHBA was not
3.1. Regression analyses The relationship between the MFP and the components of the milk in trial 2 is expressed by the following equation: MFP 5 2 0.413 2 0.487 MU (mmol / l) 2 0.0182 milk protein (%) 2 0.00095 milk (kg); R 2 5 0.685. With regards to this study whenever milk urea was measured, BU was positively related to MU over the three experimental periods. The relationship between MU and BU is expressed by the following equation, which is similar to results found by other authors (Roseler et al., 1993; Gonda and Lindberg, 1994): BU (mmol / l) 5 1.675 1 0.817 MU (mmol / l); R 2 5 0.769. When one changes the components of the DM in
Fig. 5. Mean serum levels of K, Na and Cl in the three periods (P1, P2 and P3) separately for trials 1 and 2. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
G. Campanile et al. / Livestock Production Science 55 (1998) 135 – 143
Fig. 6. Mean blood glucose (Gluc) and total cholesterol (Chol) levels in the two trials and mean blood NEFA and BHBA levels in trial 2 in the three periods. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
measured) while blood glucose decreased. The greater quantity of protein fed in the diet, increased protein synthesis, which requires greater energy availability. Energy requirements were probably not met, because the energy level in the diet was not varied (Table 2). This could have caused a ‘‘relative’’ energy deficiency which could explain the increase of BHBA derived from lipid catabolism, evidenced by the transient increase in blood lipoprotein levels (Fig. 6). Moreover, the imbalance between energy used for milk production and energy intake is calculated at an estimated deficiency of 0.107 UFL per day. As a results of energy imbalance BCS decreased (although not significantly) 0.25 points in P2 and increased 0.64 points in the following period (P3), when excess energy of 0.25 UFL per day was estimated (Table 2). NEFA blood levels did not significantly increase (Fig. 6). Elevated insulin blood levels ( . 6 U / ml) characteristic of midlactation, did not permit the optimum use of this metabolic pathway. As previously proposed, in trial 1 where only the protein requirements were met in P1 and P3, the further increase of protein intake in P2 triggered a more intense gluconeogenesis evidenced by the glycemia increase (P , 0.01) and the stability of plasma lipoprotein (Fig. 6). In the second trial the increase of CP/ DM in the diet resulted in greater milk yield and an improvement in milk quality and also gave rise to increased
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Fig. 7. Mean serum levels of ALT, AST, CK and GGT in the three periods (P1, P2 and P3) in trial 2. (A, B on bars: P , 0.01; a, b on bars: P , 0.05).
serum enzymatic activity (Fig. 7) which was necessary to deal with the sudden increase in biosynthetic activity.
4. Conclusion In the Italian-bred Mediterranean buffalo cow, diets of low ratio protein / energy or with low protein content DM are common since high quantities (20– 34 kg) of corn silage are used. This sometimes causes an increase in the MFP often responsible for contractual problems within the dairy industry. In most cases an increase in diet protein concentration can normalize the MFP. Similar results have been found in Jersey cows (Peterson and Freeman, 1966). In those cases, higher MFP values were found in subjects fed low protein level diets while the same diets did not alter the MFP in Holstein cows (Peterson and Freeman, 1966). Those results and ours suggest that there may be one or more minor factors which affect the MFP. It should be remembered that Jersey and buffalo cows yield milk with a higher dry residue than the Holstein. In buffalo cows, as well as in dairy cows, dietary protein characteristics and P/ E ratio influence urea levels in blood and milk. This ratio (P/ E) must be further evaluated considering that the buffalo cow adapts itself to the lack of protein easier than the dairy cow (Bertoni et al., 1993b). It is important to remember that low values of BU and MU were
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reported only when we passed from high to low protein content diets in both trials. Smith (1969) reported that deficiency of nitrogen in ruminants decreases the efficiency of urea clearance by the kidney, increasing ruminal return and decreasing hematic levels. In agreement with Oltner and Wiktorsson (1983), we show that in buffalo cows the urea determination in milk can be used as a valid method of estimating the P/ E ratio but it cannot replace conventional feed analysis. From these results we also conclude that the buffalo cow diet should ensure a sufficient protein percentage, not only to meet requirements, but also taking into consideration the energy level, above all in diets rich in fermentable energy. Moreover if the task was only to meet requirements, then low protein concentrations would suffice (9% of DM) but these often cause alterations of the MFP.
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