UNIVERSITY OF AGRICULTURE, FAISALABADDepartment of Rural Home Economics

Synopsis for M.Sc. Degree in Home Economics (Food and Nutrition)

TITLE: EFFECT OF EXOGENOUS OXYTOCIN ON MINERAL CONTENTS OF MILK

Name of the student Saba Maqbool

Regestration No. 2008-ag-347

ABSTRACT

Milk is lacteal secretion of mammary gland and is an essential diet especially of newly

born babies and for old age group because it contains all essential nutrients e.g. protein,

carbohydrate, fat, vitamin and minerals. Oxytocin, a hormone released from the pituitary

gland, causes the uterus to contract and thereby initiates the process of parturition, or

childbirth. Oxytocin plays a major role in lactation mainly by its action on milk ejection

via the contraction of myoepithelial cells. The effect of oxytocin on milk production and

the presence of oxytocin receptors on different epithelial cells suggest that this hormone

may play a role in mammary epithelial cells. Exogenous oxytocin is injected daily before

milking to increase milk production. Milk of 6 Nili Ravi buffalo will be collected as

control and treated samples from livestock farm of University of Agriculture, Faisalabad.

Milk samples will be analyzed to determine changes in minerals due to oxytocin. Data

obtained for each attribute will be subjected to statistical analysis.

UNIVERSITY OF AGRICULTURE, FAISALABADDepartment of Rural Home Economics

Synopsis for M.Sc. Degree in Home Economics (Food and Nutrition)

TITLE: EFFECT OF EXOGENOUS OXYTOCIN ON MINERAL CONTENTS OF MILK

Date of admission: 15-09-2008

Date of Initiation (Research) After approval

Probable Duration (Research) 6 months

PERSONNEL:

Name of the student Saba Maqbool

Registration No. 2008-ag-347

SUPERVISORY COMMITTEE:

Mrs. Naheed Abbas : (Chairperson)

Miss. Asmah Lodhi : (Member)

Prof. Dr. Zia-ur-Rehman : (Member)

1. INTRODUCTION:

Milk is one of the oldest foods known to man and it is defined as the physiological

secretion from the mammary gland of mammals (Nickerson, 1999).

Milk is a whitish liquid containing proteins, fats, lactose, and various vitamins and

minerals that is produced by the mammary glands of all mature female mammals after

they have given birth and serves as nourishment for their young.

Milk is an opaque white liquid produced by the mammary glands of mammals. It

provides the primary source of nutrition for young mammals before they are able to

digest other types of food. The exact components of raw milk vary by species, but it

contains significant amounts of saturated fat, protein and calcium. Cattle's milk has a pH

ranging from 6.4 to 6.8, making it slightly acidic.

Milk is a food of high nutritional quality, relatively low cost, with high palatability and

digestibility. It is almost complete food because it contains all essential nutrients e.g.

protein, carbohydrate in the form of lactose, fat, vitamins and minerals. (Komorowski and

Ealy, 1992). Milk and other dairy products, therefore, comprise an important source of

food for all age groups. Pakistan is the 5 th largest milk producing country in the world

with annual production of about 42,199 billions liters (Govt. of Pakistan, 2008). The milk

business has been growing rapidly for the past few years, especially in the country like

Pakistan.

Milk is the normal secretion of mammary glands of all mammals. Its purpose is to

nourish the young of the species. The nutritional needs of species vary and so it is not

surprising that the milk of different mammals differ in composition. The principal

constituents of milk are fat, protein, milk sugar (lactose) and minerals of milk vary not

only in amounts among the different animal species but also with the exception of

lactose, somewhat in chemical, physical and biological properties (Norman and

Hotchkiss, 1996).

Buffalo milk is commercially more viable than other milk for the manufacture of fat-

based and SNF-based milk products, such as butter, ghee and milk powders because of its

lower water content and higher fat content. Proteins of buffalo milk, particularly the

whey proteins, are more resistant to heat denaturation. Dried milk products prepared from

buffalo milk exhibit higher levels of undenatured proteins when processed under similar

conditions. The presence of higher levels of various bioprotective factors, such as

immunoglobulins, lactoferrin, lysozyme, lactoperoxidase as well as bifidogenic factors,

render buffalo milk more suitable than cow milk for the preparation of a wide range of

special dietary and health foods.

The variation in milk consumption depends on so many factors like genetics, stage of

lactation, management practices, parity, diet, age, udder health and season (Haenlein,

2003).

Milk is the natural food for human. Regular intake of milk is beneficial as a complete

food, easily given, readily digested and observed. It is indispensable food for adults. As a

good source of principal nutrients it can be recommended. Buffalo milk contains 4.5%

carbohydrates, 7.5% protein and 103 Kcal/dl energy (Passmore and Eastwood, 1987).

Significantly, cholesterol content of buffalo milk is 0.65 mg/g. Buffalo milk is superior to

cow milk in terms of important minerals, namely calcium, iron and phosphorus which are

higher by 92%, 37.7% and 118% respectively than those present in cow milk. Buffalo

metabolizes all the carotein into vitamin A, which is passed on to milk as such.

Minerals have many roles in the body including enzyme functions, bone formation, water

balance maintenance, and oxygen transport. Minerals contribute to the buffering capacity

of milk, the maintenance of milk pH, the ionic strength of milk, and milk's osmotic

pressure.

Minerals of milk are divided into two main groups: macrominerals and trace elements.

All contribute to the well-being of offspring and are important for growth of most organs,

particularly for mineralization of bone matrix. The seven macrominerals of milk are

important to these processes. Three of the seven (Ca, P, Mg) are required to develop bone

and are present in milk to a large degree complexed with protein in micelles. Potassium

(K), sodium (Na), and chlorine (C1) are found mainly as ions in the soluble fraction of

milk (Holt, 1985).

Milk is a good source of calcium (Ca), magnesium (Mg), phosphorus (P), potassium (K),

selenium (Se), and zinc (Zn). Many minerals in milk are associated together in the form

of salts, such as calcium phosphate. In milk approximately 67% of the calcium (Ca), 35%

of the magnesium (Mg), and 44% of the phosphate (P) are salts bound within the casein

micelle and the remainder are soluble in the serum phase. The fact that calcium and

phosphate are associated as salts bound with the protein does not affect the nutritional

availability of either calcium or phosphate. Milk contains small amounts of copper (Cu),

iron (Fe), manganese (Mg), and sodium (Na) and is not considered a major source of

these minerals in the diet.

Calcium (Ca) and phosphorous (P) are the major minerals found in milk. These minerals

are required in large quantities by the rapidly growing neonate for bone growth and

development of soft tissues. Calcium (Ca) and phosphorous (P) mostly are associated

with the casein micelle structure. Milk also contains most other minerals found in the

body.

Zinc (Zn) is a nutrient required for many proteins involved in DNA synthesis, protein

synthesis, mitosis and cell division. Adequate zinc (Zn) supply is particularly important

during the periods of rapid neonatal growth and development as illustrated by

observations of early neonatal death associated with low milk zinc (Zn) levels in lethal

milk. Copper (Cu) plays an important role as a cofactor for enzymes that generate cellular

energy, cross-link connective tissue and mobilize cellular ion (Lopez et al. 2002).

Adequate iron (Fe) intake is essential for optimal growth, hematopoiesis and cognitive

development during infancy. Iron (Fe) deficiency anemia is the most common deficiency,

estimated to affect 1-2 billion people worldwide. Iron (Fe) in milk is bound to lactoferrin,

transferrin, xanthine oxidase, and some to caseins. Moreover, good understanding of the

properties of milk mineral is important for fundamental research and for development of

dairy products. The quality of fermented and non fermented milk products such as

yoghurt, cheese, butter and evaporated milk for various purposes are influenced

significantly by compositional factors (Lindmark et al. 2003).

The mineral fraction, cations (calcium, magnesium, sodium and potassium) and anions

(inorganic phosphate, citrate and chloride) play an important role in the structure and

stability of casein micelles (Gaucheron, 2005; Holt, 1985). Thus structure and stability of

casein micelles is closely related with mineral fractions. Thus, the knowledge of minerals

concentration in milk samples is of particular important.

Oxytocin is a hormone released from the posterior pituitary gland, causing a contraction

of the myoepithelial cells around alveoli and small ducts of mammary gland (Linda et al.

1993). Thus oxytocin is generally considered to increase milk production by enhancing

milk ejection. The magnitude of the increase from oxytocin injection is quite variable,

ranging from 10-12% of milk production in some studies but showing non significant

effects on milk production in some others. The factors that control milk production vary

and are dependent on dosage and time of oxytocin injection (Linda et al.

1993).

The discovery of oxytocin and the elucidation of its role in the neuro-hormonal milk

ejection process allowed for managing the milking process with an exogenous hormone.

Alterations in both milk and fat yield were obtained by milking a second time,

immediately following the primary milking, with the aid of oxytocin. Results varied from

significant increase in milk production and fat yield to no change either in milk yield or

fat yield. Disparate results can be explained by the varied experimental designs

employed, with majority of work involving small sample sizes, alternating treatments and

short treatment periods. Variations in dosage and timing of injections have contributed to

the confusion regarding effects of exogenous oxytocin. Studies have involved injections

prior to hourly milking or injections up to 1 hour before milking. Such design do not

mimic normal physiology of lactation in which oxytocin is released into blood stream due

to normal milking stimuli, binds to myoepithelial cells receptors in the udder and elicits

milk ejection. Only a few studies administrated oxytocin immediately prior to milking,

increases in milk production were reported when oxytocin administration occurred within

minutes of normal milking. Milk increase was 11.6% than those not receiving oxytocin

but no alteration in milk consumption (Nostrand et al. 1991).

Exogenous oxytocin is becoming common in Pakistan. The purpose of this study is to

solve the question which is raised about the effect of oxytocin injections on the minerals

of milk.

OBJECTIVES:

The study will be conducted to achieve the following objectives.

1) To check the effect of oxytocin on mineral composition of Nili Ravi buffalo milk.

2) To determine chemical composition of normal and oxytocin injected milk.

2. REVIEW OF LITERATURE:

Linzell and Peaker (1971) investigated that when goats were milked each hour after being

given a dose of synthetic oxytocin within the range thought to be released by the

pituitary, there was a progressive rise in milk yield becoming statistically significant by 5

hr. The effect was reduced if the milk was not removed from the gland each hour.

Oxytocin treatment and, to a lesser extent, frequent milking without oxytocin, altered

milk composition. Na, Cl and non-casein protein increased; potasssium (K) and lactose

decreased. Oxytocin infusions permitted the leakage of lactose from milk to plasma and

sucrose from plasma to milk. In some goats’ very small doses of oxytocin caused changes

in milk. The increase in the rate of milk secretion following milk removal is probably of

greater physiological significance than the small changes in milk composition and

supports Levy's idea of a local negative feed-back via a chemical component of milk.

Linzell and Peaker (1974) investigated the effects of oxytocin on milk secretion and the

permeability of the mammary epithelium in rabbits. A single dose of 100 m-u. oxytocin

no significant effects on milk composition were evident but after 1 u. milk sodium (Na)

and chlorine (Cl) were significantly increased. Twenty-four hr after 1 u. oxytocin, sodium

(Na) and chlorine (Cl) were decreased while K, lactose, fat and protein were increased.

During an I.V. infusion of oxytocin milk sodium (Na) and chlorine (Cl) increased while

K and lactose decreased. The passage of [(14) C] sucrose, 24Na and (36) Cl from blood

to milk also increased. These effects of oxytocin are discussed in relation to the

permeability of the mammary epithelium and the pathways for ion movements, and to

other studies on milk composition in the rabbit involving the administration of oxytocin

to aid in the evacuation of milk.

Sagi et al. (1980) subjected 12 Holstein cows to four stimulation routines for the effects

on milking performance: a).No stimulation b).Manual stimulation c).Manual stimulation

with delayed milking and d).Intravenous infusion of 0.75 IU of oxytocin. In an added

experiment, effects of the first two treatments of milking performance and release of

oxytocin and prolactin were measured. Milk yield, fat, and protein contents were not

affected by any treatment. Machine-on times were shorter and peak and average milk

flow rates higher for manually stimulated and oxytocin infused cows. Mean oxytocin

concentration in stimulated cows peaked at 2 min compared with 5 min for unstimulated

cows. No difference in time of prolactin released was detected. The timing of oxytocin

release, rather than maximal concentration, could be the most important factor affecting

milking.

John and Antonie (1988) found the concentration of protein and major cations (Ca, K,

Mg, and Na) that were measured by Udy dye-binding and atomic absorption procedures

in Califorinia market milks. Samples of nonfat (n = 47), low fat (n = 48), whole (n = 55),

extra rich (n =25), buttermilk (n = 36) were collected from retail stores. Mean protein

concentrations were; nonfat 3.53%, whole 3.16%, extra rich 3.21%, and butter milk

3.37%. Mean calcium concentration were; non fat 117mg%, low fat 129mg%, extra rich

113mg%, and butter milk 113 mg% and butter milk 115mg%. Mean potassium

concentration s were; nonfat 169mg%, low fat 185 mg%, whole 160mg%, extra rich 157

mg%, and butter milk 17omg%. Mean magnesium concentrations were; non fat 15.0 mg

%, low fat 16.3 mg%, whole 14.2 mg%, extra rich 14.0 mg%, and butter milk 15.9 mg%.

These mean values are in good agreement in those previously reported and can be used

by processors.

Allen (1990) used the oxytocin to find the association among milk composition, secretion

rates and cell electrolytes concentration. It was found that milk yield declined. Sodium

(Na) and Potassium (K) ratio increased with oxytocin dose, lactose decline and fat

remained unchanged.

Morrissery and Flynn (1990) found that milk is a significant source of many minerals

required for normal growth, development, maintenance and metabolism throughout the

life cycle. Milk helps in bioavailability of calcium (Ca), copper (Cu), magnesium (Mg)

and zinc (Zn) their absorption and utilization.

Nostrand et al. (1991) used eighty-four Holstein cows determine to the effects of

exogenous oxytocin on milk production and health. Cows were assigned at parturition by

parity group to treatments: 1) oxytocin group, animals received an injection of 1 ml (20

IU) of oxytocin at each milking throughout lactation and 2) control group, animals

received no injection. Oxytocin injections were given in the thigh region within 3 min

following the initiation of udder preparation and immediately prior to machine

attachment. Cows were milked in a parlor, and milk yield was recorded at each milking.

Milk samples were collected from each cow biweekly for milk composition

determination. Individual lactations were modeled using Woods' lactation equation;

resulting coefficients were analyzed using ANOVA. The oxytocin group produced 849

kg more milk during the lactation than the control group, with a significant difference

occurring after peak milk yield. This suggests that exogenous oxytocin maintained

greater persistency during lactation. No significant differences existed for milk fat or

protein percentages. The use of exogenous oxytocin at milking increased lactation milk

production with no apparent effect on health.

Anderson (1992) studied the comparison of trace elements in milk of 4 species. The

amount of trace elements founded in cows milk were iron (Fe) ‹ 0.2ppm, copper (Cu)

0.05 ppm, aluminum (Al) 0.45 to 0.10 ppm and manganese (Mn) ‹0.04 ppm.

Manganese (Mn) was low relative to bodily needs.

Flynn (1992) outlined the nutritional roles, recommended intake and hazards of

deficiency or excess of 20 mineral and trace elements e.g. Na, K, Ca, Cl, Mn, P, Fe,

Mn, Cu etc that are considered to be nutritionally essential to man. The content,

chemical form and bioavailability of these minerals are considerd in both human and

cow milk.

Maurya and Ludri (1992) showed that oxytocin did not affect milk yield or residual milk

yield after milking. Feeding during milking has been shown to influence milk production

and milking time as well as secretion of pituitary hormone oxytocin. He injected 20

Murrah buffaloes with 0-14 IU oxytocin before milking, did not affect milk yield. Milk

let-down time decreased. Percentage milk fat was higher for the 5 or 10 IU oxytocin

injections than for lower doses. Residual milk yield was 2.1-3.8% of the total yield.

Linda et al. (1993) studied the effect of daily oxytocin injections before and after

milking on milk production, milk plasmin and milk composition. Saline injection was

given before milking as a control. Oxytocin increased milk production by 3%. The

effect on milk plasmin activity, fat, protein, SCC and lactose was non significant and

may indicate that effect of oxytocin is not manifested through an effect on cell

remodeling.

Knight (1994) examined eight cows in mid lactation. They were divided into two

groups, control half and test half. The test half was milked immediately after oxytocin

administration while control half milk 3 hour before oxytocin. The milk yield in test

half was greater. This support the established view that oxytocin act by enhancing the

milk ejection reflex, and refutes the claim that the hormone has a direct stimulatory

action on mammary metabolism.

Bansode et al. (1996) found that increase in milk yield was not significant after oxytocin

injection but there was some change in milk composition after oxytocin treatment.

3. MATERIALS AND METHODS:

Collection of Milk Samples:

Milk samples of 6 Nili Ravi buffaloes will be collected as treated samples from the

livestock farm of University of Agriculture Faisalabad and 6 animals will be selected for

control samples. Analysis will be performed in the laboratory of Rural Home Economic

and Food Technology, University of Agriculture, Faisalabad.

Sample Preparation:

Milk will be analyzed for their mineral composition including both electrolytes and trace

elements. Before the estimation of minerals all samples will be subjected to wet

digestion.

Wet Digestion:

5 gram of milk sample will be taken into 100 ml conical flask. 10 ml of concentrated

nitric acid will be added. The contents of flask will be heated until the brown fumes will

become white. After cooling, 5ml perchloric acid will be added. The contents of the flask

will be heated vigorously, till volume will be reduced to 2-3 ml. The contents will be

diluted up to 100 ml by adding distilled water. This digested and diluted sample solution

will be used for the estimation of electrolytes and trace elements.

Standard Preparation:

Commercially available Na and K standard having 150 mmol/L, Na and 5mmol/L k will

be used. This standard will be diluted in ratio of 2:50 in redistilled water.

For the measurement of zinc (Zn), copper (Cu) and iron (Fe) a multi standard will be

used which is diluted with redistilled water to prepare 0.1, 0.2, 0.4 ppm standards.

Absorbance of each standard will be divided by its concentration and thus a mean value

will be obtained. This value will be multiplied by absorbance of wet digested sample and

volume of dilution to estimate, the concentration of trace elements in sample.

Electrolytes (Na and K):

Na and K concentration will be estimated by flame photometer.

Trace elements:

Zn and Cu will be determined by atomic absorption spectrometer.

Statistical analysis:

Means and standard error of mean will be calculated for tabulation of data. Data thus

obtained will be subjected to two and three way analysis of variance technique. In case of

any significant difference means will be separated and student T test will be applied to

find the difference between two means.

4. References:Allen,J.C., 1990. Milk synthesis and secretion rates in cows with milk composition

changed by oxytocin. J. Dairy Sci., 73(4): 975-984.

Anderson,R.R., 1992. Comparison of trace elements in milk of four species. J. DairySci., 75(11): 3050-3055.

Bansode,P.D., A.M.Mantzi, B.T.Deshmuk and B.A.Talvelkar, 1996. Effect ofintramuscular injection of oxytocin on milk production and its constituents. Ind.J. Dairy Sci., 49(10): 718-720.

Flynn,A., 1992. Minerals and trace elements in milk. Advances in Food and Nutrition Research., 36(3): 209-252.

Gaucheron,F., 2005. The minerals of milk. Repord. Nutr. Dev. 45(7):473-483.

Govt.of Pakistan, 2008. Economic survey 2007-08. Government of Pakistan, FinanceDivision,Economic Advisor’s Wing, Islamabad, Pakistan.

Haenlein,G., 2003. Nutritional value of dairy products of ewe and goat milk. Accessed

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Lindmark,M.H., R.Fonden and H.E.Pettersson, 2003. Composition of Swedish dairymilk. Int. Dairy J., 6(2): 409-425.

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Maurya,V.P and R.S.Ludri, 1992. Effect of oxytocin administration on milk let down time, milking rate and composition of milk in buffaloes. Ind. J. Anim. Sci., 62(3):210-214.

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