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A lot of research work has been carried out on the different aspects of milk including composition; oxytocin induced changes in milk, mode of action, possible effects, doses, stage of lactation etc. The most pertinent literature related to the present study i.e., biochemical profile within bovine mammary secretions is influenced by an exogenous oxytocin treatment during lactation period has been reviewed under the following headings: 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 Factors affecting milk composition Effect of lactation stages on milk composition Residual/complementary milk Oxytocin Oxytocin and milk yield Effect of oxytocin on milk composition Electrophoretic pattern of protein fractions of milk Milk enzymes

2.1

Factors affecting milk composition

The composition of milk is not fixed since many factors influence the end

product. These variations can be related to genetics and environment, level of milk production, stage of lactation, disease, season, locality and age of the cow (Gopalkrishan and Lal, 1994). The milk composition also varies within the cows from milking to milking (Friggens and Rasmussen, 2001).

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The composition of milk also differs within species. The lactose content of milk is moderately constant between dairy breeds, protein varies to some degree but fat varies widely. For example, it was reported that Guernesy (5.0%) and Jersey (5.5%) breeds contained the highest milk fat as compared to Holstien (3.5%) that contained the lowest milk fat percentage (Hurley, 1997). The age of the cow is closely related to the number of lactations, as an increase in number of lactations is associated with decrease in fat and solid not fat (SNF) content of milk. A survey of lactation records of Holstein shows that milk protein contents decreases 0.1-0.5 units over a period of five lactation or 0.02-0.05 units per lactation (Rook, 1961). Feed and diet composition are important factors that can cause changes in milk composition. Protein concentration can be changed to some extent but lactose scarcely (Sutton, 1989). Although other diseases can affect milk components level and distribution, mastitis has been the predominate disease studied. Mastitis results in a reduction of milk fat and increase in milk proteins that has been attributed to the influx of blood-borne proteins (Shuster, 1991). Mastitis is also associated with increase in the concentrations of different enzymes in milk (Auldist et al., 1995).

2.2

Effect of Lactation stages on milk composition

Lactation stage is a major factor affecting the characteristics of milk componets

such as fat content, protein, macro-mineral contents, and some of the enzyme activities but not on the casein: protein ratio or phosphorus content (Schutz et al., 1990; Decaen and Adda, 1970; Gue!guen, 1971; Benslimane et al., 1990). 2.2.1 Physicochemical composition The protein content in cow milk rises above the average from 21st week of parturition. Increase during the last 10-12 weeks of parturition is rapid. The casein content of cow varies during the first 60 days and shows an increasing trend after 200 days in lactation. The albumin level of cow milk varies from 0.23% to 0.36% but
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increases after 175 days of lactation as influenced by the stage of lactation. The globulin content also varies from 0.12% to 0.38% (Srivastava, 1993). According to Coulon et al. (1997) milk pH, calcium and the urea content are higher in late lactation. Beata (2008) while comparing the daily milk yield, its chemical composition in milk depending on the lactation stage reported that fat and protein content in milk during test milking at successive lactation stages showed a growing tendency and the highest concentration of these nutrients was identified between the 200th and 300th lactation day in cows milk. Afterwards these contents decreased rapidly even lower than those at the beginning stage of lactation. Total solids and solids not fat are also affected by stage of lactation. A study on trend of SNF in Holstein cows during whole milking cycle indicates the lowest SNF during second month followed by an increase to the eight month and a rise in nine and ten months of lactation (Ozrenk and Inci, 2000). Occurrence of changes in milk content during lactation; the decrease of milk yield accompanied by an increase in fat and protein content is the usual description of milk secretion (Kolb, 1987). In contrary, fat and protein curves had been reported with continuous declined shape and also with the standard shape as some disagreement has been reported in earlier studies (Schutz et al., 1990; Stanton et al., 1992; Pollott, 2004). Curves for milk and milk components were normally regarded as independent curves though, these curves are linked, as milk is a mixture of fat, protein, lactose, vitamins, and minerals, either dissolved or suspended in water. This connection was previously studied at phenotypic and genetic level. Milk yield, fat yield and protein yield curves follows the standard lactation curve in 19.3% of lactations, whereas fat and protein percent have reversed standard shape. High changeability of shapes is contributed by early phase of lactation with the fat and protein yield curves (Silvestre et al., 2009). 2.2.2 Fatty acids The fatty acids composition of milk is related to lactation stages. The lactation stage effect is noticeable and mainly linked to lipid store mobilization in early lactation,
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but it only lasts a few weeks each year (Chilliard et al., 2003). Both lactation stage and energy balance contribute to variation in milk fat composition and change the activity of different fatty acid pathways (Stoop et al., 2009). At initiation of lactation, cows are in negative energy balance, causing mobilization of adipose fatty acids and incorporation of these long-chain fatty acids into milk fat (Belyea and Adams, 1990). Concomitantly, de novo synthesis of short-chain fatty acids by mammary tissue has been inhibited by high uptake of long chain fatty acids (Bauman and Davis, 1974). Eastridge and Palmquist (1988) studied the pattern of fatty acids in milk fat at 1, 4, 8, and 12th week of lactation, as a proportion of those occurring at 16th week. Two points are striking; firstly, the proportion of short-chain fatty acids, except for C4:0, is low in early lactation, and these fatty acids increase, reaching more than 90% of maximal proportions by 8th week of lactation. This increase is consistent with the release of inhibition by adipose mobilization, which is largely completed by 4th to 6th week of lactation (Gamsworthy and Huggett, 1992). Second, the synthesis of short-chain fatty acids is inhibited to different degrees in a pattern that shows increasing inhibition from C6:0 to C12:0. Lynch et al. (1992) observed the same pattern of change in de novo synthesis of fatty acids over an entire lactation in cows on a bovine somatotrophin. An interesting cycling of milk fatty acids composition was observed in cows injected with recombinant bovine somatotrphin at 14 days intervals. Within a single injection interval, the percentages of C8:0, Cl0:0, C12:0 and C14:0 were significantly lower at 5th day post injection than at 12th day post injection, but C18:1 showed the opposite pattern. It was further observed that the concentration of cholesterol in milk fat changes with stage of lactation in the same manner as the fatty acids that are synthesized de novo from acetate (Lynch et al., 1992). Smith et al. (1978) observed that dietary protected tallow produced fatty acids pattern similar to that of early lactation. Synthesis of C4:0 is not inhibited at all, which is consistent with its unique origin from two known pathways independent of the inhibitable acetyl-coenzyme A (CoA) carboxylase pathway (McCarthy and Smith, 1972). Similar trends in fatty acids distribution as a percentage of total fatty acids were shown by other
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researchers and the variation in actual synthesis of individual short-chain fatty acids during lactation was relatively small when expressed as grams of fatty acids per kilogram of milk rather than as proportions (Karijord et al., 1982; Syrstad, 1982). Effect of lactation stages through thin layer chromatography was significantly analysed by Armughan and Narayanan (1981) indicating that with progressive lactation the major changes from cholestrol fat have been increased in lower fatty acids and declined in oleic acid. The effect on influence of fatty acids composition has also been studied. Kgwatalala et al. (2009) reported that Canadian Holstein cows had higher C18; total, lower C10, C12, C14, and conjugated linoleic acid index, respectively during early lactation compared with the subsequent lactation stages. Early lactation was characterized by higher concentrations of oleic acid (C18:1 cis-9), vaccenic acid (C18:1 trans-11), linoleic acid (C18:2), monounsaturated fatty acids, total polyunsaturated fatty acids, and lower concentrations of capric acid (C10:0), C10:1, lauric acid (C12:0), C12:1, myristic acid (C14:0), myristoleic acid (C14:1), palmitic acid (C16:0) and total saturated fatty acids when compared with the subsequent lactation stages. Neither the stearoyl-CoA desaturase 1 genotype nor the stage of lactation had an influence on conjugated linoleic acid concentrations in milk. Oxytocin release, intra-mammary pressure and milking characteristics in dairy cows has been changed during lactation were studied by Mayer et al. (1991). Pre-milking baseline intra-mammary pressure had its maximum value in early lactation until month 4 and then decreased to approximately 50% of its initial level. Ejection pressure followed a similar pattern, but dropped only to approximately 75% of its maximum. This was due to the constant elevation of pressure increase, reaching its highest level in late lactation. Time from commencement of stimulation until maximum pressure exceeded 1 min in almost all instances even in early lactation and increased throughout lactation.

2.3

Residual /complementary milk

Residual milk under review means the milk which may not be removed from the

udder due to certain reasons. At the start of milking about 15-25 % of the total amount of
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milk in the udder is not removed during milking. Residual milk is also called as complementary milk. Percentage of residual milk after milking as reported by Schmidt (1971) is 12.8% for machine stripped cows, 14.0% for non machine stripped cows, 16.8% for cows receiving oxytocin subcutaneously and 12.2% for cows that were measured for one year. Residual milk decreases in proportion to milk yield as lactation progresses; that is the percentage remains same throughout the lactation period (Schmidt, 1971). The amount of residual milk obtained by injecting 10IU of oxytocin intravenously varied roughly as the normal milk yield within breeds. The fat content of the residual milk was highest at the peak of lactation and decreased as lactation advanced. The relationship between quantity and fat test in residual milk was the reverse of that found with normal milk, i.e., high yields of residual milk were associated with higher fat tests than were low yields (Swanson and Hinton, 1951).

2.4

Oxytocin
Oxytocin (Greek word: "quick birth") is a nine amino acid peptide (Cys-Tyr-Ile-

Gln-Asn-Cys-Pro-Leu-Gly) with molecular mass of 1007 Daltons. It is synthesized in hypothalamic neurons and transported down axons of the posterior pituitary gland into blood to stimulate contraction of myoepithelial cells causing ejection of milk. The synthetic oxytocin is quite commonly used in human as well as veterinary medicine.

2.4.1 Mechanism of oxytocin

Milk ejection is an active transport of alveolar fluid into the cisternal compartment. It consists of contraction of myoepithelial cells that encircle the alveoli like a basket and transport of the milk through the milk duct system. Tactile stimulation of the mammary glands through a neuroendocrine reflex arc causes a natural reflex called milk ejection (Crowley and Armstrong, 1992). From the neural lobe of the pituitary gland by the milking stimulus, oxytocin released into circulation and induced milk discharge during lactation. A cow having approximately 40 liters of blood would have to release about 0.4 to 2.6IU of endogenous oxytocin to establish the range of concentrations (Gorwit, 1979; Sagi et al., 1980). In response to elevated oxytocin blood concentrations,
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binding to the oxytocin receptors of the myoepithelial cells causes alveolar contraction (Soloff et al., 1980). As a result, alveolar milk is forcefully shifted into the cisternal space. It causes a rapid increase of pressure within the cistern (Bruckmaier and Blum, 1996) and an enlargement of the cisternal cavity size (Bruckmaier and Blum, 1992). Nevertheless, alveolar milk cannot be ejected if milk is not simultaneously removed from the udder due to the limited cisternal space (Bruckmaier et al., 1994; Bruckmaierl et al., 1997). Eighty percent of milk stored in the udder regarded as alveolar fraction, is available after milk ejection that is stimulated by the release of oxytocin and myoepithelial contraction. Tactile teat stimulation causes milk ejection, either manually or by the milking machine. The time from the beginning of a tactile stimulation to the occurrence of the milk ejection lasts from forty seconds upto more than two minutes and increases with decreasing level of udder filling. Thats why, in end production stages of lactation, cows need a longer pre-stimulation (Bruckmaier, 2005).

2.4.2 Role of exogenous oxytocin

There are many factors can disturb milk ejection. It has been reported that a disturbed milk ejection is due to a decreased secretion or complete absence of oxytocin from the pituitary gland (Bruckmaier, 2005). Oxytocin release from the pituitary gland is inhibited in dairy practice during different types of emotional stresses or for several weeks after parturition in cows. Inhibition of milk ejection is caused by milking in unfamiliar surroundings which could be eliminated by small doses of oxytocin injections (Bruckmaier et al., 1993). Anthony et al., (1959) used oxytocin to aid milk let-down. These different techniques do not measure the same trait. During early stages of lactation, milk consumption is not an accurate estimate of the total milk production of a cow (Gifford, 1953). A complete milking by machine or by hand provides no estimate of milk consumption. Oxytocin treatment did not have a significant effect on milk consumption or on total milk production (Schwulst et al., 1966).

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Action of endogenous oxytocin is responsible to remove upto 90% of stored milk from cows during normal machine milking. Then, administration of oxytocin (intravenous) at supraphysiological dosage (10IU) can remove remaining residual milk (Knight, 1994; Bruckmaier, 2003). Many reports have shown that the use of exogenous oxytocin can enhance milk production (Graf, 1969; Gorewit and Sagi, 1984; Nostrand et al., 1991; Ballou et al., 1993). Consequences of oxytocin injections in increasing milk yield varied ranging from 3% to 15% that could be significant or non significant (Sagi et al., 1980; Nostrand et al., 1991; Ballou et al., 1993; Knight, 1994). Increase in milk production, associated with the injection of oxytocin, can only be explained by the stimulation of myoepithelial cell contraction to obtain residual milk, thereby limiting the negative effects of feedback inhibitor of lactation or increased intra-alveolar pressure on milk secretion (Knight, 1994). Direct stimulation of mammary blood flow could explain the galactopoietic effect of oxytocin (Fleet et al., 1993) and could result from direct stimulation of mammary tissues (Ballou et al., 1993). Oxytocin treatment may be involved in mammary cell maintenance and metabolism, in addition to causing myoepithelial cell contraction and milk letdown (Zamiri et al., 2001). Kimura et al. (1998) and Lollivier et al. (2001) also reported that lactating mammary epithelium might be an additional target for oxytocin and oxytocin may supposed to be another task other than milk ejection in mammary gland.

2.5

Oxytocin and milk yield

Milk yield in dairy cows is controlled by many factors, such as hormonal status,

genetics, nutritional state, environment and milking frequency. Most of the studies have been focused on the effect of exogenous oxytocin on the ejection of total milk, or residual milk from the udder (Donker, 1958; Graf, 1969; Morag and Griffin. 1968 and Peeters, et al., 1960). Jones (1967) removed cisternal milk and then observed the ejection of milk from the fine ducts and alveoli after exogenous oxytocin administration. Usually this is done by intravenous injection of 10IU of oxytocin or injection of 20IU either by
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subcutaneously or intramuscularly. Though, 0.5IU (intravenous) can be as effective as 16IU might work (Nostrand et al., 1991). Twice daily injections of oxytocin instantly after normal milking followed by remilking resulted in significantly greater milk yield, but, the percentage of milk collected at the normal milking declines over time, whereas the percentage of residual milk increases. Cows were subjected to an injection of 1ml (20IU) of oxytocin at each milking throughout lactation reported by Nostrand et al. (1991). Administration of exogenous oxytocin to cows for a full lactation increased milk yield by 11.6% over cows not receiving oxytocin. It appears that most of the increase in milk yield occurred during the declining phase of lactation. Donker et al. (1954) reported that an intravenous dose of 1.5 to 3.0IU of oxytocin was sufficient to bring about effective removal of residual (complementary) milk. Graf (1969) observed that 10IU of oxytocin was needed to obtain maximum yields of residual milk. By contrast, Morag and Griffin (1968) found doses as low as 0.5IU of oxytocin as effective as 16IU in obtaining residual milk. This discrepancy may be attributed to differing criteria used by various workers in describing "milk ejection". Alternatively, the intensity of milk ejection may be proportional to the duration and intensity of exposure to oxytocin (Thompson et al., 1973). Apparently, even minute amounts of exogenous oxytocin can be effective in eliciting milk ejection. Effectiveness depends on the definition of proper milk ejection and the time prior to milking at which the hormone is administered. Curto et al. (1972) reported that 0.04IU of oxytocin was sufficient to induce milk ejection in some cows. Analysis of milk flow and yield data showed that multiparous cows were less responsive to incremental doses of 0.05 to 0.2IU of oxytocin than primiparous cows (Ewy and Barowicz, 1977). From milk flow patterns, the lowest dose of exogenous oxytocin that elicited milk ejection in cows was 0.10IU. Small doses of exogenous oxytocin (less than 0.3IU) given occasionally did not inhibit release of endogenous oxytocin in response to milking stimulation (Sagi et al., 1980). Five doses of oxytocin (0.5, 1.0, 1.5, 2.0, 3.0IU) were injected through the subcutaneous abdominal vein (milk vein) of 15 Holstein Friesian cows one minute before
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machine attachment. Total milk yield was slightly higher for cows receiving 2.0 and 3.0 IU oxytocin. Administration of 2.0 or 3.0IU of oxytocin to cows significantly decreased the total residual milk yield as compared with other doses. As compared with lower doses, a dose of either 2.0 or 3.0IU of oxytocin applied intravenous caused slightly higher milk yields but does not affect milk flow dynamics. Oxytocin doses (2.0 or 3.0IU) reduced total residual milk yield, suggesting that extraction of milk is more efficient (Gorweit and Sagi, 1984). Small doses of exogenous oxytocin, administered may mimic stimuli that normally promote milk ejection in cows. Efficacy of these amounts depends on criteria to determine milk ejection. Cleverly et al. (1968) found that 0.1IU of oxytocin administered to cows elicited increased intramammary pressure equivalent to that during milking. Donker (1958) found that 0.12IU oxytocin was needed to elicit normal ejection in one cow; residual milk yield was an index. Peeters et al. (1960) measured quantities of milk ejected from the left forequarter of cows after IV injection of oxytocin. They reported that the threshold concentration needed to elicit milk ejection was approximately 0.02IU. By observing the presence or absence of bimodal milk flow patterns, Sagi et al. (1980) reported that 0.02IU oxytocin elicited milk ejection in 9 of 16 cows receiving this treatment and that 0.10IU induced milk ejection in all cows. Increasing the dose to 0.30IU resulted in greater peak milk flow rates and shorter machine on-time. Exogenous doses of oxytocin (10IU or more) were considered to be supraphysiological (Gorewit et al., 1983). However, doses less than 10IU did not consistently yield more than 75% of residual milk (Thompson et al., 1973). Supraphysiological (1, 10, 100, or 1000IU) doses of oxytocin were used by Allen (1990) after a normal milking to investigate the association among milk composition, secretion rates and cell electrolyte concentrations. However, in many reports, workers used extra physiological oxytocin doses (1020IU) (Nostrand et al., 1991; Ballou et al., 1993; Knight, 1994) and even 40IU (Dill et al., 1974). Milk ejection, milk production and milk quality in dairy cows are influenced by harmful effects of oxytocin large doses and could elucidate discrepancies in earlier reports as well as milk yield approached a constant level

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with repeated injections of oxytocin, indicating a constant rate of secretion (Lane et al., 1970; Allen, 1990; Bruckmaier, 2003). The issues of benefit and safety of oxytocin at different concentrations during bovine lactation is not thoroughly understood. Lefcourt and Akers (1983) questioned the physiological need for oxytocin for efficient milk removal in cows. Oxytocin appeared to be quite effective in amounts down to 1.5IU per injection based on the early assay, although fright or excitement raised the threshold level to more than four times this amount, as noted by lacks of response to repeated injections on certain occasions (Donker et al., 1954). Acute injections of oxytocin can reduce subsequent milk yield by an amount greater than the gain of complementary milk. Frequent injections of large doses of oxytocin hinder with normal secretory activity of mammary epithelia and inhibit the normal milk ejection reflex. Continuous administration of oxytocin (100 or 200IU/day) decreases milk yield (Graf et al., 1973). Increase in milk sodium and somatic cell count suggests additional negative effects on milk quality. Although rules made by Pennsylvania dairy herd improvement association have recently been altered to allow use of oxytocin on test day (Anonymous, 1988) such practice should be approached with caution. In contrast, chronic administration of high doses of oxytocin (100IU four times daily for 28 days) did not alter milk yield, percentage of protein or somatic cell count (Stewart and Stevenson, 1987). Intramuscular injection of 20IU oxytocin prior to each milking for 60 days postpartum tended to increase average and peak milk yields (Nostrand et al., 1987). Calf suckling and oxytocin injections are commonly used for pre-milking stimulus in dairy buffaloes under field conditions (Qureshi and Ahmad, 2008). In rural circumstances, due to different milking temperaments and in case of death of young calves murrah buffaloes are being injected intramuscular with oxytocin hormone in spite of the ban on administration of oxytocin injections. Determination of the cisternal and alveolar components of milk is essential to be studied, to investigate the effects of oxytocin on mammary health, milk production and composition. Buffaloes were injected with oxytocin at the dosage level of 2.5IU per 0.5ml and other group of buffaloes
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received 5.0IU per ml injection of oxytocin intramuscularly at the hip region for a period of one month with control group. There was no significant change in cisternal, alveolar and total milk yield by exogenous oxytocin administration (Bidarimath and Aggarwal, 2007).

2.6

Effect of oxytocin on milk composition

The use of exogenous oxytocin in herds for milk production reasons is presently

prohibited by the United States Food and Drug Administration; thus, its use as a management tool is illegal and not recommended. Administration of exogenous oxytocin to cows for a full lactation increased milk yield by 11.6% over cows not receiving oxytocin, but no apparent difference in milk composition (Nostrand et al., 1991). The effect of oxytocin on milk composition studied by several scientists is discussed in literature as under the following subheadings:

2.6.1 Fat
Nutritional aspects of milk fat have received increasing importance now a days. Milk is an example of oil-in-water emulsions. The milk fat exists as small globules or droplets dispersed in the milk serum. It consists of monoglycerides, diglyceride, triglycerides, fatty acids, sterols, carotenoids and vitamin A, D, E and K. Fatty acids make up to 90% of the milk fat made up of globules with diameter ranging from 0.1 to 10 mm depending on breed (Mulder and Walstra, 1974; Mehaia, 1995; Attaie and Richter, 2000; Michalski et al., 2001). Formation of the milk fat globules takes place in the secretary cells of the mammary glands. Increase in the lipid droplets size occurs during the transportation of that lipid globule, because of dropletdroplet fusion. At the site of the apical plasma membrane, the lipid droplets are secreted from the epithelial cell. Among whole duration of the secretion process, the lipid droplets are covered by the plasma membrane and finally pinched off into the milk channel (Dylewski et al., 1984; Deeney et al., 1985). Phospholipids, lipoprotein, glycolipids and enzymes, e.g. xanthine oxidase, g-glutamyl transpeptidase and alkaline phosphatase formed a membrane around the milk fat globules
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whereas the interior part of the milk fat globules is mainly consists of triglycerides. The milk fat globule membrane provides protection to the triglycerides through lipolysis and oxidation process. Fat content, milk yield, lactation stage, fatty acid composition and activity of g-glutamyl transpeptidase are the factors affecting the size distribution of milk fat globules. Different factor influence the milk quality, stability and technological properties; among one of them is the size of milk fat globules. St-Gelais and his collogues explained that the larger surface area of small milk fat globules have a higher water binding capacity and the thinner casein strands are formed in cheeses with small milk fat globules. On the contrary, they found that low-fat Cheddar Cheeses with large fat globules are best in texture, flavour and colour as compared with cheeses produced with small or conventional milk fat globules (St-Gelais et al., 1997). In addition to size of milk fat globule, milking frequency also affects milk composition. A three times milking per day has been indicated to stimulate a slight, but significant, decrease in milk fat content compared to two times milking per day (Barnes et al., 1990). Correspondingly effects of once-daily milking, increased fat, protein content and somatic cells and similarly increased influx of serum protein and ions in milk due to change in permeability of the tight junctions between the mammary epithelial cells. Lactose efflux also increased in the blood (Claesson et al., 1959; Carruthers et al., 1993; Stelwagen et al., 1994; Stelwagen and Lacy-Hulbert, 1996). The residual milk apparently includes constituents which normally find their way into subsequent milkings, and following oxytocin treatment the subsequent milking was low in milk and fat (Adams and Allen, 1952). Oxytocin appeared to be quite effective in amounts down to 1.5 per injection based on the early assay, although fright or excitement raised the threshold level to more than four times this amount, as noted by lacks of response to repeated injections on certain occasions (Donker et al., 1954).The changes in residual milk during the first 10 months of lactation have been studied in ten cows of the Jersey and Holstein-Friesian breeds.

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It is widely accepted that milk fat percentage increases from first-drawn to lastdrawn milk and is extremely high in residual milk (Johansson et al., 1952; Whittlestone, 1953). Other components of milk vary, but not as much as milk fat. Mielke and Bernhardt (1964) observed a change from 0.4% fat in first drawn milk to 9.4% in last drawn milk. The composition of milk secreted and ejected within a short period after removal of residual milk has received limited attention. Johansson (1952) reported changes in composition of milk after three oxytocin injections, 25IU each, were given at 15 minute intervals following normal milking. This resulted in decreased milk fat content after the first injection. Swanson and Hinton (1951) stated that no milk could be secured with a second injection immediately after the first injection of oxytocin (10IU). Correspondingly different scientist described the effect of milking in relation to oxytocin. They reported that both milk and fat yield significantly varied according to second time milking that must be immediately after primary milking and with the aid of oxytocin (Adam et al., 1952; Knodt and Petersen, 1944). Supraphysiological doses of oxytocin were injected to cows with level of 1, 10, 100, or 1000IU oxytocin after a normal milking; to investigate the relationship among milk composition, secretion rates, and cell electrolyte concentrations. It was found that milk fat yield remained unchanged in proportion to oxytocin dose (Allen, 1990). In another study, cows were assigned at parturition by parity group to treatments. These animals received an injection of 1ml (20IU) of oxytocin at each milking throughout lactation with control group (Nostrand et al., 1991). Milk fat yield increased while total residual yield decreased with either 2.0 or 3.0IU oxytocin administration as investigated by Gorweit and Sagi (1984). Five doses of oxytocin (0.5, 1.0, 1.5, 2.0, 3.0IU) were administrated through the subcutaneous abdominal vein (milk vein) of 15 Holstein Friesian cows 1 min before machine attachment. Milking hourly with oxytocin brought about increases in the rates of secretion of several milk constituents, particularly during the latter part of the trials. The fat secretion rate was particularly high after an early period of low secretion. The trend shown by the butterfat percentages was erratic, but there was first a definite drop followed by a marked upward trend from the 30th hour,
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tending to plateau slowly after the 70th hour (Donker et al., 1954). The purpose of this investigation was to determine compositional changes in milk associated with repeated injections of oxytocin. Milk fat determined in normal milk followed by four successive intravenous oxytocin injections (20IU) at 20 minute intervals and observed that fat (%) increased through first oxytocin sample, then decreased to approximately the original fat level in normal milking (Lane et al., 1970). Residual milk was higher (P<.01) in fat and positive correlations (P<.01) existed for fat (%) between the normal and residual fractions for all milk components when forty cows were injected with 10IU of oxytocin in the jugular vein and to remove the residual milk. Injection of oxytocin into 16 cows prior to milking for five consecutive milkings caused a significant reduction (P<.01) in fat of milk obtained during the post-treatment period. Injection of oxytocin after every three consecutive milkings showed that the decrease in fat content and increase in milk yield took place both in the normal and the residual fractions (Natzke and Schultz, 1966). A study by Bidarimath and Aggarwal (2007) proved that fat % in cisternal, alveolar and total milk decreased significantly (P<0.01) by 11.8% and 21.3% in buffaloes groups receiving 2.5IU per 0.5ml and 5.0IU per ml oxytocin, respectively. When milk samples from three groups of buffaloes were collected at 15 days interval on 0, 15, 30 and 45 days with oxytocin injection (2.5IU per 0.5 ml) and (5.0 IU per ml) intramuscularly at the hip region for a period of 1 month with control group. Immediately following each milking 10IU of oxytocin were injected intravenously and the residual milk obtained. During the nine day period of oxytocin injection the normally drawn milk was lower in fat than during the control period, testing 5.24% as compared to a normal of 5.72%. The fat production during the 30 hours period was 11.0% less than the rate of fat production during the three day preliminary period. From the above it appeared that by removing the milk from the gland at frequent intervals it may be possible to retard the passage of blood fat into the gland sufficiently to decrease the amount of fat secreted by the gland (Shaw, 1942).

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2.6.2 Fatty acids composition of milk

Fatty acid composition is known to change with the stage of lactation of the cow (Parodi, 1974). However, no information is available for any species regarding the effect of lactation on triacylglycerol composition. Milk fatty acid composition has a number of effects on milk quality, including aspects such as its physical properties (e.g. melting point and hardness of butter, crystallization and fractionation of milk fat) as well as its nutritional properties (e.g. potential effects of specific fatty acids on human health). Fatty acids composition also affects the organoleptic properties of milk (due to factors such as the effect of free short-chain fatty acids and oxidative changes in fatty acids). Milk fatty acids have a dual origin: they are either taken up from plasma lipoproteins (Chilliard, 2000) or they are synthesized de novo in the mammary gland from acetate and 3-hydroxybutyrate. Lipoprotein lipase permits triglyceride hydrolysis and fatty acids uptake by the mammary gland. In addition, the mammary gland uses plasma non esterified fatty acids released by adipose tissue as a source of long-chain fatty acids for milk lipid synthesis. The fatty acids mainly stored as triglycerides in ruminant adipose tissue are C16:0, C18:0 and Ccis9-18:1. For this reason, lipid mobilization occurs in early lactation, when the energy balance is negative, induces a quick increase in stearic and oleic acids in milk. The mammary glands cannot convert C16:0 into C18:0 by extending the carbon chain. Moreover, totally distinguished secretary mammary cells exhibit high delta-9 desaturase activity, which converts stearic acid into oleic acid (cis9- 18:1) and contributes more than 50% of all the oleic acid secreted in milk (Bickerstaffe et al., 1974; Enjalbert et al.,1998). These different metabolic pathways (de novo synthesis, uptake from plasma, desaturation) contribute to the formation of a group of fatty acids that are used for triglyceride formation (97 to 98% of milk total lipids) through esterification on glycerol. Fatty acids asymmetrical distribution on the glycerol molecule influences the physical properties of milk fat and the digestibility of certain fatty acids (Chilliard et al., 2000).

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Ely and Petersen (1941) demonstrated that oxytocin is the hormone that ensures the ejection of milk, with the myoepithelial cells being the mammary receptors of this neuroendocrine reflex. Mielke and Bernahrdt (1964) reported an extremely variable response in amount of cistern milk in the gland, whereas the amount of alveolar milk, as measured by forced release by oxytocin, was constant from milking to milking. Using intrammary pressure techniques. Chen et al. (1966) determined that about one-half of the total milk was in the alveoli and duct system before milking. Lawson and Graf (1969) used oxytocin to affect an increase in intramammary pressure before milking; however, Merriman and Kitts (1965) could produce only a slight increase with oxytocin immediately after milking. Changes in the composition of residual bovine milk were significant when injections of oxytocin were administered reported by Lane et al. (1970) and Dill et al. (1972). Milk fat content increased progressively during normal milking and reached a maximum, representing a three-fold elevation when the first residual milk was removed under oxytocin influence. Fat content subsequently decreased with oxytocin treatments, this exists for an effect of this hormone on milk fat synthesis. Milk fat is derived eventually from fats, carbohydrates, and proteins in feed. There are several routes for conversion of these nutrients into milk fat. Most evidences suggest that long-chain fatty acids are transported intact into the mammary system (Dimick and Patton, 1965; Glascock et al., 1966). Conclusive evidence is available to indicate that short-chain fatty acids are synthesized in the mammary system by condensation of acetate units (Kurnar et al., 1965). Dill et al. (1974) worked on influence of repeated oxytocin injections to animals on composition of cow milk fat and reported that monoglycerides content increased markedly with oxytocin treatment while phospholipid content was depressed slightly and no significant effect of oxytocin was observed on milk fatty acids composition. With a probable difference in rates for synthesis mechanism, composition of milk fat produced during forced milking through repeatedly oxytocin injections might vary from that in the normal milk.

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2.6.3 Protein
Protein was determined in milk from normal milking, followed by four successive intravenous oxytocin injections at 20-minute intervals. Protein was significantly (P < 0.01) lower in milk removed after the injection of oxytocin than in the samples taken during normal milking. Milk yield approached to a constant level with repeated injections of oxytocin, indicating a constant rate of secretion. Gorweit and Sagi (1984) found that higher doses of oxytocin before machine attachment decreased percent milk protein in Holstein cows milk. Allen (1990) reported that Supraphysiological doses of oxytocin have been used to investigate the association among milk composition, secretion rates, and cell electrolyte concentrations. Groups of 8 Holstein cows were injected intravenously with 1, 10, 100, or 1000IU oxytocin after a normal milking. Yield and concentration of protein declined with increasing oxytocin doses. The decline in protein contents has been noted by Donker et al. (1954) in the experimental than in the preexperimental samples; total protein, casein and serum protein, calculated on fat free basis in the experimental samples fluctuated considerably from one hour to the next. There was no definite detectable trend. Johansson (1952) reported changes in composition of milk after three oxytocin injections, 25IU each, were given at 15 minute intervals following normal milking. The protein content decreased constantly from the first 200cc of milk to the last oxytocin injection whereas, Stewart and Stevenson (1987) pointed out that chronic administration of high doses of oxytocin (100IU four times daily for 28days) not altered milk yield and percentage of protein. Significant reduction (P<.01) in protein(%) of milk obtained during the post-treatment period due to injection of oxytocin (10IU) into sixteen cows prior to milking (Natzke and Schultz, 1966). According to Wheelock et al. (1965) after injection of oxytocin and removal of residual milk, concentrations of whey proteins (probably serum derived) are increased. This change in milk composition persisted for several milkings. The study was conducted on eighteen lactating Murrah buffaloes injected with oxytocin intramuscularly in 83.7214.75 days after calving injected with

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oxytocin intramuscularly showed protein(%) increased significantly (P<0.01) in experimental groups (Bidarimath and Aggarwal, 2007).

2.6.4 Lactose
From the normal and residual milk fractions of forty cows, quarter samples from one fifty nine quarters were collected, to explore differences in milk composition and mastitis-screening tests. They were then injected with 10IU of oxytocin in the jugular vein and milked again to remove the residual milk. No difference was found between normal and residual milk for lactose content when calculated on a fat-free basis. Natzke and Schultz (1966) showed that concentration of lactose declined with increasing oxytocin doses. In order to determine whether the decreased milk lactose secretion was caused by a decline in synthesis or leakage into blood plasma and subsequent renal clearance, milk and urine samples were collected for 24 hours periods before and after 100IU of oxytocin. Urinary lactose accounted for 2.73.19and 12.9 8.9% of the total collected on the days before and after oxytocin. This increase (46.7g/d) was only one-fifth of the 25% decline in total lactose recovery, suggesting that reduced mammary lactose synthesis is primarily responsible for lower milk yield (Allen, 1990). Lactose values also showed considerable fluctuation from hour to hour within the range without detectable trend and with no difference between the experimental and the pre-experimental milks (Donker et al., 1954). The composition of milk secreted and ejected within a short period after removal of residual milk has received limited attention. Johansson (1952) reported changes in composition of milk after three oxytocin injections, 25IU each, were given at 15 minute intervals following normal milking as lactose percent decreased constantly from the first 200 cc of milk to the last oxytocin injection. Swanson and Hinton (1951) stated that no milk could be secured with a second injection immediately after the first injection of oxytocin (10IU). Lactose concentration decreased in the next milking after injection of oxytocin and removal residual milk. This change in milk composition persisted for several milkings (Wheelock et al., 1965). It was determined in milk from normal milking, followed by four successive intravenous oxytocin injections at twenty minute intervals.
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Lactose was significantly (P < 0.01) lower in milk removed after the injection of oxytocin than in the samples taken during normal milking (Lane et al., 1970). Effect of chronic oxytocin treatment on the mammary gland immune system was investigated by Werner-Misof et al. (2007). Cows in the oxytocin group were injected with 50IU oxytocin (5ml) and this chronic oxytocin administration resulted in the increased level of lactose in blood and decreased in milk. A numerical decrease was observed for lactose levels from day 2 to day 7 in response to the oxytocin treatment.

2.6.5 Ash
Changes in composition of milk after three oxytocin injections (25IU each) studied by Johansson (1952) who reported ash percentage decreased constantly from the first 200cc of milk to the last oxytocin injection.

2.6.6 Solids not fat

Compositional changes in milk associated with repeated injections of oxytocin studied by Lane et al. (1970). Results showed that Solids-not-fat was significantly (P < 0.01) lower in milk removed after the injection of oxytocin than in the samples taken during normal milking when it has been determined in milk from normal milking, followed by four successive intravenous oxytocin injections at 20 minute intervals. Rate of secretion approached the rate needed to synthesize the milk produced daily by these cows. The changes in residual milk during the first 10 month of lactation have been studied in ten cows of the Jersey and Holstein-Friesian breeds by injecting 10IU of oxytocin intravenously varied roughly as the normal milk yield within breeds. The monthly average changes in the non-fat solids of the various milk samples are plotted. In all months the residual milk was lowest in solids not fat percentage. There was a slight upward trend of all solids not fat values as lactation progressed (Swanson and Hinton, 1951). Solids not fat content of milk decreases with age of the cow. Within any given lactation, solids not fat content is relatively high in the first month, drops to a low level in the second, then rises as lactation progresses. Cows that conceive generally show a
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steeper rise in solids not fat content than cows that remain open through their lactation. The pregnancy causes a decline in milk yield, therefore percent of solids not fat increases.

2.6.7 Total Solids

Total solids in milk indicate all solid contents present in milk of any species. Determination of total solid content of milk is of quite importance as these affect product quality and different attributes of dairy products. The maximum total solids were reported in sheep milk 19.73% followed by buffalo milk 18.36%. The total solids in different species of cow milk have been observed by Ozrenk and Ini (2000). The observed values were 12.11% and 14.54% for Holstein and Jerseys breeds of cow milk. Enb et al. (2009) made comparative study of cow and buffalo milk and milk products and reported investigated values were 13.40 and 12.10% for buffalo and cow milk respectively. Johansson (1952) reported that total solids and ash percentage decreased constantly from the first 200 cc of milk to the last oxytocin injection and also observed changes in composition of milk after three oxytocin injections, 25IU each, given at 15 minutes intervals following normal milking.

2.6.8 Minerals in milk

All twenty two minerals considered to be essential to the human diet are present in milk. Mineral content of fresh milk reported by Webb et al. (1974) are sodium (350900mg/L), potassium (1100-1700mg/L), chloride (900-1100mg/L), calcium (11001300mg/L), magnesium (90-140mg/L), phosphorus (900-1000mg/L), iron (300600g/L), zinc (2000-6000 g/L), copper (100-600g/L) and manganese (20-50g/L). In dairy practices oxytocin is widely used for increase in milk yield and has become popular as a treatment for mastitis caused by environmental pathogens such as Coliforms or Streptococci. Although it is reported that oxytocin can be as effective as antibiotics (Guterbock et al., 1993 and Knight et al., 2000) there is no information on the mode of action of exogenous oxytocin (Hillerton and Semmens, 1999). There are some significant results reported by scientists (Linzell and Peaker, 1971a; Linzell et al., 1975
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and Allen, 1990) as increasing numbers of somatic cells, sodium chloride and electrical conductivity as well as decreasing concentrations of lactose in milk and increasing blood lactose and potassium levels after oxytocin administration. After injection of oxytocin and removal of residual milk, concentration of sodium and chloride has been increased. This change in milk composition persisted for several milkings (Wheelock et al., 1965). Immediately following a single dose of 100mU of oxytocin has no significant effect on milk composition has been evident but after 1IU of oxytocin, milk sodium and chloride has been significantly increased. The passage of sucrose, sodium and chloride from blood to milk also increased. While milk yield remains relatively high, the concentrations of lactose and potassium in milk decreased while those of sodium and chloride increased (Gachev 1963, 1965, 1971; Cowie 1969; Linzell and Peaker 1971; Peaker and Taylor 1975). Oxytocin increases the permeability of a paracellular pathway, probably through 'tight junctions', allowing substances to pass directly between blood and milk down their respective concentration gradients. This suggested that the rise in sodium and chloride could be partly due to contamination of the milk already in the teat by tissue fluid expressed from the tissues by compression during manual milking. This idea was supported by the finding that, in three goats with circulating labelled albumin, more protein bound iodine was found during frequent milking in the milk of the milked gland than in the milk from the cannulated one. The most constant change detected on frequent milking is a fall in potassium concentration, but this is often accompanied by a rise in sodium and chloride and a fall in lactose. In cows similar changes have been produced by the distension, due to leaving the milk in the udder for long periods and by oxytocin (Mackenzie and Lascelles, 1965; Wheelock et al., 1965) depends on the magnitude of the doses usually employed. It is also well known that the same changes occur in the milk secreted by diseased glands, and in very low yielding glands in late lactation. Early workers also showed that after 2-3 days of fasting of animals there was a marked fall in lactose concentration and a compensatory rise in sodium and chloride (Overman and Wright, 1927; Gowen and Tobey, 1932).
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Allen (1990) also reported that doses of oxytocin of 1IU or greater altered milk sodium and potassium concentration, presumably by increasing the permeability of the mammary epithelial tight junctions to small molecules, allowing electrolytes to move between the milk and interstitial space down their electrochemical gradient. Milk sodium increased in proportion to oxytocin dose. Increased sodium, chloride and decreased lactose concentration in milk also result from supraphysiological doses of oxytocin, apparently due to tight junctions between mammary epithelial cells becoming leaky (Linzell and Peaker 1971; Linzell et al., 1975). Another study conducted by Allen (1988) on lactating mouse mammary tissue or isolated alveoli were incubated in solutions containing high sodium and low potassium concentrations, the cells lost potassium and gained sodium. This was attributed to high sodium and potassium permeability of the apical plasma membrane that maintains the Na: K ratio in milk proportional to the ratio in cells (Peaker, 1977). If bovine mammary cells behave in the same way, increasing the sodium concentration in milk would be expected to result in increased sodium in the cells. Adverse effects of elevated sodium on cell function have been demonstrated in several cell types. Increases in intracellular Na:K ratios in cultured fibroblasts decreased the rate of synthesis of protein and DNA (Ledbetter and Lubin, 1977). Similar effects were seen in lymphocytes (Quastel and Kaplan, 1970). It seems possible that increasing the Na:K ratio in lactating mammary cells can inhibit synthesis of proteins necessary for milk formation. The change in cell Na:K could be the result of opening tight junctions, permitting paracellular sodium influx into milk. Perhaps with chronic oxytocin the mammary cells are able to maintain normal sodium and potassium concentrations by increasing the activity of Na+-K+-ATPase on their basolateral membranes as frequently demonstrated in cultured (Rayson, 1989) cells. Alternatively, the myoepithelial cells may become less sensitive to the effects of oxytocin in cows given chronic high doses, and milk removal may occur through greater response from alternative mechanisms (Lefcourt and Akers, 1983).

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Milk samples were collected from the normal and residual milk fractions of forty cows, to determine differences in milk composition. They were then injected with 10IU of oxytocin in the jugular vein and milked again to remove the residual milk. Residual milk was higher (p<0.01) in chloride percent. Milk production and chloride percent increased significantly (p<0.01) as a result of the oxytocin injections (Natzke and Schultz, 1966). Werner-Misof et al. (2007) evaluated effect of chronic oxytocin treatment on the mammary gland immune system in fourteen healthy cows were treated with intramuscular oxytocin administration, the control group were injected with 5ml of saline (9g/L) for eight days (Day 18) before each milking. Conclusively, in treated animals electrical conductivity, sodium, chloride and potassium levels increased whereas lactose concentration was decreased in milk. A numerical decrease was observed for lactose levels from day 2 to day 7, whereas sodium and chloride levels increased numerically within the first 3 days in response to the oxytocin treatment. A numerical increase was observed for electrical conductivity levels between day 2 and day 9. An increase in milk sodium and great falls in lactose and potassium concentrations during frequent milking were observed however, changes were primarily due to large doses of oxytocin. The study suggested that alveolar milk is little different from teat milk and minor changes in milk composition occur during storage in the ducts (Zaks, 1964; Zaks et al. 1965).

2.7

Electrophoretic patterns of protein fractions of milk

Proteins can be separated by many techniques of which the most common are

various types of chromatographic and electrophoretic techniques. According to solubility at pH 4.6, the milk protein fraction can be divided into caseins insoluble at this pH and whey proteins which are soluble. Caseins are quantitatively the most important milk protein components representing 75-80% of total milk proteins. This protein complex, known as a micelle comprises of four different caseins (s1, s2, and k-caseins) which are held together by non-covalent interactions and appear as a highly stabilized dispersion
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in milk (Veloso et al., 2002). Caseins belong to the group of phosphoproteins which are heat stable because they do not coagulate when subjected to high heat treatment (Fox et al., 2003). The four casein fractions that make up whole casein (CN) have similar molecular weights (19,000 to 25,230) and distinct differences in their amino acid sequences and their degree of phosphorylation (1,5, 8 to 9, and 10 to 13 phosphate groups per molecule for K-CN, -CN, s1-CN, and s2-CN, respectively) (Eiger et al., 1984). Proteins after removal of casein by acid precipitation are collectively called as whey proteins that represent about 18 -20% of total milk protein. Whey proteins contain two well defined groups of proteins which could be fractionated by saturated MgSO4 or half saturated (NH4)2SO4. The precipitate was referred as Lg and soluble protein as lactalbumin. The lactoglobulin fraction consists mainly of Ig. The lactalbumin fraction of bovine milk contains three main proteins, -Lg, -La and blood serum albumin. Thus whey contains four major proteins, -Lg, -La, BSA, and Ig in 50%, 20%, 10% and 10% of whey protein fraction respectively -Lg is the principal whey protein in bovine, ovine, caprine and buffalo milk, although there are slight interspecies differences. -Lg does not occur in human, rat or mouse milk in which -Lac is principal whey protein. BSA is a quite large molecule; normal bovine milk contains a low level of BSA, presumably as a result of leakage from blood. Mature milk contains little quantity of Ig but in clostrum its level is high and its level decreases postpartum (Fox and McSweeney, 1998). First step in process of fractionation is separation of whole casein from whey in skim milk (Farrel, 1975). The electrophoretic techniques currently used in the study of milk proteins are sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDSPAGE), alkaline urea PAGE, acid urea PAGE and these has allowed identification of the milk casein and whey components. PAGE is a powerful technique for the separation of protein. Separation by PAGE is based on overall protein charge and molecular size. A modification of PAGE known as SDS-PAGE uses the anionic detergent SDS to minimize the influence of protein charge in the PAGE separation, allowing estimation of protein molecular weight (Whitney et al., 1977; Pharmacia, 1980, 1982 and Podislo, 1981). Zonal electrophoresis
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in starch gel (SGE) containing 7M urea was used to resolve casein into zones or bands. The two principal bands were s1 and -casein. Incorporation of urea was necessary to dissociate extensive intermolecular hydrophobic bonding. Owing to the presence of intermolecular disulphide bonds, K-casein resolves poorly on SGE or PAGE unless it is reduced usually by 2-mercaptoethanol or it is alkylated (Fox and McSweeney, 1998). The proteins from the bovine and caprine milk showed different profiles. In both, the five major correspondings s1-CN, -CN, K-CN, -Lg and -Lac were observed. (Tomotake et al., 2006). In bovine whey, there are five major protein fractions with defined molecular weights, i.e., immunoglobulins (Igs), mainly IgG, BSA, -Lac, -Lg and PP (Whitney, 1977; Eiger et al., 1984). The effect of oxytocin and milk removal on secretion in the goat was studied by Linzell and Peaker (1971) who reported decrease in non-casein protein after oxytocin administration because of leakage between tight junctions connecting secretory cell. Kindstedt et al. (1991) studied the effect of long term bovine somatotropin treatment on nitrogen (protein) distribution in Jersey milk and reported decrease in protein, true protein, casein as a proportion of total and true protein when taken at day 8 of the injection cycle. Literature is not available on effect of oxytocin on protein fractions on lower molecular weights.

2.8

Milk enzymes
Many indigenous enzymes have been identified in milk from 1924 to 1970 (Fox

and Kelley, 2006). These enzymes are essential as indicators of the adequate pasteurization of milk (alkaline phosphatase and g-glutamyl transferase) or of mastitis (acid phosphatase) and lactoperoxidase and lipase are considered to be important for the stability and quality of milk.

2.8.1 Alkaline phosphatase (ALP)

Phosphatase in milk was recognized first in 1925 by Demuth, then characterized as an alkaline phosphatase indigenous to milk by Graham and Kay (1933), it became considerable when it was shown that the timetemperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those
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required to kill Mycobacterium tuberculosis (target micro-organism for pasteurization). The enzyme is readily assayed, and for the high temperature short time pasteurization of milk, a test procedure was developed as a routine quality control test based on the inactivation of alkaline phosphatase (Graham and Kay, 1933; Whitney, 1958). Between individuals and herds, alkaline phosphatase activity of cow milk varies significantly and it also varies along with lactation stages (minimum at first week and maximum at twenty eight weeks). Phosphates activity varies inversely with milk yield and it is independent of fat content, breed and feed (Haab and Smith, 1956).

2.8.2 Acid phosphatase (ACP)

Huggins and Talalay (1948) reported first the occurrence of an acid phosphomonoesterase (ACP) in milk and confirmed by Mullen (1950), who reported that acid phosphatase was optimally active at pH 4.0, and was very heat-stable (heating at 88oC for 10min is required for complete inactivation). Acid phosphatase activity strongly inhibited by fluoride and it is not activated by Mg2+ but it is activated slightly by Mn2+. In milk, level of acid phosphatase activity is only ~2% that of alkaline phosphatase. Acid phosphatase activity becomes maximum at 5 to 6 days post partum, then decreases and remains at a low level to the end of lactation. Specific activity is higher in cream but almost 80% of the acid phosphatase of milk is found in the skimmed milk (Andrews et al., 1992). Kitchen (1985) reported that there is only one isozyme of acid phosphatase in milk that is strongly attached to the milk fat globule membrane and is not released by non-ionic detergents.

2.8.3 Lactoperoxidase (LP)

The most widely recommended industrial application of the (Lactoperoxidase) LP system in food production is in the dairy for the preservation of raw milk during storage and transportation to processing plants. In cow milk, lactoperoxidase is the second most abundant enzymes after xanthine oxidase (Pruitt and Kamau, 1991; Wit and van Hooydonk, 1996). Its concentration in cow milk is around 30mg/L constituting about 1% of the whey protein (Reiter, 1985). The lactoperoxidase concentration is low in cow
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colostrums, unlike other antibacterial proteins however, it increases rapidly to reach a maximum after 3 to 5 days postpartum (Reiter, 1985). Variations in enzyme level were reported to depend on the sexual cycle of the cow, season, feeding regime and breed (Reiter, 1985; Kussendrager and Hooijdonk, 2000). Cow milk contains from 1.2 to 19.4U/mL lactoperoxidase and is about 20 times richer in peroxidase activity than human milk (Gothefors and Marklund, 1975). Lactoperoxidase activity in cow milk ranged from 1.5 to 2.7U/ml with an overall mean of 2.3U/ml (Fonteh et al., 2002). However, Reiter (1985) reported lower (1.42U/ml) lactoperoxidase activity in cow milk.

2.8.4 Lipases
Lipases are biologically an important group of enzymes since they are associated with fat metabolism. The purification and study of lipases is desirable, because it would contribute to our understanding of the properties and function of these enzymes (Chandan and Shahani, 1963). Dorner and Widmer (1932) were perhaps the first to indicate that milk lipase is associated with casein. Since then, the conclusive evidence for the location of lipase in casein has been presented, revealing that the milk fractions rich in casein were also high in the lipase activity. Tarassuk and Frankel (1957) reported that cow's milk contained at least two different lipase systems; one is membrane lipase, which is irreversibly adsorbed on the fat-globule membrane material when freshly drawn milk is cooled; the other is plasma lipase, which is associated with casein. The membrane lipase is present in higher concentration in milk from cows late in lactation and those maintained on dry feed. Recently, Skean and Overcast (1961) observed that casein obtained by centrifugation of skim milk at 50,000 rpm possessed nearly twice as much lipase activity as the casein separated by acidification. They observed that only the fastest moving casein fraction, casein, possessed lipase activity. Extensive research work has been carried out on different aspects of milk including factors affecting milk composition (breeds and species, stages of lactation,
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feeding management, health status of the animal, season, locality and level of milk production), oxytocin induced changes in gross composition of milk, oxytocin mechanism, possible effects and doses of oxytocin/milk yield etc. However the major emphasize of the present research was to evaluate the effect of oxytocin administration on gross composition, minerals, fatty acid composition, protein fractions, enzymes and thiocyanate content at three lactation stages (mature milk, peak production and end production) in milk of Sahiwal cows. Many scientists reported that regular administration of supraphysiological doses of oxytocin ultimately decreases the milk yield but their findings regarding the composition are contradictory. Some of the authors revealed that fat, protein and some other constituents decrease in concentration on oxytocin administration while other authors reported that oxytocin injections have no effect on milk compositions. In western countries, the use of oxytocin is very low. In Pakistan there is indiscriminate use of oxytocin during milking. Therefore very little work is done on the fatty acid profile, milk enzymes and protein fractions. So the information derived from this important research work will be helpful for the dairy farmers, processors and consumers for their awareness and intended used.

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