Metabolic Disorders in the Parturient Buffalo

Water buffaloes response to lactation is different from cattle; metabolic disorders related to parturition or production have a low incidence[1]. The levels of glucose and calcium are fairly stable during early lactation in buffaloes [2,3] and it has been reported that the metabolites like glucose, calcium, phospholipids, magnesium and insulin are higher in buffaloes compared to cattle [4]. A number of studies have demonstrated the effect of time since calving on the metabolic profile of buffaloes [5]. It was reported that during peak lactation buffaloes metabolize body reserves to supplement the lower amounts of bloodstream lipids [6], however, this lipid mobilization was lower compared to cattle [1]. It has been shown that during the first two months after parturition, the glucose concentration decreased [7], whereas non-esterified fatty acids (NEFA) and cholesterol increased in lactating buffaloes [7,8]. However, buffaloes have less intense negative energy balance compared to dairy cattle probably because of a lower milk yield [5,9]. Serum triglyceride concentrations increase during lactation and show a positive correlation with milk fat levels in buffaloes [10] probably because buffaloes produce fattier milk. Increase in liver triacylglycerol and NEFA are observed during late pregnancy and after parturition in buffalo similar to cows, however, the increase in buffalo was lower compared to cows with no indication of fatty liver in the parturient buffalo [11]. Low incidence of ketosis in buffalo can partially be explained by a lower milk yield and less intense negative energy balance at parturition and a lower thyroid activity in lactating buffalo [12]. The rumen physiology of the buffalo appears to be different from cattle [13]. With a ruminal pH of 6.28 [14] the buffalo has a good ability of buffering capacity and adaptation of rumen microorganisms to different conditions of energy and protein in the diet [13]. Moreover buffalo rumen has a capacity for improvement in the digestibility of non-structural carbohydrates with high fibre [15]. It is probable that there are some yet poorly understood mechanisms that contribute to a low incidence of ketosis in buffalo.

Milk fever has been mentioned as an important metabolic disorder of the transition cow with mortality of 5 - 10% cows affected with this disorder [16]. Although calcium and phosphorus contents of milk are low during early lactation in both cattle and buffalo [17] yet milk fever is less prevalent in the buffalo and contrarily phosphorus deficiency parturient hemoglobinuria is common in buffaloes compared to cattle [18-20]. The reasons for such a disparity are poorly known and are only partly explainable on the basis of a lower milk production in the buffalo. Likewise hypomagnesemia or grass tetany and downers syndrome are common in cattle but uncommon in the buffaloes with exceptionally few reports on these two disorders [21-24]. In this chapter we describe the metabolic disorders in parturient buffalo; milk fever, ketosis, hypomagnesemia and parturient hemoglobinuria.

1. Milk Fever (Parturient Paresis)

Milk fever is a metabolic disease commonly seen in high producing dairy buffaloes [25-28], similar to that seen in dairy cattle, and is characterized by general muscle weakness, incoordination and depressed consciousness within a few days before or after calving [29]. Compared to cattle blood calcium levels show limited variability in buffalo during pregnancy, lactation and dry milk period [1,30]; higher levels were recorded during the last month of pregnancy and lower ones at the end of lactation [3,8]. Although deficiency of calcium was recorded in the soil and forages fed to buffaloes in many studies [31-35] clinical disease was not recorded. The almost constant serum levels of calcium, phosphorus, parathyroid hormone and the low concentration during late pregnancy and early lactation in buffaloes in one study indicated that buffalo need to utilize only a little of their endogenous mineral resources [30]. It has also been mentioned that buffaloes are able to meet their requirement of calcium from the coarse roughages whereas on similar roughages cattle show negative balances [36] indicating that buffaloes have an inherent better capability for mineral retention. All these factors probably contribute to a lower incidence of milk fever in the buffalo. In buffalo species calcium excess is considered to alter the Ca/P ratio during the dry milk period, including parathyroid hypoactivity which would cause the increasing of magnesium level and the decreasing of calcium one at the beginning of lactation due to non-immediate mobilization of calcium by the bones [4]. The altered Ca/Mg ratio favors vaginal and/or utero-vaginal muscle relaxation responsible for uterine atony and increased incidence of vaginal/uterine prolapse [5,37].

1.1. Incidence

A survey of 9,432 buffaloes having calved at private and organized farms in the state of Punjab revealed an incidence of parturient paresis as 3.1%, with 80% of cases occurring between July and December [28]. In a recent study in which 1000 pregnant Kundhi buffaloes were followed for parturition in Pakistan, showed a milk fever incidence of 6.7% [38]. A high milk fever incidence is recorded in adult buffaloes and is more common in dry areas where straws and stovers are the staple roughage. The incidence of milk fever observed during the 2nd to 7th lactation in buffaloes was 5.1%, 18%, 33.3%, 25.6%, 10.3%, and 7.7% [28].

Milk fever has been recorded to occur during the last few days of pregnancy or within 1 - 3 days of parturition [39-41]. Exceptionally it may occur even up to 8 weeks after calving [28]. Buffaloes in their 3rd to 5th lactation are more likely to develop the condition [39,41,42] with 60% being affected during the 3rd lactation, probably due to peak lactation.

1.2. Etiology

A decrease in serum calcium levels usually occurs in buffaloes at the time of calving [8] and is greater in animals prone to milk fever. Usually three factors are responsible for the maintenance of the calcium level in the body fluids namely absorption of dietary calcium from the intestine, calcium mobilization from skeletal reserves and excretion/reabsorption from the kidneys. Any disturbance in the function of one or more factors may initiate the development of milk fever. Serum calcium decrease may occur due to either one or a combination of the following factors [29].

Excessive Calcium Drainage in the Colostrum:

The amount of calcium in the colostrum is dependent on the volume of colostrum secreted. When the loss of calcium in colostrum is much higher than the normal capacity for its absorption from the intestine and mobilization from the long bones, a decline in serum calcium is likely.

Impaired Absorption of Dietary Calcium from the Intestine:

Loss of appetite is seen in some animals at the time of parturition and these animals are more likely to be affected because of the reduced calcium absorption from the intestine.

Mobilization:

Slow and insufficient mobilization of calcium from the skeletal reserves during the terminal stages of gestation is a possible cause for hypocalcemia.

1.3. Calcium Homeostasis and Milk Fever

The maintenance of calcium pool in the plasma and extracellular fluids is dependent on parathyroid hormone (PTH), vitamin D and calcitonin. The animal receives its requirement of daily calcium by absorption from the intestines. Calcitonin favors mineralization of the bone whereas the parathyroid hormone favors demineralization of the bones when Ca requirement in the plasma is increased, whereas the absorption of Ca from the intestine or its loss in urine is dependent upon 1,2,5 dihydroxyvitamin D (vitamin D) secreted from the kidneys [43] (Fig. 1). Calcium homeostasis during early lactation in dairy cows has been explained [44]. Most cows are in negative Ca balance during the early weeks of lactation because more Ca leaves the body via milk, endogenous fecal loss, and urine than is absorbed from the diet, in part because the intestinal mechanisms for absorbing Ca are not fully adapted to lactation [45] and also because dry matter intake is less than favorable. To maintain normal plasma Ca, the negative Ca balance is met by resorption of bone Ca stores and absorption of Ca from the intestine. Bone Ca mobilization is stimulated by a concerted effort of PTH and vitamin D, but intestinal Ca absorption is controlled by vitamin D alone [44]. During the dry period, these mechanisms for replenishing plasma Ca are relatively inactive [45]. Thus, nearly all cows experience some degree of hypocalcemia during the first days after calving, before the intestine and bone adapt to lactation. The adaptation process begins with dramatic increases in the plasma concentrations of PTH and vitamin D at the onset of hypocalcemia. About 24 h of vitamin D stimulation is required before intestinal Ca transport is increased significantly [46]. Bone Ca resorption (recruitment and activation of osteoclasts) is not significantly increased until after about 48h of PTH stimulation [47]. In cows with milk fever, these adaptation processes can be even more prolonged. For these cows, the mammary drain of Ca causes extracellular and plasma Ca concentrations to fall, even to the point of eventual death, before adaptation of intestine and bone can occur.

Figure 1. Calcium homeostasis mechanisms.

Similar to studies in cows [48,49] a few studies [50,51] addressed the dietary cation/anion ratio on the development of milk fever in Nili Ravi buffaloes in Pakistan. Addition of anions to the diet (or preferably reducing the cation content of the diet) reduces the alkalinity of the diet, reducing the metabolic alkalosis and perhaps initiating metabolic acidosis [44]. Buffaloes fed anionic diets during the prepartum period had improved digestibility, blood pH and serum bicarbonates were low and increased as the diet was more cationic [51]. Buffaloes fed anionic diets had a low incidence of parturient hypocalcemia [50]. It has been demonstrated that addition of anions to the diet does not increase apparent intestinal absorption of Ca [52,53]. However, studies [54] indicate that bone and perhaps renal tissues are refractory to the effects of PTH in the alkaline state, and the stimulatory effects of PTH are enhanced during metabolic acidosis. It has been shown that the acid-base balance of the cow can influence vitamin D production, which is dependent on PTH, and influences the degree of hypocalcemia experienced at parturition. Cows fed a diet high in K and Na+ prior to parturition (inducing metabolic alkalosis) had lower plasma vitamin D concentrations at parturition than did cows fed a diet high in Cl and SO4 (inducing metabolic acidosis), despite more severe hypocalcemia in the cows on the alkalotic diet [55]. The dietary cation-anion ratio during pregnancy and parturition thus appears to be an important factor in the development of the disorder. A previous radiographic study on healthy pregnant buffaloes revealed sub clinical metabolic osteopathies without change in blood parameters [56] suggesting that subclinical hypocalcemia is more likely in buffaloes.

1.4. Clinical Pathology

Following a sudden drain of calcium into milk, the body reserves are decreased and hypocalcemia occurs. It has been mentioned that cows with milk fever have reduced smooth and skeletal muscle contractions [16]. This decreases the ruminal motility, feed intake and milk production. The immune functions of affected cows are suppressed [57] and affected cows are at greater risk of developing mastitis, retained placenta and metritis [16]. Cardiac functions may be affected in the more severe cases resulting in death of untreated cows. Buffaloes affected with milk fever are likely to develop similar changes yet little descriptions are available, probably due to a low incidence of the disease. Hypocalcemia was recorded in studies on buffaloes affected with milk fever, with serum calcium ranging from 4.68 – 5.92 mg/dL [28,39,41,42,58-60] compared to values of 8.40 - 10.0 mg/dL in healthy lactating buffaloes [4,28]. The levels of AST (liver enzyme aspartate aminotransferase), ALT (a hepatocellular leakage enzyme), and NEFA were elevated in buffaloes with milk fever. Lecithin cholesterol acyl transferase estimations have been recently suggested as a good indicator for milk fever in buffaloes [59].

1.5. Clinical Findings

Milk fever is commonly observed during the immediate post-partum period [39,41,58] but few cases have been recorded in non-pregnant, non-lactating buffaloes [61]. The incidence of milk fever in buffaloes is lower than in cows, however, the clinical manifestations are comparable [28,39,41,42]. Although hypocalcemia (Calcium 5.2 mg/dL) was detected in 6 buffaloes in a recent study, no clinical signs, except poor milk production, were evident [8]. Buffaloes with milk fever have lower (P<0.01) rectal temperature (99.26 vs. 100.5°F) [41,60], pulse rate (37.36 vs. 48.38), respiration rate (13.4 vs. 21.8/min) and depressed ruminal movements (1.6 vs. 7.6 every 5 min) compared to healthy animals [29]. The affected animals develop anorexia and are disinclined to move. Buffaloes evidence tremors, frothy saliva, turning of head towards flank (Fig. 2) and recumbency (Fig. 3) [62]. The muzzle is dry and the body and extremities are cold to the touch [41]. The pupils are dilated and the animal appears to be depressed. The intensity of the heart sound is reduced and the pulse is weak [29]. The animals are either unconscious or semiconscious. As the disease progresses, the buffalo lays either in sternal or lateral recumbency [42] and muscle tremors, grinding of teeth and flank watching are common. In one study [28] 25.6%, 43.6% and 30.8% of buffaloes with milk fever were in the standing posture, sternal recumbency and lateral recumbency. When treatment is delayed, the disease progresses rapidly and the animal becomes deeply unconscious. The condition deteriorates rapidly during a period of 12-24 hours and ultimately death takes place due to respiratory failure [29].

The economic losses due to milk fever in buffaloes are due to loss of milk production and therapeutic costs [63]. The total loss estimates is around Rs.665.74 rupees (Indian rupees) per affected buffalo [63].

Figure 2. A buffalo with milk fever.

Figure 3. A buffalo with milk fever in lateral recumbency.

1.6. Diagnosis

Evidence of recent parturition with sudden emptying of the udder and the associated symptoms such as recumbency, low body temperature, head turning towards the flank, are sufficient to presume hypocalcemia as the diagnosis. Calcium blood profiles can confirm the preliminary diagnosis, however, therapy should be instituted immediately and not wait for laboratory results.

1.7. Treatment

The usual therapy suggested is the prompt yet slow IV administration of 20% calcium borogluconate (300 - 450 mL) [25,39,42,64]. The infusion should be warmed to body temperature before administration in cold weather. Buffaloes respond to IV calcium therapy regaining normal posture, feed intake and milk within 2 - 3 h of therapy and calcium levels return to normal [63]. Relapses observed after 24 - 48 h can be successfully treated by repeating the same therapy [39]. A satisfactory approach to avoid relapses could be the administration of a 20 - 25% solution of calcium borogluconate partly intravenously (300 - 350 ml) and partly subcutaneously (200 - 250 ml). Alternatively, an infusion of a combination of calcium and magnesium is satisfactory [39]. Oral calcium supplements and vitamin D injections are suggested. In animals with continued excessive drainage of calcium in milk, calcium borogluconate may be administered 2 or 3 times at intervals of 12 hours. In milder cases oral supplementation of calcium preparations such as calcium chloride and tricalcium phosphate (100 g daily) or commercially available preparations, are suggested for a couple of days [29,65]. Preventive approaches for milk fever in cattle suggest the prepartum administration of vitamin D injections [66] or oral supplementation of anionic salts [16]. Similar approaches have been documented to be useful in buffaloes [50].

2. Ketosis

Ketosis is a metabolic disorder that is characterized by elevated concentrations of the ketone bodies β-hydroxybutyrate (BHBA), acetoacetate and acetone in blood (hyperketonemia), urine, and milk. The disease mainly occurs in early-lactation dairy cows and buffaloes when body reserves are used to support lactation. Excessive mobilization of fatty acids from adipose tissue during postpartum negative energy balance and their conversion to ketone bodies by the liver leads to higher levels of ketone bodies in the blood with development of clinical ketosis. Similar to cattle, the disorder can have a clinical and subclinical presentation in dairy buffaloes [67]. Clinical ketosis occurs frequently compared to subclinical ketosis and affects individual animals in a herd; buffaloes show a decrease in feed intake, weight loss, a drop in milk yield, depression, and occasionally nervous signs.

2.1. Etiology

The etiology of ketosis appears to be multifactorial and complex and it is possible that the same factors that affect dairy cows may also affect buffaloes although scientific evidence for this is lacking. It has been mentioned that ketosis occurs in cows when physiologic mechanisms for adaptation to negative energy balance fail [68]. Failure of hepatic gluconeogenesis to supply adequate glucose for lactation and body needs may be one cause of ketosis; however, poor feedback control of non-esterified fatty acid release from adipose tissue is another likely cause of ketosis [68]. All dairy cows generally experience a period of negative energy balance and fat mobilization after calving, but not all animals develop hyperketonemia, and even less develop clinical ketosis. Individual variation exists in the metabolic adaptation to negative energy balance. Negative energy balance is considered less intense in the buffalo because of a lower milk yield [1,69]. The development of ketosis seems not to be associated with the extent of the negative energy status alone; however, inadequate metabolic adaptation appears to contribute to the development of ketosis [68]. There are many factors that can result in the development of ketosis during early lactation. A high body condition score (= larger fat stores) and large fat mobilization around parturition increase the risk for ketosis in early-lactation cows [70-72]. Any disease in the peripartum period that results in reduced feed intake increases the risk of cows developing ketosis. For example, lameness [73] and milk fever [74] were associated with an elevated risk for the disease. Besides the several "cow factors", environmental influences also play an evident role in the etiology of ketosis. The feeding strategy in the dry weeks and early-lactation period is critical for the prevention of hyperketonemia at dairy farms [75,76], which can be illustrated by studies showing that nutritional factors such as prepartum energy intake [77] and postpartum nutrient composition of diets [78] influenced the metabolic status of cows and the prevalence of ketosis during early lactation. Concisely, ketosis is a multifactorial disease, and several cow and environmental factors contribute to the etiology.

The increased demand for glucose during peak lactation or during the initial stages of milk production in high yielding animals restricts the oxaloacetate availability for metabolic reactions. This results in diminished activity of the tricarboxylic acid cycle and hence energy production and reduced rate of acetyl CoA catabolism. Restricted energy production results in increased breakdown of depot fats causing more production of acetyl CoA. The excess acetyl CoA is directed towards ketone body synthesis (Fig. 4). Because of the diminished capability of the extra-hepatic tissues to catabolize acetyl CoA, the blood ketone concentration is elevated causing ketonemia and the appearance of ketone bodies in urine causing ketonuria. Ketonemia and ketonuria together are referred to as ketosis. The composition and quantity of feedstuffs used for animals may be responsible for the development of ketosis.

Figure 4. The formation of ketone bodies. 

2.2. Incidence

The first description of ketosis in buffalo appears to be that of Ramiah [79]. Screening of 3,395 adult female buffaloes for ketosis by the Rothera’s test in Maharashtra (India) revealed an incidence of 2.85% [80] and a nearly similar incidence was recorded in other studies on 600 [81] and 500 buffaloes [82]. Likewise, studies on 342 buffaloes in Tamil Nadu revealed an incidence of 2.92% [83]. A higher incidence of 3.1% [84], 4.8% [85], 5.29% [86-87], 8.11% [88], 9.2% [89], 23.45% [90] and 29.9% [91] has also been recorded.

The usual age of affected buffaloes varies from 8 - 10 years [81,82,84,85,87-89,92] and affected buffaloes are in their 3rd to 4th lactation [81,82,86,92-95].

Most studies depict that the condition develops within a month of parturition [81,82,85,87,90]. A higher incidence during winter months [82,87,89,94-95] appears to be related to a higher calving rate during this season [96].

2.3. Clinical Findings

Ketosis in buffaloes has been described to be either clinical [67,80,82,87,97] or subclinical [67,92,95,99] with clinical signs being less intense in the subclinical form [67]. The incidence of clinical (70%) and subclinical (30%) ketosis was described in Egyptian buffaloes in a recent study [67]. Clinical ketosis has been recognized to be of 2 types: the wasting form and the nervous form, with the wasting form being more common (87.6%) and the nervous form being less frequent (12.4%) [80].

Reduced intake of concentrates and slightly lowered milk production are the first observed signs of clinical ketosis in buffaloes [67,80,100-101] which progresses to complete anorexia, marked reduction in milk production, ruminal stasis and loss of body condition (Fig. 5) [67,82,96]. Acetone-like fruity smell is evident in the breath, milk and urine in a high [82,96,98,102-105] or a small proportion [67] of affected buffaloes. Constipation is evident with the progression of the disease and buffaloes may pass hard mucus covered feces [87,102,106]. Grinding of teeth and licking of body is common in affected buffaloes [82,96]. After sometime, signs of pica may be observed in a large proportion of affected buffaloes [96]. Animals may evidence signs of stiffness, they are dull and unwilling to move and their body coats become rough and dry [100].

Figure 5. A buffalo with poor body condition and ketosis.

Buffaloes with subclinical ketosis have a poor food intake and poor milk production. Diagnosis is usually made by testing their urine or serum for ketone bodies and blood glucose levels [65,99].

The nervous form of ketosis is observed only in a small proportion of buffaloes [80,107-108]. An affected buffalo moves aimlessly, may press its head against a wall and sometimes moves in circles [29]. A staggering gait and moderate tetany may be observed [100]. Apparent blindness and muscle twitching may occur [108].

2.4. Clinical Pathology

Little changes do occur in the hematology of buffaloes with ketosis. Most workers found no changes in hematology except slight neutropenia [82,102,106].

Significant hypoglycemia was a consistent finding in buffaloes with ketosis [81,92,109-111] along with ketonemia and an increase in serum cholesterol, total lipids and triglycerides (Table 1).

The insulin levels in ketotic buffaloes are low [88,89,104] and urine ketone is significantly higher [87]. The levels of oxidants like NO and malondialdehyde are high in buffaloes with ketosis [67,105]. Similarly, serum beta-hydroxybutyrate (BHB) levels are high in buffaloes with ketosis [67,103]. Hyperketonemia in buffaloes with ketosis is considered to originate because of stress [67].

2.5. Clinical Diagnosis

Recent parturition and typical refusal of concentrates by buffaloes coupled with reduced milk production should raise an alarm about the presence of ketosis in buffaloes. Similar to cattle, the diagnostic test most commonly used for confirmation of clinical [80-82] and subclinical [65,92,95,99,112] ketosis in buffaloes is the Rothera's test performed on milk or urine. The Rothera's test and a large number of commercially available strips are based on sodium nitroprusside (for example, Ketostix, Bayer or Ketocheck powder, Great States), however, these have been considered suitable to detect only acetoacetate and acetone in dairy cows [113,114]. The gold standard test for ketosis in blood is the estimation of β-hydroxybutyric acid as this ketone is considered to be more stable in blood compared to acetone or acetoacetate [114]. Commercial strips for detection of (BHB) in urine or milk are popular for use in cows however their availability in buffalo raising countries is minimal. A few recent studies in buffaloes evaluated (BHB) in ketotic buffaloes [67,105]. Herd screening of buffaloes for ketosis is not popular and a buffalo-side test which detects clinical or subclinical ketosis with reasonable accuracy needs to be developed and made available. The estimation of blood glucose would reflect the status of glucose and aid in the diagnosis. Studies on glucose tolerance tests have been conducted on buffaloes [115-117] and have suggested diminished glucose utilization in ketotic buffaloes compared to healthy buffaloes. A recent study concluded that there is an unexpected glucose tolerance to acute IV glucose loading in water buffalo compared with other ruminants [117]. Lecithin cholesterol acyltransferase (LCAT) has been shown to be a good predictor for ketosis in Egyptian buffaloes [59,105].

2.6. Therapy

Therapy in ketosis is oriented towards the restoration of glucose levels in affected buffaloes. This is usually achieved by daily IV administration of 20 - 25% glucose (0.5 g/kg) till recovery [80-82,87,89,95,101,118]. Two to three such treatments are usually sufficient for recovery from anorexia and a return to normal milk production [92,95]. In order to promote gluconeogenesis, IM administration of dexamethasone (0.05 mg/kg) [119] or triamcinolone acetonide (0.05 mg/kg) [80-82,90,92] are suggested.

Figure 6. A commercial strip for detection of ketosis.

Figure 7. A commercial BHB strip for ketosis.

Low insulin [120] in affected buffaloes (Table 1) has led to the IM administration of bovine insulin (0.5 U/kg) along with 20 - 25% glucose with a better result [80,82,89,99,101] in affected buffaloes, probably because of a better utilization of intravenously administered glucose. A few studies suggested that ketotic buffaloes would benefit from the administration of an anabolic steroid, nandrolone laureate 125 mg [65], or nandrolone phenylpropionate 100 mg IM [111].This would favor a prompt increase in blood glucose and a decline in high levels of serum cholesterol within 2 - 3 days of therapy [65].

Oral administration of gluconeogenic precursors such as 200 g of glycerol, 60 - 80 g of sodium propionate [87,90,92,101,121] or 250 g of propylene glycol mixed with water [100], have been suggested for 3 - 5 days for mild cases of ketosis or as a supplement in buffaloes after IV glucose therapy.

3. Hypomagnesemic Tetany of Dairy Buffaloes

Hypomagnesemic tetany of dairy buffaloes is a rare disorder of lactating as well as non-lactating pregnant buffaloes fed on a large quantity of young tender herbage [29]. The incidence of this disorder is extremely low in buffaloes compared to cattle, probably because buffaloes have significantly higher blood values of magnesium compared to cattle [4,122] and rarely are buffaloes exclusively raised on grasses as are cows. Physiologic values of serum magnesium vary from 2.90±0.18 to 3.13±0.27 mg/dL during early and mid-lactation in buffaloes [10,65].

3.1. Etiology

In pregnant and lactating buffaloes the developing fetus and drainage in milk; demand a higher requirement of the mineral magnesium. A low level of magnesium in the feedstuffs and lack of mineral supplementation in the diets of these animals make them vulnerable to the condition [100].

3.2. Clinical Findings

The clinical manifestation of the disorder has been described [29]. In acute cases, clinical signs appear suddenly. Affected animals suddenly stop eating, show uneasiness and stand cautiously attentive. These signs are associated with the twitching of muscles. The animal may appear frightened or "mad". The gait is staggering; later the sick animal falls and moves its limbs in spasms. Death may occur in less than 4h after the appearance of the clinical signs [21]. There may be a sudden rise in body temperature after the tetanic attack. The pulse and respiration rates are increased. In sub-acute cases, a gradual loss in appetite results in an unthrifty condition [123]. A loss of 22 - 55 kg in body weight of pregnant animals and about a 27% drop in milk yield of lactating animals has been recorded [22].

3.3. Treatment

Magnesium sulfate or magnesium borogluconate (25 g) dissolved in about 400 - 500 ml of sterile distilled water is suggested to be administered subcutaneously at a very slow rate [29]. The treatment should be followed by daily oral feeding of 50 - 60 g magnesium oxide for a period of 7 - 10 days. If the response is poor, slow IV administration of calcium-magnesium borogluconate is suggested [100].

4. Parturient Hemoglobinuria (Red Water)

Parturient hemoglobinuria is an acute life threatening disease of high yielding buffaloes and cattle characterized by hypophosphatemia, intravascular hemolysis, hemoglobinuria, and anemia [124-126]. A higher incidence has been recorded for buffaloes compared to cattle [18-20]. The disorder is highly fatal owing to intravascular hemolysis and severe anemic anoxia [127]. The condition is mostly observed during advanced pregnancy or within a month of parturition [128].

4.1. Epidemiology

The disorder has been recorded in buffaloes in India [124,127,129-141], Pakistan [19,125,142-155], Egypt [105,156-159], Brazil [160-161], Sri Lanka [29] and Iran [162].

The condition is recorded in adult buffaloes with higher frequency during advanced pregnancy (8 - 9 months) and in the post-partum period (1 - 60 days postpartum) [29,124,127,140,155,162,163]. A higher incidence during pregnancy [126,164-165] and during the postpartum period [140] has been demonstrated in different studies. The disease was considered to be more prevalent in stall-fed compared to grazing buffaloes [145]. The highest incidence of the disease appears to occur during the 3rd – 5th lactations [18,164,166].

4.2. Incidence

The overall incidence of the condition is low. In most countries the disorder is sporadic and of low incidence affecting high producing buffaloes, however, in certain areas in India (Haryana, Rajasthan, Uttar Pradesh and Punjab), the disorder is much more prevalent [167]. Shah et al., [149] recorded an incidence of 6.5% in a survey of 1987 buffaloes in Lahore (Pakistan). In Punjab (Pakistan) the incidence was recorded to vary from 0.02% – 4.4%[18,168]. In another survey in Maharashtra (India), the incidence was 0.70% [140]. A seasonal incidence of the occurrence of the disorder has been recorded in many studies with some studies recording a higher incidence during winter [140,153], while others recorded a higher incidence during summer [131,166,169]. The seasonal incidence is probably related to the availability of phosphorus in the soil and the diets fed to buffaloes [124,126,152].

4.3. Risk Factors

Pregnancy beyond 6 months and lactation are considered the putative risk factors for the development of the disorder with the relative risk and odds ratio for pregnancy and gestation being 1.71 and 3.77 respectively[163]. It has been mentioned that buffaloes once affected with parturient hemoglobinuria, are 21 times more likely to have low serum profiles of phosphorus and are thus likely to suffer from the condition again [125]. A recent study found that age (≥7 years), pregnancy (≥7 months), lactation (≥3), postpartum period (≤60 days), previous history of parturient hemoglobinuria and ingestion of cruciferous plants, were significant risk factors for the disorder in buffaloes, whereas feeding of cottonseed cake, use of mineral mixture or drugs, previous history of disease other than parturient hemoglobinuria were non-significant factors in the development of the disease [170].

4.4. Etiology

The etiology of parturient hemoglobinuria appears to be multifactorial yet poorly understood. However, a consistently low serum phosphorus in affected buffaloes [124,131,133,134] reflects a deficiency of phosphorus as the putative cause for the condition. Dietary deficiency of phosphorus has been considered a common cause of the condition [124,139,162,171-174]. Feeding of sugar cane tops, sugar beet, kale, mustard, cabbage and Lucerne [29] or berseem (Trifolium alexandrinum) [125,126,146,151,156,158] gram/lentil straw [173], sorghum straw [140], cabbage and turnip leaves [175] and wheat/ rice straw [174] precipitate a deficiency in phosphorus either due to their low content of phosphorus or due to presence of inhibitory factors such as metallic ions which interfere with the absorption and assimilation of dietary phosphorus [29]. Areas where the soil is deficient in phosphorus would produce crops deficient in phosphorus [125,152]. High levels of molybdenum [126,131,176] or potassium in the feed or high salt content in the water [160] can exacerbate phosphorus deficiency. Higher potassium was recorded in roughages and soil in rice growing areas of Pakistan with concurrent low calcium and phosphorus in the serum of buffaloes raised in these areas [177]. Similar findings were also recorded in an arid area in India with recommendations for supplementing the buffalo diets with phosphorus and calcium [178]. Therefore, the dietary cation/ anion ratio seems important in maintaining the homeostasis of phosphorus and calcium in the serum. Copper deficiency is also an etiological factor of post-parturient hemoglobinuria as its deficiency reduces the activity of the copper-containing enzyme superoxide dismutase, which is a part of the erythrocyte protective mechanism against oxidative stress [179,180]. Low copper was recorded in buffaloes affected with parturient hemoglobinuria [126,131,135]. Ingestion of cold water was shown to precipitate hypophosphatemia in a pregnant Egyptian buffalo [156].

4.5. Pathogenesis

The mechanism of intravascular hemolysis and the associated anemia in response to deficiency of phosphorus continues to be poorly understood. All animals suffering from hypophosphatemia do not develop intravascular hemolysis [158,181]. There is an association with hypophosphatemia and a low dietary intake of phosphorus, and it is presumed that the drain of lactation causes further depletion of phosphorus reserves [140]. The dependence of mammalian red blood cells on glucose metabolism as the main source of energy for viable function and structure makes them highly vulnerable to factors that inhibit the glycolytic pathways. The deficiency of phosphorus has been documented to result in depletion of ATP [182-184]. The reduced ATP levels along with decreased membrane phospholipids (which help in maintaining the shape and integrity of red cells) were considered responsible for the change in the shape of red blood cells with resultant echinocytosis/ sphero-echinocytosis, which are prone to hemolysis [167]. Cruciferous plants are known to cause hemoglobinuria because of their high s-methyl cysteine sulfoxide content which is converted by ruminal microflora to dimethyl-sulfoxide (DMSO) [151]. Once absorbed into circulation, the DMSO causes precipitation of hemoglobin leading to hemolysis (Yates, 1990). Sugar beets, alfalfa, and berseem are thought to contain saponins that are known to have a lytic action on erythrocyte membranes [151,185,209]. The hemolytic action of saponins is believed to be the result of the affinity of the aglycone moiety for membrane sterols, particularly cholesterol, with which they form insoluble complexes [209].

It has been observed that during advanced gestation, more phosphorus and calcium are required for the developing fetus. The absence of supplementation leads to hypophosphatemia [166]. Moreover, high calcium to phosphorus ratio observed during gestation in buffaloes [147], results in decreased phosphorus absorption from the intestinal tract and ultimately leads to hypophosphatemia. Phosphorus deficient soils are common in dry tropical countries like India and Pakistan [19,124,154]. Although many soils are naturally deficient in phosphorus, heavy leaching by rain and constant crop removal also contribute to phosphorus deficiency in the soil. It was noticed that fodders grown on phosphorus deficient soils are consequently low in phosphorus content, and thereby prolonged feeding on such fodders can lead to hypophosphatemia [33]. A significant decrease in erythrocyte count, hemoglobin concentration, and hematocrit in affected buffaloes, indicating severe anemia, was recorded in buffaloes with hemoglobinuria [135]. Intravascular hemolysis probably occurs due to an impaired glycolytic pathway and the depletion of ATP in erythrocytes results from phosphorus deficiency [186]. A subnormal concentration of ATP predisposes red blood cells to altered functions and structure, causing a loss of normal shape, and an increase in fragility, ultimately leading to hemolysis. In vitro studies on buffalo erythrocytes revealed that the uptake and fate of glucose was dependent on the concentration of phosphorus in the medium. When erythrocytes were incubated in a synthetic medium deficient in phosphorus, all classes of cellular phosphates including ATP were significantly depleted and these changes could be reversed by addition of phosphorus to the medium [186]. Erythrocytes from parturient hemoglobinuria affected buffaloes revealed significant depletion of phosphorus, ATP and acid labile phosphates [184]. In vitro incubation of these erythrocytes in a medium containing phosphorus replenished the cellular phosphates [184].

A possible mechanism for parturient hemoglobinuria could be the drastic reduction in the glutathione content in red blood cells, as recorded in buffaloes with parturient hemoglobinuria [128,136,137,187,188]. A low activity of glucose-6 phosphate dehydrogenase [139], red cell catalase [128] and a significant increase in Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic-pyruvic transaminase (SGPT) and alkaline phosphatase, was recorded in buffaloes with parturient hemoglobinuria suggesting possible alterations in these enzymes in precipitating hemolysis. Oxidative stress has been suggested as a possible putative factor for hemolysis [127,189-191].

Development of the disease in pregnant buffaloes during advanced pregnancy is presumed to be due to the increased demands of the developing fetus [18,164] coupled with dietary deficiency [165]. It also appears that from the seventh month onwards, pregnant buffaloes have a significantly wider ratio of blood Ca/P compared to those in the earlier stages of pregnancy [147].

Figure 8. Suggested pathogenesis of parturient hemoglobinuria.

4.6. Clinical Findings

The first notable clinical sign in affected buffaloes is the passage of red to coffee-colored urine [124,126,130,135,140,145,154,166,169] within 20±10 days before or after parturition and rectal temperature of 101°F to 103°F [19,192]. In most cases the appetite is normal but milk yield is significantly reduced [126] and anorexia is marked as the disease progresses. Constipation is common and feces are hard and black-tinged [166]. Excessive formation of hemosiderin and its deposition in the gastrointestinal mucosae in buffaloes with hemoglobinuria could be responsible for gastrointestinal disturbances such as ruminal stasis, constipation, straining and dark coloration of the feces [166,193]. Body temperature is slightly higher initially but then tends to become subnormal [19,124,140,145]. Heart rate is slightly increased. The mucous membranes of the conjunctiva and vulva are discolored or pale in appearance and affected buffaloes may have a foul breath and frothy urine [155]. As the disease progresses, jaundice and weakness may be evident with recumbency [18,126,135,193]. Labored breathing and jugular pulsation can be observed during the terminal stages of the disease. Clinical signs mostly appear after 4 months of pregnancy [124] in buffaloes and some buffaloes may abort [145]. Heuer and Bode [125] observed that feces might be firm and dry or fetid and occasionally buffaloes may be diarrheic. According to these authors, an elevated body temperature (up to 40°C) in the early stages is a variable sign; at later stages of the condition, low temperature is common. The disease is most commonly observed during summer when animals are fed straw from wheat or paddy, or stoves of maize, sorghum and pearl millet, which are all very poor sources of phosphorus. The duration of the disease varies from 3 - 9 days [153] with mortality up to 15%. Death may occur within a few days. In non-fatal cases, convalescence requires about 3 weeks and recovering animals often show pica. Recovered animals may continue to evidence decreased appetite or respiratory distress [194]. In a recent study, 1/13 hemoglobinuric buffaloes concurrently developed ketosis [195]. Rough hair coat and a stiff gait are noticed in some affected buffaloes [196], some buffaloes may become recumbent. Gangrene at the tip of ears has also been observed in pregnant hemoglobinuric buffaloes [165].

4.7. Clinical Pathology

In marginal phosphorus-deficient areas, normal non-lactating animals in an affected herd may have serum inorganic phosphorus levels within the normal range. Lactating buffaloes in an affected herd may have moderately low levels of phosphorus (below 4 mg/dL) without any clinical sign [199] or low values (2 - 3 mg/dL) with limb paralysis as a clinical sign [200]. However, in Romanian buffaloes the phosphorus values were recorded as 2.68±1.26 and 2.28±0.92 in pregnant and lactating buffaloes without any clinical change [201]. Buffaloes with hemoglobinuria have extremely low levels of 0.97 - 2.6 mg/dL of phosphorus (Table 2). Blood glucose, bilirubin, creatinine and serum alkaline phosphatase are elevated in affected buffaloes [140,154,165,171,172] with possible jaundice due to intravascular hemolysis. The serum enzymes AST, ALT and ALP are significantly elevated in buffaloes with parturient hemoglobinuria [202]. Hemoglobinuria affects the functional activities of different body organs, mainly liver, heart and kidneys [129]. Erythrocyte count, packed cell volume and hemoglobin levels are also greatly reduced (Table 3). Heinz bodies may be present in erythrocytes and perhaps Howell Jolly bodies too ([135,140]. Affected buffaloes evidence macrocytic hypochromic anemia with neutrophilia, higher creatinine and serum urea nitrogen [140,165]. The urine is dark red-brown to black in color and usually moderately turbid. No red cells are present in the urine. Urine analysis reveals a pH of 8.0 - 9.0 and urine samples are positive for protein and hemoglobin [130,140] and sometimes ketones and bile pigments are present too [166,203]. Due to probable overload in the liver, serum SGOT, SGPT and ALP levels are increased in affected buffaloes [202].

4.8. Diagnosis

Post-parturient hemoglobinuria is characterized by an acute hemolytic anemia in buffaloes calved within the preceding 4 weeks. Other causes of acute hemolytic anemia are not confined to the post-calving period. Laboratory tests are usually necessary to confirm the diagnosis and to eliminate hematuria as a cause of urine discoloration. The presence of hemoglobin in the urine can be verified by the benzidine test or using commercially available dipstick strips (Fig. 9). Upon centrifugation of the dark coffee colored urine, the red blood cells settle at the bottom in hematuria samples whereas in hemoglobinuria samples the color remains unchanged. The differential diagnosis of red urine in buffaloes was summarized by [151]. In a recent study [105], lecithin cholesterol acyltransferase (LCAT) was found to be low in buffaloes with hemoglobinuria and was suggested as a predictor for parturient hemoglobinuria in buffaloes. Significantly lower activity of LCAT was recorded in 37 Egyptian buffaloes 4 weeks prepartum, of these, 23 developed ketosis and 14 developed parturient hemoglobinuria after parturition. The levels of SGOT, SGPT and ALP have been suggested as diagnostic indicators for hemoglobinuria in buffaloes [202].

Figure 9. Urine dipstick for hemoglobin in urine showing positive color change.

4.9. Mortality

The mortality rate recorded in different studies vary from 12% [124] to 15% [126,149,153], however, exceptionally high mortality of 53.5 - 63.4% was recorded in studies in Pakistan [18,145]. The mortality is dependent upon the time of initiation of therapy since the onset of disease. In buffaloes receiving therapy early mortality is low [29].

4.10. Necropsy Findings

The blood is thin and icterus is widespread throughout the body. The liver is swollen, and fatty infiltration and degeneration are evident. Discolored urine is present in the bladder. The kidneys and liver are pale enlarged and congested with deposition of hemosiderin [124,126,132]. The epicardium and endocardium show hemorrhages and the lungs are emphysematous and edematous [124,126,133,140,166]. Death usually occurs due to anoxic anoxia.

4.11. Treatment

Sodium acid phosphate 60 - 80 g dissolved in 300 mL of distilled water as a 20% solution, administered IV is the usual therapy for affected buffaloes [124,127,135,140,165]. This is followed by a similar dose administered SC [135] and oral supplementation of phosphorus (60 g), copper sulfate (3-5 g) and cobalt sulfate (100 mg) for a period of 7 - 10 days. Two to four IV therapies are required for complete recovery [140]. The intravenous injection of phosphorus in the form of monobasic sodium phosphate 60 g in 300 ml of sterile distilled water followed by oral administration of the same dose, twice daily for 3 days, has often been effective [29]. Inorganic phosphorus injections are suggested to be administered IV [151]. Ten percent glycerol phosphate calcium (1 mL/kg IV) was suggested in one study [198] along with twice-daily oral supplementation with 100 - 120 g of dicalcium phosphate with good recovery in affected buffaloes.

In severe cases, blood transfusions may be a useful supportive therapy [100,151]. Blood collected from slaughtered buffaloes supplemented with 2.5 g of sodium citrate (per liter of blood), penicillin and streptomycin is suggested for transfusion in buffaloes [151]. Oral supplementation of copper sulfate (2 g in 500 mL of water) has been advocated for hemoglobinuric buffaloes in areas with high content of molybdenum in fodder [131]. Alternatively, copper glycinate (1.5 mg/Kg dissolved in 540 mL normal saline and infused IV) has also been suggested [151,198]. Supplementation of iron and other drugs would depend upon the general condition and requirements of the animal [165,195,196]. Reoccurrence of the condition in recovered buffaloes has been recorded in 18.3% to 27.4% buffaloes [126,164]. Antifibrinolytic drugs have been suggested for the therapy of hemoglobinuria on the basis of increased fibrinolytic activity in affected buffaloes [205]. Epsilon amino caproic (EACA) (20 g dissolved in 540 mL of 5% dextrose and administered IV till recovery) and para-amino methyl benzoic acid (300 mg dissolved in 540 mL of 5% dextrose and administered IV till recovery) have been shown to be 90 - 92% effective [206,207]. Similarly, Botropase (a blood coagulant and antifibrinolytic drug prepared from the venom of snake; Bothrops jararaca) has been suggested as a supplement to sodium acid phosphate therapy in buffaloes with good recovery rates [151,208]. Oxygen releasers like inosine (0.5 g in 5 - 10 mL of dilute HCl mixed with 540 mL of 5% dextrose administered IV once daily for 2 - 3 days) have also shown promise in buffaloes that continue to evidence respiratory distress despite a clear urine [194].

Antioxidants have been suggested for therapy of hemoglobinuria in buffaloes because of the possible oxidative stress that may be present in affected buffaloes [127,137,189,191] with vitamin C being the common antioxidant used. Ascorbic acid either at 5 g [165,188,194,207] or 7.5 g infused daily IV along with 500 mL of normal saline[189] were found to be 68.5 and 82% effective, respectively. Other antioxidants like vitamin A and E have also been suggested to be helpful [195].

4.12. Prevention

An adequate intake of phosphorus according to the requirements for maintenance and milk production should be ensured, particularly during late gestation and early lactation. A decrease in the incidence of the disease was reported after copper supplementation of cattle in a copper-deficient area [179]. However, in one study on buffaloes, copper was found to be normal in buffaloes with hemoglobinuria [197]. Because of hemolytic saponin and the low phosphorus contents in cruciferous plants like Berseem [157], it is suggested to feed mixed with other fodders to reduce the incidence of hemoglobinuria. Diet or water supplementation with a source of phosphorus is suggested in deficient areas [151]. This includes the addition of sodium acid phosphate 30 g/animal/day or bone meal 100 g/animal/day or the use of commercially available preparations [151].