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Haematological profile and techno-patologies
Campanile G., Di Palo R.,  d'Angelo A.*
Department di Scienze Zootecniche, Università Federico II - Napoli
*Departement di Strutture, Funzioni e Tecnologie Biologiche


The inter-relation between metabolic pattern and diet during the different physiological phases in the buffalo are discussed herein, highlighting the modification of the endocrine-metabolic pattern in the most critical phases of productive life (early lactation, growth etc.).
In the buffalo the lesser milk yield is caused by a lower negative energetic balance at early lactation compared to the cattle. This causes reduced fat mobilization characterized by lower NEFA haematic levels.
Moreover, the buffalo adapts itself better than bovine to the greater energy:proteins ratio and to the lower protein concentrations in the diet. Such a diet, over a long period of time, does not lead to a reduction in the blood and milk urea levels. High protein concentrations, on the other hand, at lactation-onset do not show reduced azotemia (typical at early lactation) to meet proteic requirements and does not cause an excessive rise in haematic ammonia.
Particular attention should be paid to mineral content during the dry milk period as this could be one of the causes of uterine and vaginal prolapse. This is due to alteration of uterine-vaginal muscular tone following the change in Ca:Mg haematic ratio immediately post-partum.

 Key words: buffalo, nutrition, metabolic profile


The study of haematic constants help in identifying possible nutritional errors in species of zootechnical inerest (Payne et al.1970) Moreover, they provide useful indications on metabolic profile within the different physiological phases of the subject. Zicarelli et al. have carried out studies with this objective on Mediterrean buffaloes, bred in Italy, from 1980 onwards. Since then many other researchers have contributed to the improvement of the buffalo diet troughout the different productive stages (i-e,growth,dry milk period ,lactation ). The aim of this work is to describe metabolic profile inter-relations during different physiological phases, highlighting biological mechanisms which are at the basis of productive activity in the subjects.

Energetic Metabolism

Buffalo lipomobilization, which increases NEFA (as observed in the bovine milk cow) (Bertoni and Lombardelli, 1991; Chiesa et al., 1991) begins towards the end of gestation (Campanile et al 1995). At early lactation, haematic concentrations of NEFA are high, but never reach the levels found in the bovine milk cow, peaking at about 20 days post-partum (fig. 1). It than decreases (about 110 days post-partum ) returning to the haematic levels registered in the initial dry milk period phase.At this point in time (about 110 days) an increase in insulin plasma levels is noted. This indicates endocrine signal passage from the catabolic phase to the anabolic (Campanile et al., 1995) on the lactation curve (fig. 1).

It is already well-known that lactation start is characterised by a negative energy balance which is more intense in the buffalo than in the bovine. We can therefore confirm that buffaloes, even if fed a well-balanced diet reach a "physiological" hyponutritive condition as they are unable to ingest the quantity dry matter (D.M.) needed to meet requirements. The organism responds to this emergency by raising GH levels and lowering those of T4 (functional hypothyroidism).

Figure 1. NEFA, B-OHB and insulin haematic levels at different days from calving.

  NEFA,B-OHB                                               calving                                                                                                Insulin

 The growth hormone reacts by reducing insulin activity, or at least sensitivity of adipose tissue towards this hormone. This mechanism facilitates mobilization of the adipose tissue and the  muscular proteins which provide energy and the necessary precursors in order to maintain glucose haematic levels within values which allow the subject to carry out its phisiological activity. Low glycemia values have been found in buffaloes which intake less than 1020 Kcals per litre of milk of NEl (net energy for lactation). In these buffaloes whose NEFA and -OHB were not analysed, haematic levels turned out to be inferior compared to what is deemed normal in these species (55 60 mg\dl) (Zicarelli et al., 1982) and inversely correlated with the quantity of milk produced, but positively with distance from calving (Zicarelli et al., 1982; Eltohamy et al., 1994). Other studies have shown an increase of this haematic constant following a rise of T3 and T4 values in cold climates especially in winter (Campanile et al., 1994).

In buffaloes, towards the end of the lactation curve, it is interesting to note a possible increase of -OHB (fig. 1) in a phase characterised by lesser fat mobilization and a reduction in the quantity of short chain fatty-acids present in milk fat. These events (noticed also by other autors) (Bertoni, 1994) could be explained by changing the group of a poor production buffaloes which on Italian farms, is carried out after approximately 180 lactation days. In this case, it has been noticed that the increased fill value of the diet and/or to the change of the same which is administered to the less productive subjects. There are two possible reasons for this: energy requirements are not sufficently met in subjects which, at this phase, produce a milk particularly rich in fats and/or stress derived from the re-estabilishement of group hierarchy. In same cases from the 110th day of lactation, an increase of  -OHB can be observed upon increase of insulin and trygliceride haematic levels. Bertoni and coll. (1994) found a poorer use of -OHB upon raising insulin values in milk cows. Nevertheless, -OHB increase has been noticed in subjects whose daily requirements were more than sufficiently met and whose diets contained high proteic and energetic levels (14.5% - 15%  CP/DM  and 0.89 - 0.94 FMU/DM). The high proteic levels could have directed ruminal fermentation towards greater production of butirric acid  which determines a greater quantity of circulating -OHB not immediately used by the organism.

Adding supplements of protected fats both at lactation start and after  lactation peak (about  55 days postpartum) causes fat mobilization increase in buffaloes (Di Palo et al., 1997) and bovine (Sklan et al., 1994). In fact, higher NEFA and total cholesterol  plasma values are found, although the values of -OHB are lower. Protected fat ingestion should therefore stimulate production through an active mobilization of body reserves which can guarantee the udder the precursors for the synthesis of the milk components. The higher NEFA values in respect to the controls, do not indicate energy defiency, but represent a more efficient use of  body reserves. The lower -OHB values confirm this.  In fact, it means a compensated fat mobilization which positively influences the energy balance (low -OHB values), synthetic hepatic cell activity (high cholesterol levels) and milk yield.

Total cholesterol and HDL indirectly provide for evaluation of esogenous energy availability together with hepatic functionality. When the energetic quota consumed by the animals manages to meet at least part of its daily requirements the increase of NEFA, following fat mobilization, does not cause lipid liver due to trygliceride persistence in the hepatic cells.  In this case, the hepatic cells are not  capable of increasing the synthesis of cholesterol. In fact, cholesterol (as is known) indicates good hepatic tissue lipoprotein  production which transport the triglycerides synthetized by the NEFA (Nefa Acil CoA -glicerophosphate triglycerides). In buffaloes with a moderate negative energetic balance, an increase in total cholesterol and HDL within 71 days of lactation is observed (fig. 2). Then a lowering is found to coincide with the final stage of the lactation curve and the beginning of the anabolic phase as soon as the insulin (fig. 1) and triglyceride (fig. 2) haematic levels rise.

Particularly high total cholesterol hematic levels are found in buffaloes bred in rather cold climes (Campanile et al., 1994), in fact it is well known that catecholamine which are stimulated by stress factors (these include metereological factors) increase fat mobilization with  rising NEFA with the aim  of producing endogenous heat needed for thermoregulation.

Therefore, lactation and low temperatures and/or high thermal ranges are resposible for the energetic gap which probably causes fat mobilization with the introduction of NEFA in circulation. In fact, total cholesterol is higher in subjects which move more, but this does not cause energetic deficiency as the normal -OHB values prove.

Figure 2: Total cholesterol, cholesterol HDL and triglycerides haematiclevels at different days  from calving

(Total Cholesterol)                      calving - parto                                       (Choles. HDL, Triglycerides)

Many papers often highlight a good reletionship between total and HDL cholesterol and milk yield. HDL cholesterol is less influenced by the dry matter (DM) characteristic of the diet which means that it could represent a good genetic index of the galactopoietic capacity of the buffalo (Di Palo et al., 1989).

Triglycerides increase during the curve of lactation and show a positive correlation with the milk fat levels (Zicarelli, 1988). In one study on 306 buffaloes with different post-partum distances and fed 17 different diets, it emerged that triglycerides represent a good index of meeting energy requirements, in the early stages of lactation  (1-31 days) while total  cholesterol  and HDL in the intermediate phase (31-110 gg) and glycemia only after 110 days as it is positively correlated with the energy provided by the diet (Di Palo et al., 1990). It is well know, in fact, that the NEFA which are released at early lactation following intense fat mobilization, are used hepatically for the syntesis of the triglycerides only if the balance between energy absorbed in the diet and that emitted due to production is not especially deficit. Rising glycemia, on the other hand, is obtained only during the anabolic phase of lactation when energy intake is equal to, or superior to the energy release. Normal or high values of hematic glucose are responsible for the lowering of GH and the increase of the insulin:glucagon ratio which achieves a lesser or zero efficiency in activating neoglucogenesis through other molecules (Aa etc).

Proteic Metabolism .

In buffalo cows, as well as in dairy cows, dietary protein characteristics and P/E (protein:energy) ratio influence urea levels in blood and milk (Campanile et al., 1996). This ratio (P/E) must be better evaluated considering that the buffalo cow adapts itself to the lack of protein easier than the dairy cow (Bertoni et al., 1993) It is necessary to remember that low values of BU (blood urea) and MU (milk urea), compared to the cow, were reported only when passing from 12% to a low 9% protein content diets (Campanile et al., 1996). Proteic concentrations < 9%,used for a long time, did not determine low blood urea values, but rather cause milk freezing point (MFP), especially if the diet shows a high amount of fermentable energy (Campinile et al., 1996). It would seem interesting, therefore, to evaluate the protein/energy ratio in order to to obtain a correct diet. In the buffalo, such a ratio can be broader; supplying diets high in proteic concentration determine in fact, less damaging effects compared to the bovine milk cow. The buffalo, for example, makes better use of nitrogen in the diet than the bovine, even if there is a carbohydrate deficit (Langer, 1969) since buffalo ruminal behaviour is more favourable toward the growth of micro-organisms which use non proteic nitrogen.

BU and MU levels are positively influenced by the CP/NSC ratio also in the buffalo. In fact, the greater availability of fermentable energy, which can be used by ruminal microflora, could allow a better use of ammonia at ruminal level for microbial protein synthesis. Therefore, a smaller quantity of urea could be produced (Journet et al., 1975).

In our experiment (Campanile et al., 1996) the length of the period during which a diet with low protein concentration, as previously stated, was used, 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 they were lower immediately following a sharp reduction in the protein contribution of the diet. Prolonging the absorption of low protein concentrations induced the subjects to maximise the use of the fermentable organic matter for the production of bacterial proteins (Bertoni et al., 1993); such a condition brought about the activation of a normal aminoacidic breakdown which aided in keeping urea levels high. Following sharp reduction in the protein content of the diet, a decline in circulating urea was verified. This was possible when a fermentable energy supply was present, which, in contributing to the increase of insulin levels, reduced or blocked the normal aminoacidic breakdown, and, therefore, reduced the urea levels in circulation.

In the dairy cow decreasing BU was registered exclusively in diets with low nitrogen levels together with low energy levels (Ndibualonji et al., 1995). This, on the other hand, is normal if the low nitrogen levels were associated with high energetic levels. Smith (1969) reported that nitrogen deficiency in ruminants which live in tropical areas reduces renal clearance of the urea, increases the return to the rumen and decreases hematic levels. This, in turn, would promote better recycling of the urea in the digestive tract and a better proteic synthesis by the ruminal bacteria (Houpt, 1970).

Zicarelli (1994) stated that the low proteic content of tropical forage, meets the daily nutritive requirements of the buffalo vs the bovine more efficiently due to the broader E/P ratio of the milk. Moreover, the more efficient recycling of the urea in buffaloes explains their greater suitability and/or adaptability to tropical and subtropical areas.

Azotemia is influenced by days in milk and the diet, in fact, in buffaloes which intake low proteic concentrations, it's progress reflects the modifications of dry matter intake during lactation. The figure 3 shows that diets with 13.5% CP/DM in subjects which yield more than 20 kg of ECM (equivalent corrected milk), the azotemia presents at between the 11th and 32th day, low values which go back up between the 70th and 110th day when dry matter intake increases. An accurate examination of the data suggest that between the 11th and 70th day, dry matter intake is comprised of between 13 and 16 kg and therefore, the proteic requirements are not met. This leads to a greater fat mobilization (NEFA increase) especially in the first days of lactation (Fig. 3). It has also been noted that low azotemic values, in bovine milk cows, cause a greater GH incretion which stimulates fat mobilization, inhibits insulin activity and makes nitrogen derived of tissue so as to guarantee the necessary quantity of energy and protein required for production purpose (Ndibualonji et al., 1995). On farms which administer higher proteic concentrations which we consider more suitable for meeting daily proteic requirements in the period of lesser dry matter intake, especially in the more productive subjects, lowering azotemia is not found (fig. 3). The greater availability of keto acids, which derive from proteic catabolism, favours glucose syntesis. Glycemia is therefore of higher in subjects with proteic excess. High glucose values and low insulin values, guarantee the udder a greater availability of glucose for lactose synthesis. This metabolic process, used by subjects with proteic excess, contributes to reducing negative glucose output, at the beginning of lactation. Spires and Clark (1979) have noted that high levels of NH3 in steers, cause a poor use of the preceeding glucose, in others words, a rapid glycogenolysis.

Interpretation of the azotemia is carried out taking into consideration many nutritive aspects and the integrity of renal parenchima. Our researchers generally evaluate creatininemia (1.5 - 2.0 mg/dl) in order to exclude renal damage, which is fairly fequent in this species.

In the Mediterranean buffalo bred in Italy, excessive proteic diets are recorded, especially in spring and early summer when pasture is abundant. Proteic excess in bovine determines ruminal hyer NH3 with an increase of ammonia and urea nitrogen levels in the blood.  On the basis of our experiences, with these diets the NH3 levels  have resulted lower than those reported by Jordan and coll. (1983) in cattle; in these buffaloes an increase of the urea levels in blood and milk and probably a greater elimination of the metabolite through urine occurred. Thus, the increase of protein intake increases hepatic microsome metabolic activity and this would favour the transformation of the ammonia of alimentary origin in urea (Visek, 1984).

High urea nitrogen levels have been observed by Zicarelli et al. (1982) in buffaloes which consume more than 60 g of digestible proteins per litre of milk. This however does not take place when 50% of the digestible proteins came from wet brewers' grains which according to recent discoveries, are not degraded at ruminal level by over 60% and are therefore endowed with a good by-passability (Di Lella et al., 1995).

Infascelli et al. (1997) found that in highly productive buffaloes (> 30 kg ECM/day), the increase of UIP obtained from an extract of Aspergillus oryzae fermentation, lead to greater milk yield and higher urea values.

Albuminemia (2.6 - 3.9 g/dl) and total proteins (7-8 g/dl) are influenced by the proteic levels in the diet only when there is excessive defficiency. These two constants together with the A/G ratio (0.65-0.85) represent, on the other hand, a good hepatic functionality index, as the positive correlations found between albumin and the milk proteic level demonstrate. In buffaloes with extensive hepatic necrosis, albumin levels lower than 2.3 g/dl were found. A reduction in the ratio is also found in acute infections.

Figure 3: Urea, Nefa, glucose and B-OHB haematic levels in buffaloes that were fed diets characterized by 13.5% (1) or 15.5% (2) CP/DM at different days in milk

            (NEFA)                                                                                                                     (Urea)

Seric Enzymes

The cytoplasmatic enzyme, GGT ( -Glutamyltransferase), is the first to increase even in conditions of slight hepatic sufferance. It is hightly correlated with SDH (Sorbitol dehydrogenase) and OCT (Ornithine carbamoyl transferase) enzymes, those which more specifically stabilize hepatic functionality, even if difficult to analyze. Only in extremely serious cases, (for example with parasitic infestions of hepatic type), it is possible to record AST (Aspartate aminotransferase) and total bilirubin increases. It has been noted (Zicarelli et al., 1982) in subjects analyzed in autumn and which feed at pasture between end-spring and early autumn, higher values of Hb (Hemoglobin), AST and ALT (Alanine aminotransferase). These enzymes seem to be present in ruminants, in greater concentrations in the muscles and heart as opposed to the liver. Moreover, they are localized at mitochondrial level for which they increase esclusively during extensive hepatic necrosis. They represent, together with LDH (Lactate dehydrogenase) and in particular CK (Creatine kinase), an index for the evaluation of muscolar cells integrity.

The determination of enzymatic activity is useful for ascertaining the validity of the use of an acidified milk for wearing buffalo calves. The evolvement of physiological conditions modifies the activity of the different apparatus in this phase, and in particular one. The passage of the fetal and neonatal phase, as well as tissue growth and change accompanied by turbulent synthesis which are verified during this life-phase, could be responsible for the progressive increase of the serum levels of ALT, AST and LDH (fig. 4) (Campanile et al., 1993). On the other hand, the growth and therefore, the progressive and ever more complete functionality of the organs, leads to greater activity of these enzymes at tissuel level and consequently to their increase at serum level. These findings togrther with normal albumin values and a progressive lowering of GGT (fig. 4) during growth, have demonstrated an evolvement of synthesis capacity and good functioning of the hepatic and renal parenchyma respectively (Campanile et al., 1993).

The progressively decreasing GGT values,  together with those of the -globulins (according to some authors) (Tradati et al., 1982), would be linked to the return within physiological limits of these two constants which rise noticeably during the colostral phase, in as far as absorbed with the colostrum.

The verification of the A/G (albumin/globulin) ratio negatively correlated with GGT ( r = - 0.417; P<0.0001) also represents a valid testimony of good hepatic functioning.

The progress of CK (fig. 4) values is of particular interest. An increase on the 30th day of life is followed by progressive reduction; this progress could represent the resultig increase of muscular tissue and functional gymnastics.

This condition would cause, therefore, a greater permeability of the striated musclolar cells and the limited liberation of CK within the circulatory system. Higher values of ALP (Alkaline phosphatase) were found in the first 40 days of life due to the more intense bone remodeling (fig. 4).

Climatic variations also influence changing hematic constants. During the hottest weather a lowering of the biosynthetic process of the organism as a consequent of the lower basal metabolism is caused by the lower T3 and T4 hematic levels.

The decreasing metabolism is accompained by lower hematic concentrations of ALT, AST, ALP, LDH, GGT and PT (Di Palo et al., 1993). Moreover, thyroid hormones, and in particular T4, determine an increase of mitochondria which swell due to the increased membrane permeability, and therefore, a greater emission of these enzymes in circulation.These hormones also stimulate proteic synthesis in various organs.

Figure 6: Average seric enzymes pattern at different ages.

Mineral Metabolism

The hematic levels of the mineral elements and their relation help to evidence, not only subclinical pathological alterations, but above all mineral deficiences. In the dry period, even minimum, but long-term definciencies (easy to ascertain as it is usual not to revise the diets of non-productive buffaloes) can cause notable damage which is reflected in the state of health of the following lactation. It has been noted that P (Phosphorus) deficiency during the dry milk period represents one of the most frequent causes of vaginal and/or uterine prolapse in this species (Campanile et al., 1989). Minerals salt supplements should be added to the buffalo diet during the dry milk period to give 45 g of Ca (calcium) and 45 g of P in order to keep the relation between these two elements near to unity. Excess Ca even if minimum causes parathiroid hipoactivity, which did not turn out to be ready, at end-gestetion and early lactation, to mobilize bone calcium. This determines excellent hematic Ca:Mg (calcium:magnesium) ratio (fig. 5) alterations which is useful at maintaining membrane potentiality and therefore permeability and excitability of the smooth muscular cells; this ratio alteration favours utero-vaginal muscolar release, which provides atony and therefore uterine prolapse (Campanile et al., 1989).

 Figure 4: Ca:Mg haematic ratio in buffaloes which have (Prol) vaginal prolapse or not (No Prol)

In subjects whose diets were P deficient before calving, it was observed that calcemia below 10 mg/dl decreased again to 8 mg/dl upon calving and phosphatemia were normal (5-6 mg/dl) . Integration with P raises calcemia speeding up the glands which regulate homeostatis raising the values to 10mg/dl during the dry milk period and to around 9mg/dl immediately after calving (Campanile et al., 1989). In subjects whose dietary intake involves a Ca:P ratio nearing unity, an increase of ALP (fig. 6) during the initial phases of lactation is proof of speedy parathyroid activation (Campanile et al., 1995). In these subjects a limited oscillation of Ca and Mg values was observed. High phosphorus hematic values were noted (Zicarelli et al., 1982), together with higher Cu (copper) levels, with subclinical metabolic acidosis in progress in diets characterized by high quantities of silage and/or concentrates (> 59%). Metabolic acidosis is responsible for both a greater incidence of uterine prolapse due to hypotonic hydratation of the organ before calving (Zicarelli et al., 1982), and a greater incidence of endometritis in the first 60 days from calving (Campanile et al., 1991).

Circulatory emissions of organic acids, which can be found during subclinical acidosis, determines a lowering of the immune system resistence of the organism; which leads to uterine colonization of the pathogens responsible for flogistic phenomenon, and therefore, hypofertility. Moreover acidosis determines hepatic sufferance proved by the higher values of GGT and LDH, and caused by the greater effort by this organ in trasforming lactic acid into propionic acid (Campanile et al., 1991).


Hematic and milk deficiencies of B-carotene are found in buffaloes, probabily due to quick transformation into vit.A (Zicarelli et al., 1986). Vitamins A and E present the same trend of total cholesterol, in as far as they increase upon increasing distance from calving to later reduce after 120 days from lactation. Their average values are 597 g/l of vit.A and 175 g/l of the vit E (Zicarelli et al., 1986).

 Figure 5: ALP, Ca, P and Mg haematic levels at different days from calving

   (ALP, U/L)                                                                                                                (Ca, P, Mg, mg/dl)


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