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BYANURADHA VERMA

(2015V13M)

Thesis submitted to Lala Lajpat Rai University ofVeterinary and Animal Sciences in partial fulfillment

of requirement for the degree of

MASTER OF VETERINARY SCIENCEIN

LIVESTOCK PRODUCTION MANAGEMENT

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SSCCIIEENNCCEESS,, HHIISSAARR--112255000044 ((HHAARRYYAANNAA))

22001177

CERTIFICATE-I

This is to certify that this thesis entitled "EFFECT OF CHELATED MINERALS

SUPPLEMENTATION ON GROWTH AND REPRODUCTIVE PERFORMANCE OF

MURRAH BUFFALO HEIFERS" submitted for the degree of MASTER OF VETERINARY

SCIENCES in the subject of LIVESTOCK PRODUCTION MANAGEMENT to the LALA

LAJPAT RAI UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES, HISAR is

a bonafide research work carried out by ANURADHA VERMA, Adm. No. 2015V13M

under my supervision and that no part of the thesis has been submitted for any other degree.

The assistance and help received during the course of investigation has been fully

acknowledged.

(Dr. S.K. CHIKKARA)Major advisor

Principal ScientistDeptt. of Livestock Production Management,

LUVAS, HISAR, HARYANA- 125004

CERTIFICATE-II

This is to certify that this thesis entitled "EFFECT OF CHELATED MINERALS

SUPPLEMENTATION ON GROWTH AND REPRODUCTIVE PERFORMANCE OF

MURRAH BUFFALO HEIFERS" submitted by ANURADHA VERMA, Adm. No.

2015V13M to the Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, in

partial fulfillment of the requirement for the degree of MASTER OF VETERINARY

SCIENCE in the subject of LIVESTOCK PRODUCTION MANAGEMENT has been

approved by the Student's Advisory Committee after an oral examination on the same.

EXTERNAL EXAMINER MAJOR ADVISOR

HEAD OF THE DEPARTMENT

DEAN, POST-GRADUATE STUDIES

ACKNOWLEDGEMENTS

First of all, I bow my head before my parents for their boundless blessings and support, whichaccompanied me in all endeavors.

It gives me immense pleasure to express my profound sense of gratitude to my esteemed majoradvisor, Dr. S.K. Chikkara, Principal scientist, Department of Livestock Production Management for hisvaluable guidance, close supervision, concrete suggestions, peerless encouragement, constructive criticismand sustained interest all through entire span of investigation and preparation of this manuscript. I amindeed greatly indebted and thankful to my co-major advisor respected Dr. Subhasish Sahu (AssistantProfessor, Deptt. of LPM), who sustained his incessant and exhilarating support to me.

It is my pleasure to express my sincere gratitude to the Dr. H.K.Gulati Professor & Head,Livestock Production Management, members of advisory committee, Dr. Sajjan Singh Sihag, (PrincipalScientist, Deptt. of ANN), Dr. S.P.Dahiya, (Professor, Deptt. of AGB), Dr.Ashok Kumar (Professor &Head ,Deptt. of VSR dean PGS Nominee and my previous major advisor Dr.S.S.Grewal for the interesttaken to give me the right perspective of my research and valuable guidance for my future ventures.

I undertake the privilege to express my deep sense of gratitude to Dr. Sandeep Gupta, (Scientist,Deptt. of VPB) for the help rendered by him in one form or other during the investigation.

Words can never express my gratitude to my family members whose endless love, affection anduntiring support and everlasting blessings bring me here. Next to God, I owe all the credit for successfulcompletion of my Master programme to my father Mr.Amar Singh Verma for his motivation, inspirationand supporting me in every adverse conditions and my mother Mrs.Banarasi Verma for her great love andsacrifices. With unbound affection I spread special fragrance of thanks to my fiance Mr.Arun Kumar forhis care and boundless affection,my sweet younger brother Mr.Ravi Kumar for his encouragement and everwilling help and my companion Ms. Jaena. This dream would not be materialized without their love,support and constant inspiration.

The valuable support rendered by my Seniors and Batch mates (Dr. Ravi, Dr.Deepak, Dr.vikas,Dr.Ramesh, Dr.Sachin, Dr.Madhur, Dr.Sujata, Dr.Sushma Dr. Neelima, Dr.Sweety) and VLDA Satish,LT Krishan and all farm labours in smooth conductance of my research work is deeply acknowledged.

Friendship needs no studied phrases, polished face or winking wiles. They are my friends, who attimes criticized, scolded and encouraged me to keep my determinacy to reach at proper decision.

I gratefully acknowledge Lala Lajpat Rai University of Veterinary and Animal Sciences forgranting funds to pursue the research work. I was blessed with a lot of friends and the moments I spenthere are the most memorable moments in my life.

Last but not least, I am indebted to those ‘dumb creatures’, the centre of my profession, theyprovided me an opportunity to serve them better.

Place: HisarDate: 29-7-2017 (ANURADHA VERMA)

CONTENTS

CHAPTER NO. DESCRIPTION PAGE NO.

I INTRODUCTION 1-3

II REVIEW OF LITERATURE 4-17

III MATERIALS AND METHODS 18-24

IV RESULTS 25-39

V DISCUSSION 40-44

VI SUMMARY AND CONCLUSION 45-48

BIBLIOGRAPHY i-vii

LIST OF TABLES

TableNo. Description

PageNo.

2.1 Effect of organic trace minerals supplementation in ruminants 7

2.2 Effect of organic trace minerals supplementation in non-ruminants 8

3.1 Distribution of experimental buffalo heifers in different dietary treatments 19

3.2 Proximate composition (% DM Basis) of feed ingredients fed toexperimental buffalo heifers 20

3.3 Percent ingredient composition of experimental concentrate mixture 20

3.4 Proximate composition (per cent) of concentrate mixture 21

3.5 Mineral composition of different concentrate mixture and roughage(%DM basis) 21

3.6 Macro-Micro elements composition of different mineral mixturesupplement in ration of buffalo heifers 21

4.1 Average body weight (kg) of all the experimental buffalo heifers atfortnightly interval 25

4.2 Average metabolic body weight (kg) of experimental buffalo heifers atmonthly interval during experimental trial 26

4.3 Average daily body weight gain (g) of experimental buffalo heifers atmonthly intervals 27

4.4 The mean values of body weight gain in buffalo heifers under differentdietary treatments. 28

4.5 Body length of buffalo heifers during experimental period 29

4.6 Body height of buffalo heifers during experimental period 29

4.7. Heart girth of buffalo heifers during experimental period 29

4.8 Abdominal girth of buffalo heifers during experimental period 29

4.9. Average dry matter intake (kg/day) of experimental buffalo heifers atfortnightly intervals 30

4.10 Average dry matter intake (kg) per 100kg body weight of experimentalbuffalo heifers at monthly intervals. 31

4.11 Feed conversion ratio (DMI/kg body weight gain) of experimentalbuffalo heifers at monthly interval. 31

4.12 Feed conversion efficiency (BW gain g/kg DMI) of experimental buffaloheifers at monthly interval 32

4.13 Effect of supplementing chelated minerals on nutrient digestibility ofbuffalo heifers

32

4.14 Effect of dietary supplementation of chelated minerals on plane ofnutrition during digestibility trial 33

4.15 Average daily water intake by experimental buffalo heifers at monthlyinterval during experiment trial. 34

4.16 Plasma calcium concentration (mg/dl) of experimental buffalo heifers atmonthly intervals 35

4.17 Plasma phosphorus concentration (mg/dl) of experimental buffalo heifersat monthly interval 35

4.18 Plasma copper concentration (mg/l) of experimental buffalo heifers atmonthly intervals 36

4.19 Plasma zinc concentration (mg/l) of experimental buffalo heifers atmonthly intervals 36

4.20 Plasma manganese concentration (mg/l) of experimental buffalo heifers atmonthly intervals 37

4.21 Plasma iron concentration (mg/l) of experimental buffalo heifers atmonthly intervals 37

4.22 Plasma cobalt concentration (mg/l) of experimental buffalo heifers atmonthly intervals 38

4.23 Total cost of feeding (Rs.) and cost of feeding per kg body weight gain ofbuffalo heifers under different treatments for experimental period 39

LIST OF FIGURES

FiguresNo.

Description PageNo.

4.1 Average body weight (kg) of all the experimental buffalo heifers atfortnightly interval

26

4.2 Average daily body weight gain (g) of experimental buffalo heifers atmonthly intervals

27

4.3 Total body weight gain (kg) of experimental buffalo heifers duringthe trial

28

4.4Fee Average dry matter intake (kg/day) of experimental buffalo heifers atfortnightly intervals

30

LIST OF PLATES

PlateNo.

Description AfterPageNo.

1. Buffalo heifers kept chained and fed individually 48

2. Body length measurement of buffalo heifer 48

3. Heart girth measurement of buffalo heifer 48

4. Water being offered to buffalo heifers in measurable bucket separately 48

5. Blood collection from buffalo heifer 48

6. Weighing of concentrate mixture 48

7. Weighing of green roughage 48

8. Weighing faecal samples during 7days digestion trial period 48

9. Automated Random Access Clinical Chemistry Analyzer 48

LIST OF ABBREVIATIONS

Abbreviation used Meaning

@ At the rate of

AA Amino acid

AAC Amino acid chelate

AAFCO Association of American Feed Control Officials

ADF Acid detergent fiber

ADG Average daily gain

AOAC Association of Official Analytical Chemists

b.wt Body weight

BW Body weight

BWG Body weight gain

Ca Calcium

CF Crude fiber

c.m. Centimeter

CP Crude protein

Co Cobalt

Cu Copper

Cr-Met Chromium-Methionine

CPI Crude protein intake

Cr Chromium

DCP Digestible crude protein

DCPI Digestible crude protein intake

DM Dry matte

DMI Dry matter intake

EE Ether extract

et al. ( Latin et alii ) – and others

FCE Feed conversion efficiency

FCR Feed conversion ratio

Fe Iron

Fig. Figure

g/kg W0.75 Gram per kg metabolic body weight

G Gram

GNC Ground nut cake

Hb Hemoglobin

ICAR Indian council of agricultural research

i.e That is

IOM Inorganic mineral

Kg Kilogram

LUVAS Lala Lajpat Rai University of Veterinary and Animal Sciences

< Less than

> Greater than

MBOTM Methionine bases organic trace minerals

Mn Manganese

Met Methionine

mg/dl Miligram per deci liter

Ml Milliliter

NCM Non Chromium-Methionine

NDF Neutral detergent fiber

NFE Nitrogen free extract

NRC National research council

OM Organic matter

% Percentage

% BW Percent body weight

P Probability

/ Per

Rs. Rupees

Sq. Square

TA Total Ash

TDN Total digestible nutrients

TDNI Total digestible nutrients intake

VFA Volatile fatty acid

Zn Zinc

ZnO Zinc oxide

Zn- Met Zinc Methionine

ZnS Zinc sulphate

ZnP Zinc peptide

1

CCHHAAPPTTEERR--II

IINNTTRROODDUUCCTTIIOONN

India ranks first in respect of buffalo population and milk production. Almost the

entire world buffalo population is in the Asian subcontinent. India, China, Pakistan, Thailand

and Indonesia are some of the major countries accounting for a large buffalo population.

Buffalo plays a pivotal role in Indian livestock industry which includes their contribution in

terms of milk, meat, skin, manure, and draft animal power. India ranks first in the world in

milk production. About 63 % of the world’s buffalo milk and 95% of buffalo milk in Asia is

contributed by Indian buffaloes (Anonymous, 2012).

Livestock sector plays a significant role in the welfare of India’s rural population .

India’s livestock sector is one of the largest in the world with a holding of 11.6% of world

livestock population which consists of buffaloes (57.83%), cattle (15.06%), sheep (7.14%),

goats (17.93%), camel (2.18%), equine (1.3%), pigs (1.2%), chickens (4.72%) and ducks

(1.94%). India has huge livestock population of 512 million nos. which mainly includes

cattle, buffaloes, goats, sheep and pigs (BAHS, 2014)

Contribution of livestock and fisheries sectors to the national economy in terms of

Gross Domestic Product (GDP) is 4.1 % and 0.8%, respectively. Agriculture and allied sector

contributed about 15.1% to the total GDP. Out of the total agricultural GDP, livestock sector

contributed about 27.25% during 2012-13. The livestock sector is an important source of

foreign exchange too and is performing well in the manner of production, value addition and

export of dairy, fishery, wool, poultry and other products. Global market for animal products

is expanding fast, and it is an opportunity for India to improve its participation in global

market. During the year 2012-13, livestock sector produced 132.4 million tones of milk, 69.7

billion nos. of eggs, 46.1 million kg of wool and 5.9 million tones of meat (BAHS, 2014). It

has been estimated that demand of milk will rise to 156 million tons by 2020 (Parthasarathy et

al., 2004).

Heifers are future herd of a dairy farm. They must be produced to replace the older

and uneconomical females of the farm through voluntary culling. When genetic trends are

positive, such replacements help in harvesting the benefits of genetic gain. Heifer production

is most expensive part of the dairy farm operation (Heinrichs, et al., 1993). It requires more

inputs for a longer period of time with no visible returns than any other farm operation.

Growth rates of replacement heifers affect economic returns on dairy farms (Cady and Smith,

1996). Lower growth rate in the early life of the heifer may be due to lack of adequate

nutrition associated with improper feeding management practices. Genetics does play its role

2

as well. This lower growth rate results in higher age at puberty and thus, higher age at first

calving in heifer, causing great economic loss to the dairy farmers, which often unrealized.

Minerals deficiency may result in delayed onset of estrus, repeat breeding and / or

infertility.(Underwood and Suttle, 1999).

In modern dairy farming, the prime aim is to achieve the puberty at an early age and

higher production. To achieve these targets, the diet must be formulated using high-quality,

easily absorbable and metabolizable nutrients. In physical terms, mineral and vitamins

constitute only a small proportion of the diet, but their importance is paramount.

Minerals play many vital roles in animal’s life. They are required in very small

amounts in comparison to other nutrients, however, their deficiency results in poor animal

health and production (Overton and Yasui, 2014). Deficiency of trace minerals in the diet

alone can reduce animal production by 20-30%. Therefore, supplementation of trace elements

in animal diets has long been practised in order to ensure their rapid growth, boost

reproductive performance and improve immune response (Overton and Yasui, 2014). Trace

mineral deficiencies can occur as a primary deficiency when mineral intake is inadequate or

as a secondary deficiency when other factors in the diet interfere with the absorption and

metabolism of the concerned trace minerals (Olson et al.,1999). One of the major

disadvantages of using such supplements is that the minerals from inorganic sources are not

fully absorbed due to antagonism and anti-nutritional factors present in the diet. In the Indian

context, there is widespread deficiency of Zn and Cu (Chander Datt and Aruna Chhabra,

2005).

The efficiency of absorption and availability of inorganic elements to the animal

depend on the dietary source, interaction with other elements and nutrients, age and

physiological status of the animal. To cope up the increasing demand of essential elements to

increase the growth rate of dairy heifers, inorganic salt based mineral mixture is supplemented

in daily ration; resulting in increased excretion, which may cause environmental pollution.

Therefore, in order to meet the increasing demand of bio-available elements and to reduce the

contamination of surface water and soil, the concept of chelated mineral / mineral proteinate

came up.

The word chelate is derived from a greek work “chele” i.e claw. A chelate is a water-

soluble organic complex that has a metal ion bound in an organic complexing agent (also

called ligand), which makes it chemically inert, ruminal stable . Chelates and other complexes

are useful in animal nutrition to protect trace minerals during digestion. However, in recent

years, chelated minerals are being supplemented in the ration as the bio-availability of these

minerals is more than their inorganic forms (Spears 1989).The goal of forming chelates is to

increase the bioavailability of minerals to the animals to support metabolic functions.

3

In India references available on the effect of supplementing chelated minerals on

performance of dairy animals are very scanty. Therefore, present investigation has been

planned to assess the effect of feeding chelated minerals to buffalo heifers with the following

objectives.

Due to the paucity of literature the present study has been planned to formulate

specific mineral mixture for buffalo calves and test their efficacy in improving growth and

health with the following objectives:

1. To study the effect of chelated minerals on growth, reproduction and nutrient utilization

in buffalo heifers.

2. To compare mineral profile of the buffalo heifers fed different levels of chelated minerals

in place of inorganic mineral mixture.

3. To evaluate the cost of feeding of different dietary treatments.

4

CCHHAAPPTTEERR--IIII

RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE

2.1 Chelated Mineral

During the last decade, concerns have increased with regard to the relationship of

mineral nutrition and reproduction in buffaloes. It is questionable whether the macro and

micro mineral bioavailability in most dairy rations is sufficient to meet the buffalo’s

maintenance and production needs in view of the improved genetic capability of modem dairy

cows. The intake of these bioavailable minerals is especially crucial in the peri partum animal.

Chelation is a biochemical process with special requirements. The word is derived

from the Greek word Chele', meaning "claw." This describes the effect of the chelating agent

to surround the metal, forming certain stable bonds. Proteins or amino acids are examples of

chelating agents. Chelated metal proteinates, which carry a neutral charge, can be absorbed as

much as 300 to 500 percent more efficiently than their inorganic counterparts.

Trace elements are so named because of the very small quantities (100 ppm or less)

present in the body and required in the diets of animals (Van Hum et al.,1992). Uneven forage

quality, variability in mineral concentrations in feeds, environmental stress, and other

nutritional deficiency factors have necessitated the use of supplemented mineral sources to

meet the demands of high producing livestock and poultry.

Modern dairy cattle fed mixtures of forage with grains and byproducts of milling, oil

feeds, brewing, distilling, citrus, sugar, and rendering industries are much less likely to exhibit

signs of trace element deficiency. Fertility (Hurley et al. 1989) and immune response

(Graham et al., 1991) may be impaired before clinical symptoms become apparent.

Deficiencies of some minerals can cause a negative effect by the lack of thrift, poor gains,

inefficient feed utilization, and reduced reproduction performance. All of these factors reduce

the productive longevity of animals and result in economic losses.

Bioavailability can be defined as the portion of the mineral that the animal can use to

meet its bodily needs. Each mineral is available in certain forms and each form is different in

its bioavailability for the animal's feeding purposes. In theory, the use of chelated minerals

will increase absorption and utilization of the mineral because of a more favorable binding or

stability constant.

Chelate Characteristics

Chelated minerals are unique in that they form a ring structure around the metal and

have coordinate covalent bonds with the metal. These bonds are shared between the metal and

nitrogen (amino) or oxygen (hydroxyl) as donor groups. At certain pH's, some metal

5

proteinates and amino acid chelates have correct sharing of bonds between metal, and oxygen

or nitrogen, and are unique in their neutral charge.

Two broad categories of sources are available to supply these elements. One category

is the inorganic sources like common oxides, chlorides, sulphates and carbonates of the

element (like zinc oxide, zinc sulphate, etc.), and these can differ in their efficacy or

bioavailability. The other category is the organic sources, often referred to and marketed as

"chelate Mineral". Different Factors affecting mineral absorption are as: Solubility of mineral

salt, pH in rumen and intestine, dietary factors (fibre, phytate and oxalate), mineral

antagonism, contaminants including heavy metals (Pb inhibits enzymatic synthesis of carrier

protein for Hb).

The proposed benefit to feeding organic trace minerals is that they should undergo

less dissociation in the reticulo-rumen, omasum, and abomasum than their inorganic

counterparts. Organic trace minerals that remain intact in the upper gastro-intestinal tract are

less likely to form insoluble and indigestible compounds than inorganic trace minerals, and

availability of organic trace minerals for absorption by intestinal tissues should be enhanced.

An ideal organic trace mineral supplement must be resilient enough to remain intact as the pH

changes throughout the digestive tract but must still be available for absorption and

metabolism by animal tissues (Andrieu, 2008). In reality, this ideal organic trace mineral does

not exist. In vitro studies have shown that organic trace minerals are more effectively

absorbed by gut tissues than inorganic trace minerals (Predieri et al., 2005; Wright et al.,

2008).

2.2 Different categories of organic trace minerals

Categories of organic trace minerals as defined by Association of American Feed

Control Officials (AAFCO, 1998) include:

Metal (specific amino acid) complexes: These are the products resulting from complexing a

soluble metal salt with a specific amino acid. For instance, one of the most common metal

complexes is zinc methionine which is produced by combining zinc sulfate and amino acid

methionine. Other such common complexes include copper lysine and manganese

methionine. These complexes are most effective and efficiently absorbed in gut among all

the organic minerals.

Metal amino acid complexes: These are characterized by a metal atom (zinc for instance)

complexed with several single amino acids. Each individual molecule is still one metal ion

and one amino acid but has a variety of amino acids in the blend. For instance for a zinc

complex in this category, the blend would include zinc methionine, zinc lysine, zinc leucine,

zinc cystine, etc.

Metal amino acid chelates: These are formed from the reaction of a metal ion from a

soluble metal salt with amino acids having a mole ratio of one mole of metal to one to three

6

(preferably two) moles of amino acids to form coordinate-covalent bonds. The molecular

size of such chelates should not exceed 800 Dalton.

Now a days trace mineral glycinate using glycine as a ligand is getting popular as

glycine is readily absorbed in gut and gets transported right into the cells. Supplementation

of zinc with glycine chelate improves the growth performance of cattle (Wang et al., 2010)

Metal proteinates: These result from chelation of a soluble mineral salt with amino acids

and/or hydrolyzed protein. The final product may contain single amino acids, dipeptides,

tripeptides or other protein derivatives. As a result, the molecular size of metal proteinates

sometimes is higher than the desired size which decreases bioavailability of minerals.

Moreover, this product has a structure that does not have a very high stability constant

because of the size of its ligand. Such molecules are easily broken apart especially with

change in pH, resulting in the loss of heterocyclic chelate ring structure. Though the metal

proteinates are less expensive, they are not much beneficial when compared with single

amino acid chelates.

Metal polysaccharide complexes: These are generally prepared by coating the metal with

polysaccharide molecules. These are larger molecules based on chains of simple sugars that

are known to be highly soluble in the digestive tract. Many studies have reported no

beneficial effect on animal performance.

Metal propionates: These result on combining soluble metals and soluble organic acids such

as propionic acid. The resultant products are highly soluble and generally disassociate in

solution.

Yeast Derivative Complexes: Other sources of organic trace elements that show promise are

mineral enriched yeast. Presently the most common is selenium yeast with selenium

complexed with a methionine molecule (selenomethionine). Chromium enriched yeast also

has gained popularity for improving animal production (Rao et al., 2012).

2.3 Process of preparing of amino acid chelates of trace minerals

Amino acid chelates of trace minerals are prepared by

chemical reaction using 2:1 molar ratio of amino acid

preferably methionine and inorganic trace mineral ions.

The basic process involved in preparing chelated mineral

is depicted (Pal and Gowda, 2015).

7

Table 2.1: Effect of organic trace mineral supplementation in ruminants

Animal Organic mineral Observations Reference

Cows Cu, Zn, Mn -Similar milk yield and composition- Similar body weight- No effect on uterine health- Similar plasma variables

Yasui et al., 2014

Cows Zn-aminoacid complex

- Decreased postpartum DMI- Increased (20%) colostrum IgG concentrations- Feed efficiency increased- Decreased services per conception- Decreased milk fat concentration

Nayeri et al.,2014

Cows Cu, Zn, Mnpolysaccahridecomplex

- Reduced no. of days in open- Increased conception rate- No change in milk yield and composition

Chester-Jones et al.,2013

Cows Cu, Zn, Mn - Increased colostrum immunoglobulins- Increased milk fat- No change in milk yield, protein and SCC- Lower calf mortality- Increased services per conception

Formigoni et al.,2011

Bulls Cu, Zn, Mn, Co - Increase in motile sperm(65.5 vs. 56.1%)- Increase in progressive sperm(47 vs. 38.4%)- Increase in sperms with rapid motility(62.3 vs. 52.8%)

Rowe et al., 2014

Rams Cu, Zn-methionine - Reduction in DMI- Increased ceruloplasmin and ALP- No effect on growth- Increased immunity

Gowda et al., 2014

Kids Cu-methionine - No effect on growth- No effect on nutrient intake and Digestibility- Increased Cu balance

Waghmare et al., 2014

Rams Cu, Zn –methionine - No effect on body weight- Reduced sperm motility- Reduced motile sperm count- No effect on semen volume- No change in nutrient intake and digestibility- Increased wool yield

Shinde et al., 2012

Ewes Cu, Zn-methionine

- Marginally lower DMI- No effect on nutrient digestibility and growth- Higher bioavailability- Reduced faecal mineral excretion

Pal et al., 2014

Kids Se-yeast - No effect on body weight gain- No change in carcass quality

Sethy et al., 2013

8

Table 2.2 : Effect of organic trace mineral supplementation in non-ruminants

Animal/bird

Organic mineral Observations Reference

Layers Cu, Mn,Zn proteinates

- Lower egg loss, higher thickness,and increased strength of the shell- No change in egg weight- No effect on feed intake, feed conversion,specific weight, and Haugh unit of eggs

Stefanello et al.,2014

Poultry Zn proteinate - No effect on tibia and liver mineral Content- Increased immune response- No change in DMI & FCR

Mandal et al., 2011

Poultry Cr propionate - No effect on intake and weight gain- Increased milk fat- Lowered serum corticosterone level in heatstressed poultry-Recoup in blood glucose level was better

Rajalekshmi et al.,2012

Poultry Cu, Zn, Mn, Fe - DMI decreased at 50% level- Increased FCR- Increased tibia mineral concentration- Similar glutathione peroxidase and ferricreducing ability in blood plasma

Rao et al., 2013

Poultry Mineral proteinates - Higher retention rate and bioavailability- Cu and Zn antagonism could be avoided- No effect on phytase- Less mineral wastage and decrease in Pollution

Ao and Pierce,2013

Quail Fe, Zn-methionine - No effect on growth- No effect on nutrient intake and FCR- Increased bioavailability

Sannamani et al.,2013

Pig Organic Zn - No effect on growth- Increased antioxidant level

Hill et al., 2014

2.4 Benefits of using chelated minerals for livestock production

The positive effects of chelated minerals on animal performance appear mainly due

to higher bioavailability as compared to inorganic sources. There are several studies in

different animal species with different sources of different mineral elements, which have

revealed notable differences in the bioavailability of organic and inorganic minerals. Studies

suggest that binding of Cu, Zn, Fe and Mn with amino acids and peptides can enhance the

bioavailability of these trace minerals, thereby leading to improved milk production, growth,

reproduction and general health status in livestock (Senthilkumar et al., 2015). The benefit

of using chelated minerals has been found to improve reproduction, health, soundness and

growth for livestock. A chelated mineral for cattle has focused on the pregnant cattle and the

young growing calves for improved immunity (less disease or sickness), reproductive

performance (shorter days open, higher conception rates, or less embryonic loss), and herd

health. Mares receiving trace mineral supplementation in a combined inorganic/chelated

product had a tendency for a reduction in the number of cycles bred and in the number of

services per mare. However, there was no effect in conception rate (Ott and Asquith, 1994).

9

It is well known that providing adequate levels of trace minerals is required for proper

immune function. Because of the increased availability of chelated products it is thought

that their use will enhance immune function (Vandergrift, 1993).

2.4.1. Body weight gain

In many of the experiments, growth performance was better in the animals

supplemented with organic minerals. Zn-methionine supplementation improved growth rates

and body weights in lambs (Garg et al.,2008). Similarly, organic sources of Se improved

growth performance in guinea pigs (Chaudhary et al., 2010) and lambs (Kumar et al.,

2009).however, no or very less improvement was seen in lambs (Kumar et al., 2009b)

supplemented with organic Se in comparison with inorganic Se. Calves supplemented with

organic trace mineral sources had a greater final weight and average body weight gain than

calves supplemented with similar levels of trace minerals from inorganic sources (Kegley et

al., 2012).

Organic trace mineral supplementation had no effect on growth performance of the

animals (Rajalekshmi et al., 2012; Shinde et al., 2012; Waghmare et al., 2014). Research

shows that organic mineral sources are more bioavailable. However production responses to

supplementation have been variable. Function of minerals are structural, physiological,

catalytic and regulatory. They are indirectly involved in the growth of animal.

Johnson et al. (1988) conducted five 28-day trials using a total of 773 newly received

calves to examine the effect of zinc methionine on health and performance. Calves were fed a

control diet or the control diet supplemented with zinc methionine to supply 360 mg

zinc/head/day. Zinc methionine supplemented calves gained 10.7% faster (0.704 vs. 0.636

kg/d) had a decreased morbidity rate (46 vs. 51%) and required 5.8% fewer medical

treatments (2.12 vs. 2.25). Zinc methionine also reduced (P < 0.03) the required medical

treatments per head (4.45 vs. 4.94) for cattle that became sick during the studies. Data from

calves detected as sick during the first 3 days of the studies were excluded.

Spears (1989) fed growing heifers a corn silage-based diet containing 24 ppm of zinc

or the basal diet supplemented with 25 ppm of zinc from zinc methionine or zinc oxide.

Average daily gain and feed efficiency were similar for control heifers and those

supplemented with zinc oxide. Heifers receiving zinc methionine gained 8.1% faster (P <

0.07) and 7.3% more efficiently (P < 0.08) than control heifers for the entire 126-day study.

Puchala et al. (1999) reported that supplementation of the diet with Zn-Methionine (1,

3 and 5 g/day of Zn-Met) increased (P < 0.07) average daily gain (65.5 versus 55.9 g/day for

control). Average daily gain (ADG) for goats receiving ZnO was lower (P < 0.04) than for

goats receiving a similar amount of Zn from 3 g/d Zn-Met (50.5 versus 62.9 g/day).

Supplementation of the diet with Zn-Met increased (P < 0.03) plasma Zn concentration (0.92

versus 0.72 mg/l for control); there were no differences in plasma Zn concentration between

10

goats receiving the ZnO supplement and goats receiving a similar amount of Zn from Zn-Met

(0.87 versus 0.92 mg/l; P > 0.56). In summary, supplementation of a Zn-adequate diet with

Zn-Met increased ADG by yearling Angora goats regardless of level of Zn-Met added.

Supplementation of 1 g Zn-Met may have positive effect on ADG and mohair growth when

diet contains about 20 ppm Zn.

Muehlenbein et al.,(2001) conducted a trial in which cows (n = 75 in 1997; n = 120 in

1998) were randomly assigned by estimated calving date and body condition score to one of

three treatments: 1) Control, control; 2) Inorganic, inorganic Cu supplement (200 mg Cu from

CuSO4); 3) Organic, organic Cu supplement (100 mg Cu from AvailaCu). In 1998, a fourth

treatment was added; 4) CU-ZN, organic Cu and Zn (400 mg Zn from AvailaZn in the

Organic diet). No differences (P > 0.10) were found in cow BW change, calf serum Cu

concentrations, calf weaning weights, or in cow 60-d pregnancy rates among treatments in

either year.

Garg et al. (2008) concluded that supplementation of 20 mg Zn/kg DM as Zn

methionine in the basal diet (containing 34 mg Zn/kg DM) of the lambs significantly

improved their growth rate and digestibility of cellulose and ADF. Also reported that intake

of dry matter (DM), organic matter (OM), crude protein (CP), digestible CP and total

digestible nutrients and digestibility of DM, OM, CP, ether extract, neutral detergent fibre and

hemicellulose were comparable (P>0.05) among the three groups.

Mondal et al. (2008) reported that body weight gain (BWG) and average daily gain

(ADG) were found higher (P<0.05) in the entire mineral supplemented crossbred male calves

but was comparable (P>0.05) between organic and inorganic supplemental group.

Bhadheri et al. (2010) studied on the effect of organic and inorganic forms of trace

minerals (Cu, Zn and Mn) at different dose levels on the growth performance of crossbred

male calves. Result revealed that supplementation of MBOTMs (methionine bases organic

trace minerals) at NRC dose level to male calves improved body weight gain and average

daily gain as compared to the calves supplemented inorganic minerals. It may be concluded

that supplementation of MBOTMs at NRC requirement in male calves can improve the body

weight gain than that of inorganic trace minerals.

Song et al. (2013) concluded that effects of Chromium-Methionine (Cr-Met) chelate

feeding for different durations on growth and carcass characteristics in the late fattening stage

of Holstein steers. Dry matter intake showed no differences among all the treatments

(p>0.05). Average daily gain was also higher in the animals fed Cr-Met chelate diets than

NCM (Non Chromium-Methionine) (p<0.05). Although no significant differences were

observed on back fat thickness, rib and eye area (p<0.05).

Gowda et al. (2014) observed that supplementation of trace minerals in combination

of inorganic and organic sources could give better results in terms of growth rate and

11

immunity of sheep and suggested to supplement the trace minerals to livestock as

combination of inorganic and organic sources except chromium which could be supplemented

through organic source only to obtain better performance.

Mallaki et al. (2015) reported that the effect of supplemental organic zinc (Zn) on

performance, nutrient digestibility, and plasma Zn status in Zandi lambs. In experiment 1, 18

male lambs (BW = 21.30 ± 0.55 kg) were fed a basal diet containing 22.8 mg Zn/kg dry

matter (DM) with no supplemental Zn (control) or 20 mg of supplemental Zn/kg of DM from

Zn sulfate (ZnS) or Zn peptide (ZnP). Average daily gain and dry matter intake were higher

for the lambs fed the diet supplemented with ZnP. Feed conversion ratio was significantly (P

< 0.05) lower in the ZnP group compared to the control and ZnS groups.

2.4.2 Bioavailability and Performance

Most of the positive effects of organic minerals on the performance of the animals

appear mainly because of their better bioavailability as compared to their inorganic sources.

Kincaid et al. (1986) reported that calves fed proteinate form of copper had higher

liver and serum level of copper than the calves fed copper sulfate which was suggestive of

enhanced bioavailability of copper from the proteinate source.

Hemken et al., (1993) Observed that results obtained in beef cattle indicated that Cu

from copper proteinate was more bioavailable compared to copper sulphate supplemented

animals.

A meta-analysis by Ao and Pierce (2013) in mineral proteinate supplemented birds

revealed higher retention rate and relative bioavailability than their inorganic counterparts,

which might be due to increased absorption and decreased urinary excretion (Mandal et al.,

2007; Pal et al., 2010). Similar observations were made in guinea pigs (Chaudhary et al.,

2010), chicks (Mandal et al., 2011; Rao et al., 2013), kids (Waghmare et al., 2014) and lambs

(Garg et al., 2008; Kumar et al., 2009b).

The bioavailability of certain minerals decrease when increased amount of other

minerals are present; e.g. copper bioavailability can be low in ruminant’s diets, especially

when Mo and S are present (Spears, 2003; Overton and Yasui, 2014). So, a form of Cu that

would not interact with tetra-thiomolybdate or S and that stays soluble in digestive tract of

ruminants would be beneficial.

Ashmead et al. (2004) recorded that amino acid chelates (AACs) were more

bioavailable than inorganic minerals (IOMs).

Nocek et al. (2006) compared inorganic and complexed trace minerals at several

supplementation levels fed over two lactations. When supplemented at 100% of predicted Zn,

Mn, Cu, and Co requirements, replacement of inorganic sulfate trace mineral salts with amino

acid complexed trace minerals increased bioavailability as measured by liver Zn and Cu

concentrations.

12

Kinal et al. (2007) reported that cows receiving 30% of needed Zn, Mn (each 315

mg/day) and Cu (63 mg/day) in amino acids-bound forms showed significant (P≤0.05)

increase in zinc and copper serum levels as compared to control group.

Mandal et al. (2007) did not find any difference in the serum Zn levels in cattle calves

supplemented with 35 mg Zn/kg DM as ZnSO4 or Zn-propionate in a basal diet containing

32.5 mg Zn/kg DM.

Garg et al. (2008) concluded that supplementation of 20 mg Zn/kg DM as Zn-

methionine in the basal diet (containing 34 mg Zn/kg DM) of the lambs significantly higher

retention and serum concentration of Zn in Zn-methionine group compared to ZnSO4 group

suggested its higher bioavailability from organic source as compared to inorganic source.

Mondal et al. (2008) reported that serum mineral concentration of zinc, copper,

manganese and iron increased linearly (P<0.05) with the increase of days due to mineral

supplementation particularly in organic mineral (T3 and T4) supplemented group. Lower dose

of organic mineral showed the similar result as inorganic mineral with required amount

therefore it reduced excretion as inorganic element and therefore reduced soil toxicity.

Absorption of trace minerals were found significantly (P<0.05) higher in organic minerals

supplemented group than inorganic group.

Siciliano-Jones et al. (2008) studied the effects of organic trace mineral supplements

on bioavailability and performance. When 250 cows were fed Zn, Mn, and Cu as either

sulfates or amino acid complexes, bioavailability (as measured by liver mineral

concentrations) was not affected by treatment. Despite the lack of a difference in liver mineral

concentrations, milk production and hoof health were improved by the amino acid complexes

of trace minerals.

Bhadheri et al. (2010) observed that supplementation of MBOTMs (methionine bases

organic trace minerals) at NRC dose level to male calves did not alter the serum macro-minerals

(Ca, P and Mg) profile, but increased serum Cu, Zn and Mn after 75 days of feeding trial.

Pal et al., (2010) The bioavailability of Zn and Cu from their methionine form was

133 and 151% relative to that of zinc sulphate in ewes.

Ao and Pierce, (2013) Furthermore, high levels of Cu or Zn supplementation as

inorganic salt in poultry diet negatively affected the efficacy of phytase in the diet which

could be overcome with mineral proteinate.

Gowda et al. (2014) recorded in sheep for improving the bioavailability of copper and

zinc through supplementation in chelated form. The average intake of dry matter was higher

in lambs fed chelated source of Cu and Zn. Activities of ceruloplasmin and alkaline

phosphatase were increased (P<0.05) on supplementation of chelated source of Cu and Zn and

growth rate amongst the lambs in three groups did not differ significantly. The immune

13

response was significantly (P<0.05) better in lambs supplemented with chelated form of Cu

and Zn.

The bioavailability estimates of organic mineral complexes among different

treatments/ experiments are sometimes inconsistent (Chester-Jones et al., 2013). There are

also species differences on the utility of organic minerals. Chelated minerals have been more

beneficially used in non-ruminants such as chicks and pigs (Ao and Pierce, 2013), possibly

due to their greater bioavailability than non-chelated forms, as they are more soluble at the

site of absorption in the non-ruminant species. They have been of less concern in the ruminant

animals, probably due to rumen microbes and their involvement in digestion. However, under

certain conditions, ruminants have responded to mineral chelates (Hackbart et al., 2010; Pal et

al., 2010; Chester- Jones et al., 2013; Rowe et al., 2014).

2.4.3 Nutrient digestibility and Nutrient intake

Usually, there is no effect on the palatability and feed intake of the animals with the

supplementation of organic minerals except a few reports of decreased feed intake in organic

mineral supplemented chicks (Rao et al., 2013) and rams (Gowda et al., 2014). This has been

consistently observed in lamb (Kumar et al., 2009b), rams (Shinde et al., 2012), ewes (Pal et

al., 2010), kids (Waghmare et al., 2014), dairy cows (Hackbart et al., 2010) and bulls

(Mandal et al., 2007) using different forms (either in single or in combinations) and dose

levels of trace minerals. In most of the above studies, digestibility of different nutrients was

also similar between inorganic or organic trace mineral supplemented animals. However,

improved nitrogen utilization was observed in lambs supplemented with organic Se as

compared to inorganic Se and unsupplemented groups (Kumar et al., 2009b; Chaudhary et al.,

2010) which was presumably due to higher bioavailability of organic Se that influenced the

thyroid metabolism resulting in improvement of metabolism via activation of thyroxine to

more active tri-iodothyronine form.

Waghmare et al., (2014) conducted a trial of 120 days on twenty male goat kids

(10.94±0.35 kg body weight; 4–5 months age) were divided into four equal groups of control

group (T1) were not fed additional Cu, while the animals of treatment groups were provided

additional 7 ppm Cu (as CuSO4) in T2, 7 ppm Cu (as Cu methionine) in T3 and 3.5 ppm Cu (as

Cu methionine) in T4 group diets. The intake and digestibility of DM, OM, CP, ether extract,

neutral detergent fibre, acid detergent fibre, cellulose and hemicelluloses were similar

(P>0.05) among the four groups. Supplementation of Cu as CuSO4 and Cu-methionine also

had no significant (P>0.05) effect on balance of nitrogen, calcium and phosphorus; feed: gain

ratio and intake of digestible nutrients. However, copper balance was significantly (P<0.05)

higher in T3 than T1, T2 and T4. The growth rate (g/d) of the goat kids in all the three mineral

supplemented groups was not different. Supplementation of 7 ppm Cu (as CuSO4 or Cu

14

methionine) and 3.5 ppm Cu (as Cu-methionine) had no beneficial effect on the growth rate

and nutrient utilization in the goat kids.

Ashry et al. (2012) concluded that inorganic metals caused a significant decline

(P<0.05) in digestibility coefficients, nutritive value, nitrogen utilization, cell wall

constituents, total VFA’s as compare to the organic metals

Mallaki et al (2015) reported that dry matter intake were higher for the lambs fed the

diet supplemented with ZnP. Feed conversion ratio was significantly (P < 0.05) higher in the

ZnP group compared to the control and ZnS groups. The results of this study showed that

feeding ZnP improves performance and digestibility of DM and could result in higher

metabolizable energy and short-chain fatty acid yield.

Garg et al. (2008) observed that intake of dry matter (DM), organic matter (OM),

crude protein (CP), digestible CP and total digestible nutrients and digestibility of DM, OM, CP,

ether extract, neutral detergent fibre and hemicellulose were comparable (P>0.05) among the

organic and inorganic zinc supplemented groups. Digestibility of cellulose and acid detergent

fibre was significantly (P<0.05) higher in Zn-meth group as compared to control group.

Jia et al. (2009) reported that Zn supplementation had influence on digestibility of

DM, CP, EE and NDF (p>0.05). Moreover, ADF digestibility in the group supplemented with

ZnMet was significantly higher than in other treatments (p<0.05).

2.4.4 Effect on milk production and milk quality

The quantity and quality of milk are one of the most important traits in the animal

production system.

Many studies (Griffiths et al., 2007; Siciliano-Jones et al., 2008; Hackbart et al.,

2010) showed a positive effect on milk yield by supplementing cows with organic trace

minerals in the long term feeding trial or at the end of short term feeding trials while others

(Formigioni et al., 2011; Nemec et al., 2012; Chester- Jones et al., 2013) reported no

differences. It appears that supplementation of organic trace minerals require a certain amount

of time before their biological effects are observed.

Rabiee et al., (2010) In a meta-analysis of 20 experiments, organic trace mineral

supplementation increased milk production by 0.93 kg, milk fat by 0.04 kg and milk protein

by 0.03 kg per day.

Moreover, Nocek et al. (2006) supplemented dairy cows with organic trace minerals

for two lactations and observed an increase in milk production compared to cows fed the same

concentration of inorganic trace minerals. The increase in milk production was greater and

response was earlier in the second lactation than in the first lactation.

Hackbart et al. (2010) reported decreased protein percentage in organic and inorganic

mineral supplemented groups than control group, however, there was no adverse effect on

total protein yield. There are many studies (Ballantine et al., 2002; Nocek et al., 2006;

15

Griffiths et al., 2007; Siciliano-Jones et al., 2008) which indicated that organic trace minerals

had no effect on milk protein percentage but enhanced the total protein yields. Ballantine et

al. (2002), Nocek et al. (2006), Griffiths et al. (2007) and Formigioni et al. (2011) also

demonstrated that cows fed organic trace minerals had greater milk fat yields than cows fed

inorganic trace minerals whereas Siciliano-Jones et al. (2008) and Chester-Jones et al. (2013)

reported no difference in fat yield. However, Hackbart et al. (2010) observed decline in milk

fat percentage but total milk fat yield was similar indicating that organic minerals

supplementation had an overall beneficial effect on milk yield and their composition.

Ashmead et al. (2004) recorded that AACs group produced 9.3% more total milk fat

(P<0.05) than the IOMs (Inorganic minerals) group due to greater milk production. First calf

heifers supplemented with (AACs) demonstrated greater mineral utilization from this source

than from (IOMs).

Ohh et al. (2005) reported increase in milk production of dairy cows when fed

organic chromium than inorgamic chromium. Besides few other citations reflecting the effect

of chelated chromium on milk production are presented in table.

Kinal et al. (2005) reported that cows receiving 30% of needed Zn, Mn (each 315

mg/day) and Cu (63 mg/day) in amino acids-bound forms were characterized by higher

(6.5%) milk production than the control animals. In these cows also a decrease of somatic cell

contents in milk (34%) was observed.

Kinal et al. (2007) indicated that the application of bioplexes of zinc, copper and

manganese in the nutrition of cows with average milk yield of 9500 kg milk for lactation had

a positive effect on the level of minerals in colostrum, milk and blood of cows. These

differences were significant in cows receiving 30% of the requirement for these elements as

bioplexes whereas the covering of cows’ requirement by 20% of trace elements from

bioplexes was less effective.

Somkumar et al. (2011) reported that Bestmin Gold treated group (Metho-chelated

organic minerals) improved the milk yield, net gain in milk and the milk fat percentage of

animals across the various stages of lactation as compared to in control and inorganic mineral

treated group of animals.

Hassan et al. (2011) reported that milk yield and fat corrected milk (FCM) were

significantly increased (P < 0.05) by feeding ZnMet rations compared with inorganic Zn

ration. The average daily milk yield were increased by 12.32% and 9.78% in 15 mg Zn

(ZnMet) ration and 25 mg Zn (ZnMet) ration than 25 mg Zn (ZnSO4) ration, respectively.

Ashry et al. (2012) also study the effect of feeding mixed chelated minerals (Mn, Cu

and Zn) methionine on dairy cow productive performance and milk yield and its components.

The treated group (chelated minerals) improved the milk yield, and the milk fat percentage of

animals across various stages of lactation as compared to inorganic minerals treated group of

animals.

16

2.4.5 Effect on reproductionThe production efficiency of farm animals is largely dependent on their reproductive

performance, and there are interactions between reproductive performance and mineral status.

Micro-minerals play a very important role in reproduction of farm animals (Overton and

Yasui, 2014).

In many of the studies, organic mineral supplementation had a positive effect on

reproductive performance (Chester-Jones et al., 2013; Rowe et al., 2014) but there are few

reports which showed either no effect or adverse effect (Shinde et al., 2012).

Rabiee et al. (2010) performed a meta-analysis of 20 experiments in cows and found

that supplementation of organic trace minerals reduced days open and number of services per

conception.

However, Griffiths et al. (2007) observed no difference in first service conception

rates and Ballantine et al. (2002) observed a positive effect.

Similarly, young cows had increased reproductive performance after receiving

organic mineral, but there was no effect in mature cows (Lamb et al., 2008). Several studies

have suggested that organic trace minerals improve various indices of reproduction, including

an increase in the percentage of cows pregnant at 150 day in milk (Nocek et al., 2006; Defrain

et al., 2009) and a reduction in days open and services per conception (Kellogg et al., 2003).

Feeding beef cows with organic mineral resulted in increased pregnancy rates during the

subsequent breeding season (Stanton et al., 2000). Significantly reduced mortality and heavier

piglets at birth and weaning were found in chelated iron fed sows as chelation helps in

increased transfer of the metal across the placenta and into the embryo. There were also fewer

stillborn piglets, more piglets weaned per parity as well as a shorter interval between weaning

and oestrus.

Rowe et al. (2014) observed an improved semen quality in bulls fed a mixture of

inorganic and organic Zn, Cu, Co, and Mn than the inorganic only group as observed by

greater percentage of motile sperm, increased percentage of progressive sperm and sperm

with rapid motility. Calving-first service and calving-conception intervals were shorter in

organic trace element group, but the number of services per conception and pregnancy rates

was similar in organic and inorganic mineral supplemented groups (Ramos et al., 2012).

Amino acid chelated trace minerals supplementation could decrease the open days by

42 days and services per conception by 42% when compared with supplementation of

inorganic mineral sources because amino acid chelated sources of minerals increase the

concentration of specific minerals in their uterine tissue. Therefore, supplementation of

minerals through organic sources may be more effective in ameliorating the reproductive

problems in ruminants.

Manspeaker et al. (1987) fed 40 first-calf Holstein heifers a control diet or the

control diet plus an amino acid chelated mineral supplement. Mineral content of the control

17

diet was not reported. The amino acid chelated supplement supplied additional iron,

manganese, copper and zinc in addition to potassium and magnesium. The study was

conducted from approximately 30 days prepartum until heifers were confirmed pregnant by

rectal palpation. Incidence of periglandular fibrosis (a pathologic response in which

endometrial tissue does not regenerate properly after parturition) was significantly lower (10

vs. 58%)in heifers given chelated minerals. Although not statistically significant, ovarian

activity tended to be higher and embryonic mortality lower for heifers fed the chelated

mineral supplement.

Kropp (1990) evaluated the effects of supplementing amino acid chelates on

reproduction in first-calf beef heifers. Beginning at approximately 45 days post-calving,

heifers were divided into two groups and supplemented with similar levels of copper, zinc,

manganese, magnesium and potassium from either amino acid chelates or inorganic forms.

Heifers were synchronized 70 days post-calving. Percentage of heifers exhibiting estrus and

conception rate following synchronization was higher (P<0.05) for heifers fed the chelated

mineral mixture. Conception rate for the entire breeding season did not differ but heifers

receiving chelated minerals conceived an average of 19 days earlier than those fed inorganic

minerals.

Bosseboeuf et al. (2006) observed that animals receiving a formulation containing

Mg, Fe, Mn, Cu, and Zn as AAC (amino acid chelate) exhibited improved fertility (P < 0.05)

compared with herd mates receiving the same minerals as inorganic metal salts (IM). The

AAC group had 75% more mature ovarian follicles with vaginal secretions indicating estrus

(P < 0.05), 80% less uterine bacterial infections (P < 0.05), 83% less peri glandular fibrosis (P

< 0.05), and 45% less early embryonic mortality (P < 0.05). Mean first conception occurred

45 days earlier in the AAC group (AAC = 90 days versus IM = 135 days; P < 0.05).

Garg et al. (2008) observed that 24 animals in experimental group exhibited estrus,

after on an average 27 days of feeding the supplement. However only 2 animals exhibited

estrus in control group during this study. So inference would be drawn that supplementing of

trace minerals, in the form of chelates, along with vitamins A, D3, and E, can help in curing

the problems of anoestrus/ repeat breeding in dairy animals.

Butani et al. (2016) conducted an on-farm trial using anoestrus and repeat breeder

buffaloes by feeding Ionic mineral mixture (T1) and mineral mixture with addition of 25%

chelated zinc (T2). Results indicated that number of days taken for return to heat of anoestrous

and repeat breeder buffaloes as well as cost of feeding reduced upon supplementation with T2

from T1 mineral mixture.

18

CCHHAAPPTTEERR--IIIIII

MMAATTEERRIIAALLSS AANNDD MMEETTHHOODDSS

3. SELECTION AND MAINTENANCE OF ANIMALS

The present investigation entitled ffect of Chelated minerals supplementation on

growth and reproductive performance of Murrah buffalo heifers” was conducted at the farm

of Livestock Production Management, College of Veterinary Sciences, Lala Lajpat Rai

University of Veterinary and Animal Sciences, Hisar for a period of 120 days. Hisar city is

situated in semi-arid region and climatic condition is sub-tropical in nature. Geographically,

Hisar is situated at 29° 10' N latitude, 75° 40' E longitude and 215.2 meters altitude.

Prior approval was taken to conduct the present investigation by the Institutional

Animal Ethics Committee. The experimental animals were kept chained and fed individually

under semi loose housing system. Experimental shed was located in east-west direction . The

animals were maintained under isomanagerial conditions and similar husbandry practices

except the different feeding treatments.

The various details of experimentation have been described in the chapter.

3. 1 Selection of Experimental Animals

3. 2 Experimental Design

3. 3 Feeding and Watering

3. 4 Chemical Analysis Of Feed Ingredients For Proximate Principles

3. 5 Body Weight Gain

3. 6 Body Parts Measurements

3. 7 Feed intake

3. 8 Digestion Trial

3. 9 Reproductive parameters

3. 10 Blood Analysis

3. 11 Estimation of mineral

3. 12 Cost of Feeding

3. 13 Statistical Analysis

3.1 Selection of Experimental Animals

Fifteen healthy Murrah buffalo heifers in the age group of 22 to 28 months were

selected from farm of the Department of Livestock Production Management of College of

Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar.

19

3.2 Experimental design

Fifteen buffalo heifers 22 - 28 months of age were selected from the buffalo farm of

Department of Livestock Production Management, Lala Lajpat Rai University of veterinary

sciences, Hisar. Before the commencement of study, all the buffalo heifers were dewormed

against parasites and sprayed against external parasites. After the preliminary adjustment

period of ten days, on the basis of nearness in body weight and age, Buffalo heifers were

weighed individually and stratified into three groups of five heifers in each group and

presented in Table 3.1. The feeding trial continued for 120 days. Experimental buffalo heifers

were kept chained and fed individually in well ventilated shed. Chelated minerals for the

experiment was procured private firm from the local market.

Table 3.1: Distribution of experimental buffalo heifers in different dietary treatmentsGroup S. No. Heifer Number Initial Body wt. (kg) Age in Months

T1

1 1102 360 27.22 1103 365 27.13 1104 326 274 1138 302 23.15 1142 282 22.3

Average 327 25.3

T2

6 1098 361 27.47 1111 334 25.98 1123 355 25.19 1132 330 23.7

10 1136 277 23.4Average 331 25.1

T3

11 1100 301 27.312 1113 290 25.813 1114 366 25.714 1117 332 25.615 1125 340 25.1

Average 326 25.9

The requirements of buffalo heifers were met by feeding concentrate mixture, green

fodder, wheat straw as per ICAR (Ranjhan, 1998). The buffalo heifers in different groups

were subjected to following treatments.

No. Group Treatment1. T1

(control)Seasonal green fodder + wheat straw + Conventional concentrate mixturehaving 2% mineral mixture

2. T2 Seasonal green fodder + wheat straw + 50% mineral mixture in concentratemixture of control group were replaced with chelated minerals.

3. T3 Seasonal green fodder + wheat straw + 100% mineral mixture in concentratemixture of control group were replaced with chelated minerals.

20

3.3 Feeding and Watering

During the entire study period, the animals were given green fodder and concentrates

mixture so as to meet their protein and energy need for growth as per ICAR (Ranjhan, 1998)

feeding standard. The chaffed seasonal green fodder (chaffed size- 2.0 to 2.5 cm) like oat and

maize (depending on the availability) were given at the rate of 15 kg/animal/day along with

wheat straw @ 4 kg/animal/day (chaffed size- 1.5 to 2.0 cm). The quantity of different feeds

given to each group was adjusted at fortnightly intervals so that the overall DCP requirements

of buffalo heifers were met according to the change in body weight. Animals were given ad

lib fresh water twice a day throughout the experimental period.

3.4 Chemical Analysis of Feed Ingredients for Proximate Principles

Before formulation of rations, the feed ingredients were analyzed (AOAC, 2005) for

proximate composition (Table 3.2). Based upon the proximate composition of feed

ingredients, the ration for the different experimental groups of animals was formulated. The

composition of the experimental diet of different treatment groups and proximate chemical

composition is presented in (Table 3.2)

Table 3.2: Proximate composition (% DM Basis) of feed ingredients fed to experimentalbuffalo heifers

Ingredients DM CP CF EE Ash OM NFEWheat straw 94.56 2.31 34.43 1.06 7.83 88.12 54.37Oat (Green) 30.54 8.99 26.26 5.68 10.36 87.25 48.71Mustard Cake 94.37 32.21 7.98 6.65 7.81 91.26 45.35Wheat 93.48 14.89 2.98 2.15 2.11 97.77 77.87Wheat bran 89.21 16.03 10.99 4.21 6.27 86.36 62.50Barley 88.23 13.41 5.53 2.12 2.40 87.12 76.54

The ingredients of the concentrate mixture were oven dried and ground in Willey

mill. Various proximate nutrients viz. dry matter (DM), crude protein (CP), ether extract (EE),

crude fiber (CF) and total ash (TA) were estimated by adopting method of AOAC (2005).

Chemical composition of the concentrate mixtures of various proximate nutrients and mineral

contents has been presented in table 3.3, 3.4 and 3.5.

Table 3.3: Percent ingredient composition of experimental concentrate mixture

Ingredient T1 T2 T3Wheat 15 15 15Barley 20 20 20Mustard Cake 30 30 30Wheat Bran 32 32 32Common salt 1 1 1Mineral mixture 2 1 00Chelated mineral mixture 00 1 2Total 100 100 100

21

Table 3.4: Proximate composition (percent) of concentrate mixture

Treatments AttributeDM OM CP CF EE NFE TA NDF ADF

T1 90.22 89.52 19.92 6.94 4.15 58.29 10.70 36.63 19.65T2 89.03 89.43 19.89 6.93 4.07 58.43 10.68 36.41 19.55T3 90.16 89.40 19.85 6.89 4.08 58.53 10.65 36.32 19.24

Table 3.5: Mineral composition of different concentrate mixture, roughage (%DM basis)

Attribute Ca(g/kg)

P(g/kg)

Cu(mg/kg)

Zn(mg/kg)

Mn(mg/kg)

Fe(mg/kg)

Co(mg/kg)

T1 25.2 15.4 11.6 58.6 56.2 270 0.10

T2 24.1 15.7 11.5 59.2 57.2 272 0.11

T3 26.2 16.0 11.4 58.8 57.4 269 0.11

Wheat straw 7.0 1.1 3.8 5.5 41.3 157 0.05

Green fodder 9.0 2.9 9.7 6.0 59.8 175 0.06

The minerals were supplemented through mineral mixture (as per ICAR, 2000) and

composition of minerals (%) is presented in Table 3.6.

Table 3.6: Macro-Micro elements composition of different mineral mixture supplementin ration of buffalo heifers

Minerals per kg. Inorganic Minerals Chelated MineralsCalcium 21% 21%Phosphorus 10% 11%Zinc 0.8% 0.9%Copper 0.1% 0.1%Manganese 0.2% 0.2%Cobalt 0.02% 0.02%Iron 0.40% 0.35%

Samples of concentrate ingredients, wheat straw, green fodder, faeces were ground

and analyzed for dry matter (DM), organic matter (OM), crude protein (CP) (method 945.05

and 944.13 of AOAC, 2005), ether extract (EE), crude fibre (CF) and total ash (method

920.39 and 942.05 of AOAC, 2005). Fiber fraction of feed, fodder and faecal sample were

analyzed by using detergent method of fiber estimation by Robertson and Van Soest (1981).

Observation

The following observations were recorded during the experimental period:-

3.5 Body weight gain

Body weight

The buffalo heifers were weighed at the start of the experiment and thereafter at

fortnightly intervals. The body weight was recorded in the morning before offering any feed

22

or water to the buffalo heifers. These body weights were used for determining the growth rate

and also for the purpose of the computing the ration for the buffalo heifers.

Calculation of body weight gain:

Weight gain = Final body weight – Initial body weight

Final body weight – Initial body weightAverage daily weight gain = ––––––––––––––––––––––––––––––

Time interval

3.6 Body measurements

Body measurements of all the buffalo heifers were recorded at the start and thereafter

at monthly intervals during the experiments. These included:-

i. Body Length (the distance from point of shoulder to the point of pin bone). It was

measured with the help of measuring rod.

ii. Body height (the distance from the floor to the highest point of withers). It was measured

with the help of measuring rod.

iii. Heart Girth (the circumference of the body over the chest of the animal just behind point

of elbow). It was taken with the help of a measuring tape.

iv. Abdominal girth (the circumference of the body over the flank of the animal just in front

of udder). It was taken with the help of a measuring tape.

3.7 Feed intake

The buffalo heifers were given weighted quantity of feed and fodder daily as per

computed ration. Daily feed intake during the experimental period was determined on the

basis of feeds and fodder offered and left over and data were compiled on monthly basis.

3.7.1 Feed conversion ratio (FCR)

On the basis of feed and fodder consumption, DM consumed by the animals were

estimated. The feed gain ratio is a measure of efficiency of utilization of feed. FCR was

calculated by the amount of dry matter intake in kilogram (Kg) required for per Kg weight

gain by animals during the trial period.

3.8 Digestion trial

A digestion trial of 7 days was conducted at the end of the experiment to know the

effect of treatments on digestibility of feed and fodder.

Weighed quantity of feed and fodder was offered to the animals at a fixed time every

day during the trial. Small representative feed and fodder samples were taken in numbered

polyethylene bags. Sampled feed and fodder were dried in hot air oven at 100±2oC for 12 hr

to determine DM% in the feed. These samples were pooled at the end of the collection period

and ground to pass through 1 mm screen and preserved in air tight polythene bags until

analyzed for proximate principles and cell wall constituents (NDF and ADF) as per standard

23

procedure.

After 24hrs of feeding the feed left in manger was quantitatively collected, weighed

and mixed thoroughly. The samples were drawn and dried in hot air oven at 100±2oC for 12

hr to determine DM% in the residue. These samples were pooled at the end of the collection

period and ground to pass through 1 mm screen and preserved in air tight polythene bags until

analyzed for proximate principles and cell wall fraction (NDF and ADF) as per standard

procedure.

Total fecal matter voided 24hr after offering feed was collected, weighed and mixed

thoroughly, and then a representative sample were drawn as per the fixed ratio decided by

weighing the feces voided one day before the collection period. For animals having fecal

outgo up to 10 kg, 1/100 part of the faeces were collected for aliquoting and for animals

having faecal outgo more than 10 kg, 1/150 part of the total faeces was collected during

collection period. At the end of collection period, the plastic container was weighed and

contents were emptied in a tray and mixed thoroughly. Dried dung samples were ground to

pass through 1 mm sieve size and analyzed for proximate principles and cell wall constituents

(NDF and ADF) as per standard procedure.

3.9 Reproductive parameters

Buffalo heifers were observed for their first heat signs, during the experimental period

and the age at first heat and conception were recorded. Number of A.I per conception was

also recorded.

3.10 Blood Analysis

3.10.1 Blood sampling

Blood samples were collected aseptically during early morning hours before feeding

and watering of the animals by jugular vein-puncture from all the fifteen animals belonging to

the three experimental groups. Approximately ten milliliter (ml) of blood was collected from

each animal and transferred immediately into a set of sterile plastic tubes without anti-

coagulant for serum mineral analysis respectively.

3.10.2 Sampling schedule

Samples were collected at the beginning of the experiments i.e. day 0, and thereafter

at monthly interval that is on day 30, 60, 90 and 120th of experiment periods.

3.11 Estimation of minerals

The contents of test tubes were held in slanting position for serum separation. The

sera were centrifuged to remove the erythrocytes present, if any. The clear, non-haemolysed

sera were then collected in clean, dry and labeled vials. These sera were preserved under

deep-freezing in capped vials for further analysis.

Serum samples were digested for estimation of minerals (copper, zinc, and

manganese). One milliliter serum mixed with 10 ml of digestion mixture consisting of nitric

24

acid and perchloric acid in the ratio of 4:1 was taken into a beaker and kept overnight for

proper digestion. Next morning beakers containing the mixture were heated using hot plate to

evaporate the contents till few drops were left. The beaker content were allowed to cool and

after cooling triple distilled water was added to the beakers and volume made up to 15 ml.

These final digested samples were kept in air tight sample bottles for further analysis of

minerals. Estimation of minerals was done using by atomic absorption spectrometer- model

Pinaacle 900T, S/N PTAS13050201 of PerkinElmer Company.

3.12 Cost of feeding

Prices of feeds and fodder, prevailing at the time of purchase (including price of

greens), were collected from the Department of Animal Nutrition of the University. On the

basis of this price, total expenditure incurred on feeding of various experimental groups was

calculated. Cost of feeding per kg gain was also computed for different groups.

3.13 Statistical analysis

Data obtained were subjected to statistical analysis as per Snedecor and Cochran (1994)

using Completely Randomized Design (CRD). All the data were subjected to ANOVA using

the General Linear Models procedure of SAS software. The mean differences among different

treatments were separated by Duncan’s multiple range tests. Consequently, a level of significant

(P<0.05) was used as the criterion for statistical significance (Duncan, 1955).

25

CCHHAAPPTTEERR--IIVV

RREESSUULLTTSS

The present study was conducted to compare the effect of supplementation of

chelated minerals on growth and reproductive performance of Murrah buffalo heifers.

Comparison was made in terms of body weight, body parameters measurements, dry matter

intake, serum mineral profile, nutrient digestibility, feed conversion ratio, reproductive

performance and cost of feeding. For above this mentioned study, fifteen buffalo heifers were

taken and then stratified randomly into three different treatment groups on the basis of

nearness in age and body weight i.e. T1 (control group, fed with conventional mineral

mixture), T2 (replacing 50% conventional mineral mixture of control with chelated minerals)

and T3 (replacing 100% conventional mineral mixture of control with chelated minerals).

4.1 Effect of supplementation of chelated minerals on the different growth parameters

4.1.1 Body weight (kg)

Table 4.1: Average body weight (kg) of all the experimental buffalo heifers atfortnightly interval

DaysTreatments

T1 T2 T3

0 327.00±16.10 331.40±14.83 325.80±13.70

15 334.80±15.72 337.60±15.13 332.20±13.94

30 341.80±16.12 346.80±14.77 340.20±14.06

45 349.20±16.27 354.80±14.88 348.20±13.53

60 359.20±16.38 365.60±14.88 357.00±14.36

75 369.00±16.72 375.00±12.93 367.00±14.56

90 378.40±16.76 386.00±15.07 376.00±14.11

105 380.40±19.99 391.20±14.31 381.00±13.86

120 389.80±16.74 398.00±13.97 387.60±14.30Each figure is an average of five values ± standard error

Average body weight (BW) of all the experimental buffalo heifers at fortnightly

interval showed increasing trend in all the three treatments throughout the experiment and has

been presented in table 4.1 and fig. 4.1. Initial average body weight of all the treatment groups

i.e. T1, T2 and T3 were 327.00, 331.40 and 325.80 kg and at the end of experiment they were

389.80, 398.00 and 387.60 kg, respectively.

No significant difference could be established between the various treatment groups

in respect to body weight (BW) in 120 days of trial.

26

Figure 4.1: Average body weight (kg) of all the experimental buffalo heifers atfortnightly interval

4.1.2 Metabolic body weight (KgW0.75)

Average metabolic BW of buffalo heifers of all the three treatments were calculated

at monthly intervals and data has been presented in Table 4.2. Initial and average metabolic

body weights (Kg W0.75) of experimental buffalo heifers were 76.83, 77.61 and 76.63 kg in

T1, T2 and T3 groups, respectively. Final metabolic body weights at the end of the experiment

for corresponding groups were 87.67, 89.07 and 87.31 kg respectively. The data did not show

any significant difference among the treatment groups during the 120 days of trial period. .

Table 4.2: Average metabolic body weight (Kg) of experimental Buffalo heifers atmonthly interval during experiment trial

DaysTreatments

T1 T2 T3

0 76.83±2.85 77.61±2.64 76.63±2.4230 79.43±2.82 80.31±2.59 79.16±2.4560 82.44±2.83 83.56±2.58 82.08±2.4790 85.73±2.86 87.03±2.57 85.34±2.40

120 87.67±2.84 89.07±2.36 87.31±2.41Each figure is an average of five values ± standard error

4.1.3 Average daily gain (ADG)

Average daily weight gain (g/day) of buffalo heifers under different treatments has

been presented in Table 4.3 and Fig. 4.2. After one month, the average daily weight gain were

493.33, 513.33 and 480.00 g/d in T1, T2 and T3, respectively. The results of the study revealed

that the average daily weight gain of experimental buffalo heifers under different dietary

27

treatment groups did not differ significantly (P>0.05) during 120 days of experiment. Average

daily weight gain at the end of the experiment, for the corresponding groups were 380.00,

400.00 and 386.66 g/d, respectively.

Table 4.3: Average daily body weight gain (g) of experimental buffalo heifer at monthlyintervals

Days T1 T2 T3

0 – 30 493.33 ±12.47 513.33±13.33 480.00±34.3230 – 60 580.00±17.10 626.66±12.47 560.00±49.8960 – 90 640.00±24.50 680.00±17.10 633.33±10.54

90 – 120 380.00±24.94 400.00±47.14 386.66±13.330 - 120 523.33±11.30 555.00±13.07 515.00±19.62

Each figure is an average of five values ± standard error

Figure 4.2: Average daily body weight gain (g) at monthly interval

4.1.4 Body weight parameter

The average initial body weight of heifers were 327.00, 331.40 and 325.80 kg, while

final body weights at 120th days of experiment were 389.80, 398.00 and 387.60 kg, in

treatment groups T1, T2 and T3, respectively. Overall, average daily body weight gain under

three treatments during the whole experimental period was 523.33, 555.00 and 515.00 g/d, in

T1, T2 and T3 groups, respectively The results of the study revealed that total weight gain and

gain per day did not differ significantly (P>0.05) in heifers fed ration supplemented with

chelated minerals as compared to inorganic mineral mixture.

28

Table 4.4: The mean values of body weight gain in buffalo heifers under differentdietary treatments

AttributesTreatments

T1 T2 T3

Initial body weight (kg) 327.00±16.10 331.40±14.83 325.80±13.70

Final body weight (Kg) 389.80±16.74 398.00±13.97 387.60±14.30

Total body weight gain (Kg) 62.80±1.36 66.60±1.57 61.80±2.35

Body weight Gain/day (g) 523.33±11.30 555.00±13.07 515.00±19.62Each figure is an average of five values ± standard error

Figure 4.3: total body weight gain (kg) during the trial

4.2 Effect of supplementation of chelated minerals on different body measurements

Average values of body length, height, heart girth and abdominal girth of buffalo

heifers under different treatments have been presented in Table 4.5, 4.6, 4.7, and 4.8,

respectively.

At the end of experiment the mean values of body length and height of heifers were

133.60, 137.20, 135.40 cm and 128.40, 129.80, 126.80 cm in treatment groups T1, T2 and T3,

respectively (Table 4.5 & 4.6). Average body length and height did not differ significantly

(P>0.05) between the treatment groups.

Similarly, the total gain in heart girth and abdominal girth were also non significant

(P>0.05) among treatment groups as shown in table 4.7 and 4.8.

29

Table 4.5: Body length of buffalo heifers during experimental period

AttributesTreatments

T1 T2 T3

Initial body length (cm) 124.40±1.97 127.20±1.88 126.20±1.72Final body length (cm) 133.60±1.86 137.20±1.93 135.40±1.50Total body length gain (cm) 9.20±0.49 10.00±0.70 9.20±1.36Each figure is an average of five values ± standard error

Table 4.6: Body height of buffalo heifers during experimental period

AttributesTreatments

T1 T2 T3

Initial body height (cm) 122.80±1.69 122.60±2.87 120.20±1.99

Final body height (cm) 128.40±1.89 129.80±2.48 126.80±2.04

Total body height gain (cm) 5.60±0.75 7.20±0.49 6.60±0.60Each figure is an average of five values ± standard error

Table 4.7: Heart girth of buffalo heifers during experimental period

AttributesTreatments

T1 T2 T3

Initial heart girth ( cm) 174.40±174.40 173.80±2.54 174.40±1.99Final heart girth (cm) 185.40±2.77 185.00±2.61 185.00±2.55Total heart girth gain (cm) 11.00±1.05 11.20±0.86 10.60±0.75Each figure is an average of five values ± standard error

Table 4.8: Abdominal girth of buffalo heifers during experimental period

AttributesTreatments

T1 T2 T3

Initial Abdominal girth (cm) 186.00±2.59 185.00±3.15 185.60±2.66

Final abdominal girth (cm) 197.40±2.16 197.80±3.25 196.80±3.40

Total Abdominal girth gain(cm) 11.40±0.68 12.80±1.02 11.20±0.97Each figure is an average of five values ± standard error

4.3 Dry matter intake (DMI)

4.3.1 Dry matter intake (kg/day)

The mean values of total dry matter intake per day (kg/d) during the experimental

period have been presented in Table 4.9 and fig. 4.4. It was observed that there was a linear

increase in DMI during progressive growth period of heifers under different dietary

treatments except at the end due to the commencement of summer, which results in reduced

dry matter intake in all the treatment groups. The dry matter intake values did not differ

significantly (P>0.05) throughout the study period.

30

Table 4.9: Average dry matter intake (kg/day) of experimental buffalo heifers atfortnightly intervals

DaysTreatments

T1 T2 T3

0 Day 7.97±0.08 7.72±0.55 7.05±0.6315 Day 7.98±0.13 7.84±0.40 7.56±0.3830 Day 8.64±0.17 8.52±0.30 8.31±0.0945 Day 8.40±0.15 8.38±0.17 8.36±0.1160 Day 8.26±0.19 8.38±0.17 8.37±0.2775 Day 8.26±0.11 8.43±0.14 8.37±0.2090 Day 8.49±0.11 8.70±0.29 8.64±0.08

105 Day 7.91±0.09 7.93±0.18 7.58±0.08120 Day 7.07±0.17 7.12±0.10 6.94±0.10

Each figure is an average of five values ± standard error

Figure 4.4: Average Dry matter intake (kg/day) at fortnight interval

4.3.2 Dry matter intake (kg) per 100 kg body weight:

The mean values of dry matter intake per 100 kg body weight in heifers of all the

three treatment groups have been presented in Table 4.10.

The statistical analysis of the data showed no significant difference of DMI per 100

kg body weight in heifers under different dietary treatment during the whole experimental

period.

31

Table 4.10:Average dry matter intake (kg) per 100 kg body weight of experimentalbuffalo heifers at monthly intervals

Days T1 T2 T3

0 2.46±0.13 2.32±0.09 2.17±0.2030 2.54±0.08 2.47±0.11 2.45±0.1160 2.30±0.06 2.30±0.06 2.36±0.1590 2.26±0.10 2.26±0.10 2.30±0.08

120 1.82±0.09 1.80±0.08 1.79±0.06Each figure is an average of five values ± standard error

4.4 Feed Efficiency

4.4.1 Feed conversion ratio (FCR)

Minimum requirement of nutrient per kg gain along with the optimum growth rate

resulting into economic rearing of animals is a desirable feature of livestock production. For

the estimation of FCR, feed intake per kg body weight gain was calculated. The average data

for dry matter required per kg gain in weight at monthly interval have been presented in Table

4.11. After thirty days FCR values were 17.61, 16.76 and 17.75 in T1, T2 and T3, respectively.

The result did not show any significant difference (P>0.05) in FCR values among the

different treatments throughout the experimental period. The FCR values at the end of

experiment, for T1, T2 and T3 treatment groups were 19.08, 18.83 and 18.17 respectively.

Table 4.11:Feed conversion ratio (DMI/kg body weight gain) of experimental buffaloheifers at monthly interval

Days TreatmentsT1 T2 T3

0-30 17.61±0.488 16.76±0.855 17.75±1.28730-60 14.32±0.368 13. 43±0.371 15.38±1.11960-90 13.42±0.654 12.47±0.263 13.69±0.218

90-120 19.08±1.325 18.83±2.027 18.17±0.728

4.4.2 Feed conversion efficiency (FCE)For the estimation of FCE, body weight gain (g) per kg of DM intake was calculated.

The average data for body weight gain (g) per kg of DM intake at monthly interval have beenpresented in Table 4.12.

After thirty days of feeding trial FCE values were 5.72, 6.06 and 5.79 in T1, T2 and T3,

respectively. The result did not show any significant (P>0.05) difference in FCE valuesamong the different treatment groups. The FCE values at the end of experiment, for T1, T2 andT3 treatments were 5.40, 5.63 and 5.58 respectively.

32

Table 4.12:Feed conversion efficiency (BW gain g/kg DMI) of experimental buffaloheifers at monthly interval

Days TreatmentsT1 T2 T3

0 – 30 5.72±0.14 6.06±0.29 5.79±0.4630 – 60 7.03±0.18 7.49±0.21 6.68±0.5160 – 90 7.55±0.34 7.99±0.22 7.33±0.1390 -120 5.40±0.40 5.63±0.68 5.58±0.22

Each figure is an average of five values ± standard error

4.5 Effect of supplementing chelated minerals on nutrient digestibility, nutrient intakeand nutritive value

4.5.1 Nutrient DigestibilityDigestibility coefficients of dry matter (DM), crude protein (CP), crude fiber (CF),

ether extract (EE), nitrogen free extract (NFE), neutral detergent fiber (NDF) and aciddetergent fiber (ADF) in heifers fed ration supplemented with different levels of chelatedmineral have been presented in Table 4.13.

The average digestibility coefficients of dry matter were 65.27, 66.98 and 64.81 percent in dietary treatment groups T1, T2 and T3, respectively. It was observed that dry matterdigestibility did not differ significantly. The digestibility coefficients of crude protein were67.25, 69.95 and 66.09 per cent for T1, T2 and T3 groups, respectively. The statistical analysisrevealed that crude protein digestibility did not differ significantly(P>0.05). The mean values ofCF digestibility coefficients were 58.57, 59.09 and 57.94 per cent in experimental heifers oftreatment groups T1, T2 and T3, respectively. Digestibility coefficients of NFE were 70.31, 72.12and 70.24 for T1, T2 and T3 groups, respectively. Statistical analysis suggested that digestibilitycoefficient of crude fiber and NFE did not differ significantly (P>0.05). Ether extractdigestibility coefficients were 69.10, 69.93 and 68.89 for T1, T2 and T3 groups. The ADFdigestibility coefficients were 48.12, 48.89 and 47.60 for T1, T2 and T3 respectively whereasNDF digestibility of T1,T2 and T3 groups were found to be 55.35, 56.41 and 53.33 respectively.Table 4.13:Effect of supplementing chelated minerals on nutrient digestibility of buffalo

heifers

ParticularsTreatments

T1 T2 T3

DM 65.27±2.77 66.98±1.20 64.81±4.90

CP 67.25±0.96 69.95±1.92 66.09±1.96

CF 58.57±1.27 59.09±0.86 57.94±1.14

EE 69.10±0.57 69.93±0.61 68.89±0.62

NDF 55.35±1.04 56.41 ±0.72 53.33 ±1.11

ADF 48.12±0.60 48.89±0.62 47.60±0.79

NFE 70.30±0.66 71.12±0.48 70.24±0.38Each figure is an average of five values ± standard error

33

4.5.2 Nutrient intake

The dry matter intake during digestion trial were 7.28, 7.35 and 7.00 kg/d for T1, T2

and T3 groups respectively (Table 4.14). Present results suggested that the dry matter intake

was almost similar in all group of animals whether T1 (control) or T2 (50% replacement of

inorganic mineral mixture with chelated mineral) or supplemented with 100% chelated

minerals (T3). The dry matter intake per 100 kg BW (kg/d) and per kg metabolic BW

(g/kgW0.75) were found statistically similar (P>0.05) in all dietary supplemental groups.

Crude protein intake (CPI) and digestible crude protein intake (DCPI) during

digestion trial were 758.28 g, 759.80 g, 756.12 g and 509.64 g , 531.33 g, 500.01 g in T1, T2

and T3 groups, respectively (Table 4.14). Similarly digestible crude protein intake was

statistically similar (P>0.05) in heifers of different dietary treatment groups.

Table 4.14:Effect of dietary supplementation of chelated minerals on plane of nutritionduring digestibility trial

ParticularsTreatments

T1 T2 T3

Dry matter intakeDMI (kg/d) 7.28±0.13 7.35±0.20 7.00±0.12DMI (Kg/100 kg BW/d) 1.88±0.09 1.85±0.05 1.83±0.09DMI(g/Kg W)0.75 83.34±2.96 82.63±1.65 81.14±3.16

Nutrient intakeCPI (g/d) 758.28±3.36 759.80±5.35 756.12±2.69CPI (g)/100 kg BW/d 196.00±8.69 191.78±6.34 196.08±6.87CPI (g)/kg W0.75 8.68±0.29 8.55±0.20 8.68±0.23DCPI (g) 509.64±6.25 531.33 ±14.28 500.01±13.64TDNI, Kg/d 5.17±0.19 5.28±0.11 5.11±0.16TDNI,(Kg/100 kg BW/d) 1.33±0.04 1.32±0.07 1.340±0.09TDNI, g/kg W0.75 59.11 ±1.09 58.70 ± 0.65 59.42±0.51

Nutritive valueCPI% 10.44±0.12 10.66±0.15 10.37±0.04TDN% 68.10±1.01 69.37±0.54 67.13±0.89Each figure is an average of five values ± standard error.

Crude protein intake (CPI) per 100 kg BW was 196.00, 191.78 and 196.08 g/d and

Crude protein intake (CPI) intake per kg metabolic BW 8.68, 8.55 and 8.68 g/d for T1, T2 and

T3 groups, respectively. The result showed that the Crude protein intake (CPI) per 100 kg

BW per day in all groups did not differ significantly (P>0.05) among different dietary

treatment groups.

Mean TDN intake (kg/day/animal) of buffalo heifers under each treatment during

digestion trial has been presented in Table 4.14. The results revealed that TDN intake was

34

5.17, 5.28 and 5.11 kg/d in T1, T2 and T3, respectively with numerically higher value for

treatment T2. TDN intake per 100 kg BW was 1.33, 1.32 ,1.34 kg/d and TDN intake per kg

metabolic body weight was 59.11 ,58.70 , 59.42 g/d for T1, T2 and T3 groups, respectively.

Both TDN intake per 100 kg BW per day and per kg metabolic BW per day did not differ

significantly (P>0.05) between treatment groups.

Nutritive value

Crude protein percentage was 10.44, 10.66 and 10.37 for T1, T2 and T3 groups,

respectively. Data indicated that supplementation of chelated minerals to buffalo heifers has

no effects on crude protein percentage and statistically similar (P>0.05) in all the three groups

of animals. Similarly TDN percent were 68.10, 69.37 and 10.37 for T1, T2 and T3 groups;

respectively; statistically TDN percent followed same trend as like CPI%.

4.6 Water intake

The average daily voluntary water intake at the starting of experiment was recorded

24.00, 23.30 and 22.40 lts. respectively. Average water intake has been presented in Table

4.15. The average daily water intake per day did not differ significantly (P>0.05) among

different dietary treatment groups at various fortnight interval periods of study.

Table 4.15:Average daily water intake by experimental Buffalo heifers at fortnightlyinterval during experiment trial

DaysTreatments

T1 T2 T3

0 24.00±3.33 23.30±2.53 22.40±1.0715 22.60±3.33 23.80±1.74 22.00±1.5130 20.40±0.67 21.40±0.51 20.80±0.8645 20.00±1.48 20.00±0.95 20.40±1.1260 24.60±1.81 25.60±1.75 26.00±1.4175 25.40±1.21 26.40±0.81 25.40±1.2190 35.70±0.77 36.20±1.02 36.40±0.51

105 38.40±1.72 39.20±1.07 36.60±2.02120 36.50±1.76 37.20±1.02 37.00±1.05

Each figure is an average of five values ± standard error

4.7. Serum mineral profile

4.7.1 Calcium concentration (mg/dl)

The Serum calcium concentration (mg/dl) of experimental animals at monthly

interval is presented in Table 4.16. The values during different months ranged between 8.28 to

10.33 mg/dl in control (T1), 8.43 to 10.55 mg/dl in T2 and 8.29 to 10.78 mg/dl in T3. The

calcium concentration in serum of experimental heifers did not vary significantly (P>0.05)

during the 120 days period of experiment.

35

Table 4.16:Serum calcium concentration (mg/dl) of experimental buffalo heifers atmonthly intervals

Days TreatmentsT1 T2 T3

0 8.28±0.15 8.43±0.16 8.29±0.1830 8.59±0.15 8.79±0.14 8.69±0.2160 9.15±0.12 9.38±0.12 9.26±0.1490 9.86±0.06 10.03±0.06 10.11±0.12

120 10.33±0.13 10.55±0.14 10.78±0.23Each figure is an average of five values ± standard error

4.7.2 Phosphorus concentration (mg/dl)

The Serum phosphorus concentration (mg/dl) of experimental animals at monthly

interval is presented in Table 4.17. The values ranged from 4.11 to 5.28 mg/dl in control (T1),

4.10 to 5.34 in T2 and 3.95 to 5.70 mg/dl in T3. Compared to day zero of experiment in all the

groups serum phosphorus concentration were increased as feeding trial advanced and it was

highest on 120th day of trial. Statistical analysis of data revealed that phosphorus serum

concentration did not differ significantly (P>0.05) in all treatment groups.

Table 4.17:Serum phosphorus concentration (mg/dl) of experimental buffalo heifers atmonthly interval

DaysTreatments

T1 T2 T3

0 4.11±0.09 4.10±0.15 3.95±0.13

30 4.29±0.11 4.40±0.11 4.22±0.09

60 4.63±0.05 4.71±0.07 4.64±0.06

90 4.96±0.12 5.06±0.08 5.13±0.11

120 5.28±0.17 5.34±0.14 5.70±0.19Each figure is an average of five values ± standard error

4.7.3 Copper concentration (mg/l)

The serum copper concentration (mg/l) of experimental animals at monthly interval

has been presented in table 4.18. The value ranges from 0.51 to 0.82 (mg/l) in control group

(T1), 0.50 to 0.85 (mg/l) in T2 and 0.51 to 0.92 (mg/l) in T3. The statistical analysis revealed

that on or after 30 days of experiment, serum copper concentration was significantly (P<0.05)

higher in treatment group T3 than T1 and T2. Compared to day zero of experiment in all the

groups serum copper concentration were increased as feeding trial advanced and it was

highest on 120th day of trial. The serum copper concentration values then followed similar

trend till end of the experiment.

36

Table 4.18:Serum copper concentration (mg/l) of experimental buffalo heifers atmonthly intervals

DaysTreatments

T1 T2 T3

0 0.51±0.01 0.50±0.01 0.51±0.0130 0.56a ±0.01 0.61b ±0.01 0.65c ±0.0260 0.63a±0.01 0.68b ±0.01 0.73c ±0.0190 0.73 a±0.02 0.78b±0.02 0.86c ±0.01

120 0.82a ±0.02 0.85b±0.02 0.92c±0.02Each figure is an average of five values ± standard errorThe mean values in a row with different superscripts differ significantly between the treatments (P<0.05)

4.7.4 Zinc concentration (mg/l)

The serum zinc concentration (mg/l) of experimental animals at monthly interval has

been presented in Table 4.19. The value ranges from 0.64 to 1.08 (mg/l) in control group (T1),

0.66 to 1.20 (mg/l) in T2 and 0.65 to 1.30 (mg/l) in T3. Data revealed that on or after 30 days

of experiment, serum zinc concentration was significantly (P<0.05) higher in treatment group

T3 than T1 and T2. Compared to day zero of experiment in all the groups serum zinc

concentration were increased as feeding trial advanced and it was highest on 120th day of trial.

The serum zinc concentration values then followed the similar trend during rest of the

experimental period.

Table 4.19:Serum zinc concentration (mg/l) of experimental buffalo heifers at monthlyintervals

DaysTreatments

T1 T2 T3

0 0.64±0.02 0.66±0.03 0.65±0.0230 0.72a±0.02 0.77a±0.01 0.84b±0.0260 0.82a ±0.01 0.86a ±0.01 0.99 b±0.0290 0.93 a ±0.01 0.98 a ±0.02 1.18 b ±0.02

120 1.08 a ±0.03 1.20 b ±0.02 1.30c ±0.02Each figure is an average of five values ± standard errorThe mean values in a row with different superscripts differ significantly between the treatments (P<0.05)

4.7.5 Manganese concentration (mg/l)The serum manganese concentration (mg/l) of experimental animals at monthly

interval has been presented in Table 4.20. The value ranges from 0.05 to 0.23 (mg/l) in

control group (T1), 0.06 to 0.24 (mg/l) in T2 and 0.06 to 0.25 (mg/l) in T3. Serum manganese

concentration on and after 60 days of experiment was significantly (P<0.05) higher in the

treatment group T3 than T1 but was comparable (P>0.05) with T2.. Compared to day zero of

experiment in all the groups serum manganese concentration were increased as feeding trial

advanced and it was highest on 120th day of trial. The serum manganese concentration values

then followed similar trend till the end of experiment.

37

Table 4.20:Serum manganese concentration (mg/l) of experimental buffalo heifers atmonthly intervals

DaysTreatments

T1 T2 T3

0 0.05±0.01 0.06±0.01 0.06±0.01

30 0.16±0.00 0.17±0.00 0.17±0.01

60 0.18a±0.01 0.20ab±0.01 0.21b±0.01

90 0.20a±0.01 0.22ab±0.01 0.23b±0.01

120 0.23a±0.01 0.24ab±0.01 0.25b±0.00Each figure is an average of five values ± standard errorThe mean values in a row with different superscripts differ significantly between the treatments (P<0.05)

4.7.6 Iron concentration (mg/l)

The serum iron concentration (mg/l) of experimental animals at monthly interval has

been presented in Table 4.21. The value ranges from 0.93 to 1.30 (mg/l) in control group, 0.94

to 1.34 (mg/l) in T2 and 0.92 to 1.42 (mg/l) in T3. Serum iron concentration on and after 30

days of experiment was significantly (P<0.05) higher in the treatment group T3 than T1 and

T2. The serum iron concentration values then followed similar trend till the end of experiment.

Table 4.21:Serum iron concentration (mg/l) of experimental buffalo calves at monthlyintervals

DaysTreatments

T1 T2 T3

0 0.93±0.01 0.94±0.01 0.92±0.0130 1.08a±0.02 1.14b±0.01 1.19c±0.0160 1.17 a ±0.01 1.21 b ±0.01 1.25 c ±0.0190 1.22 a ±0.01 1.27 b ±0.01 1.34 c ±0.01

120 1.30 a ±0.01 1.34 b ± 0.01 1.42 c ±0.00Each figure is an average of five values ± standard error.The mean values in a row with different superscripts differ significantly between the treatments (P<0.05)

4.7.7 Cobalt concentration (mg/l)

The serum cobalt concentration (mg/l) of experimental animals at monthly interval

has been presented in Table 4.22. The value ranges from 0.003 to 0.005 (mg/l) in control

group, 0.003 to 0.005 (mg/l) in T2 and 0.003 to 0.007 (mg/l) in T3. Compared to day zero of

experiment in all the groups serum cobalt concentration were increased as feeding trial

advanced and it was highest on 120th day of trial. The results of the study revealed that serum

cobalt concentration throughout the experiment is significantly (P<0.05) higher in the

treatment group T3 than T1 and T2 but T1 and T2 did not differ significantly (P>0.05).

38

Table 4.22:Serum cobalt concentration (mg/l) of experimental buffalo heifers atmonthly intervals

Days TreatmentsT1 T2 T3

0 0.003±0.000 0.003±0.001 0.003±0.00030 0.004±0.000 0.003±0.000 0.004±0.00060 0.004±0.000 0.004±0.000 0.005±0.00090 0.005±0.000 0.005±0.000 0.006±0.000

120 0.005a±0.000 0.005a±0.000 0.007b±0.000Each figure is an average of five values ± standard errorThe mean values in a row with different superscripts differ significantly between the treatments (P<0.05)

4.8 Reproductive Performance

Age at first heat

Only four heifers out of 15 from all the treatment groups came in heat during the

whole experimental period. One heifer from T1, two heifers from T2 and one heifer from T3

came in heat. Age at first heat was 916 days for T1 heifer, 809 days and 826 days for T2 heifer,

and 883 days for T3 heifer. Days taken to come in heat after start of the feeding trial was 105

days (n=1) for T1, 85.5 days (n=2) for T2 and 130 days (n=1) for T3 group.

Age at first conception

Three heifers got conceived out of four came in heat. Age at first conception was 916

days for T1 heifer, 809 days for T2 heifer, and 883 days for T3 heifer. All the three animals

from T1, T2 and T3 groups conceived in their 1st heat and one animal in T2 group which was

in heat failed to conceive during the experimental period of 120 days.

Number of A.I per conception

Number of A.I required per conception for all the three animals were one for each

animal per treatment group.

4.9 Cost of feeding

The cost of feeding of various groups of buffalo heifers in terms of total cost and cost

per kg body weight gain under different treatments was calculated from the records of feed

and fodder consumption, considering the actual price of feed and fodder and total gain in

body weight during the whole experimental period and has been presented in Table 4.23.

The total cost of feeding for a period of 120 days was Rs. 7,302, 7,809 and 8,316 in

treatments T1, T2 and T3, respectively. The corresponding values for cost per kg body weight

gain were Rs. 165.59, 163.65 and 184.56 in treatments T1, T2 and T3, respectively, which is

lowest in T2 treatment i.e. it was most economical when expressed in term of cost per unit

weight gain.

39

Table 4.23:Total cost of feeding (Rs.) and cost of feeding per kg body weight gain ofbuffalo heifers under different treatments for experimental period

Variables T1 T2 T3

Concentrate cost (Rs.) 7,302 7,809 8,316Green Cost (Rs.) 2,250 2,250 2,250Straw Cost (Rs.) 840 840 840Total Cost (Rs.) 10,392 10,899 11,406Total body weight gain (kg) 62.8 66.6 61.8Cost/kg Body weight (Rs.) 165.50 163.65 184.56

40

CCHHAAPPTTEERR--VV

DDIISSCCUUSSSSIIOONN

The discussion on effect of chelated minerals supplementation on growth and

reproductive performance of Murrah buffalo heifers in terms of body weight, body

measurements, dry matter intake, serum minerals profile, nutrient digestibility, feed conversion

ratio, reproductive performance and cost of feeding is presented in following section.

5.1 Growth parameters

5.1.1 Body weight gain

The body weight gain (kg) of experimental buffalo heifers showed increasing trend in

all the three treatments upto day 90, from day 90 to 120 though the heifers gained body

weight but leisurely as compared to first three months. (Table 4.1). The average body weights

(kg) and metabolic body weights (kg W0.75) of all the experimental animals was observed to

be statistically non-significant (P > 0.05) among various treatment groups throughout the

experiment (Table 4.1 and 4.2). However, the body weight gain during period 90 to 120 days

was less, the possible reason could be due to the change of season from winter to summer.

The result of present investigation corroborated with the findings of Muehlenbein et al

(2008) who found no differences (P > 0.10) among control, inorganic and organic

supplemented treatment groups in cow BW or body condition scores at various times

throughout the study, similarly calf birth weights did not differ significantly (P > 0.10)

among treatment groups. Similar findings were observed by Gengelbach et al. (1994) who

found no significant difference in the BW change for first-calf heifers exposed to mineral

treatments. Ahola et al ( 2004) also found no differences in BW and body condition score of

multiparous beef cows fed control (no supplemental Cu, Zn, Mn), organic (50% organic and

50% inorganic) and inorganic(100% inorganic) . It is possible that different trace minerals

enhance growth of heifers by stimulating activities of enzymes involved in nutrient

utilization. Chelates have been of less concern in the ruminant animals, probably due to

rumen microbes and their involvement in digestion (Mohanta et al, 2014)

5.1.2 Average Daily weight gain

Average daily weight gain (g/day) by buffalo heifers under different treatments has

been presented in Table 4.3. The results showed that the average daily weight gain did not

differ significantly (P>0.05) between different dietary regimen of heifers throughout the

experimental period.

The mean values of daily body weight gain during the whole period of experiment

were 523.33, 555.00 and 515.00 g/d, in T1, T2 and T3 treatment groups, respectively. The

41

result of study indicated that average body weight gain did not differ significantly (P>0.05)

due to partial or complete supplementation of chelated minerals as compared to inorganic

mineral in the three treatment groups. The findings of present study are in agreement of

earlier study who reported that the source of mineral whether inorganic or organic

supplementation did not affect the cow body weight or body condition score (Olson et

al.,1999; Muehlenbein et al.,2001; Ahola et al.,2004). Mondal et al (2008) reported no

significant (P>0.05) difference in body weight gain and average daily gain in male crossbred

calves on supplementation of organic or inorganic minerals as per NRC(2000) requirement.

Similarly, Ahola (2005) also reported that neither ADG nor DMI were affected by either trace

mineral supplementation or source throughout the growing phase in calves.

However, in contrary to the present findings, Hong et al. (2002) observed an increase

of 5.5 – 11.4% in daily weight gain of beef steer when basal diet was supplemented with

chelated minerals as compared to inorganic minerals. Similarly Bhanderi et al (2010)

concluded that supplementation of MBOTMs at NRC requirement in male calves can improve

the body weight gain than that of inorganic trace minerals.

5.2 Body Measurement

The present results revealed that there was improvement in body length, height, heart

girth and abdominal girth of the buffalo heifers in all the three treatment groups during

progressive period of growth. The body parameters of buffalo heifers did not differ

significantly (P>0.05) due to supplementation of 50% or 100% organic minerals in place of

inorganic at any stage of experiment. Since the body weight did not differ significantly

(P>0.05) among treatment groups, so also body dimensions were comparable among

treatment groups. Non-significant difference in growth performance, body measurements due

to chelated minerals supplementation was observed in the current experiment, although this

has not been well addressed in the literature.

5.3 Dry matter intake (DMI)

5.3.1 Dry matter intake per day (DMI/d)

Mean daily dry matter intakes (kg/d) during the experimental period are given in

Table 4.9 and fig. 4.4. The mean values of dry matter intake increased during the progressive

period in heifers under all the dietary treatment groups except in the last month of experiment

because of change in season from winter to summer. The dry matter intake during the whole

experimental period did not differ significantly (P>0.05) among treatment groups. The

findings of the study are in agreement with earlier reports of Lamb et al.(2008) who reported

that daily dry matter intake of hay was similar among treatment groups. Similar findings

were also reported by Mondal et al.(2008), Smith et al.(1967). However, in contrary to the

present findings Mallaki et al. (2015) found that nutrient digestibility and dry matter intake

was higher in the lambs fed with the diet supplemented with chelated mineral.

42

5.4 Feed conversion Ratio

The results of the study revealed that feed conversion ratio as well as feed conversion

efficiency of heifers were improved with the progress of experiment except at the end of

experiment due to seasonal changes (winter to summer). FCR and FCE did not differ

significantly (P> 0.05) among T1, T2 and T3 treatment groups during various intervals of

experiment. The findings are in agreement with the study of Ahola et al.(2005) who reported

no differences in gain to feed ratio between control and supplemented cattle (P=0.70) or

between ORG and ING cattle (P=0.47). Nockels (1991) compared copper proteinate and

copper sulphate in steers and found that weight gain and feed efficiency were not affected by

copper source. However, in contrary to the present findings Mallaki et al. (2015) reported

improved FCR in lambs fed chelated minerals. Similarly Dey and Garg (2004) observed

significantly improved feed efficiency in weaned albino rats given organic Zn compared to

unsupplemented and ZnSO4 supplemented groups.

5.5 Nutrient digestibility (%) and Nutrient Intake

Statistical analysis suggested that digestibility coefficient of dry matter, crude protein,

ether extract, crude fiber, NFE, ADF and NDF did not differ significantly (P>0.05) among all

the three treatment groups during the study period. The results of the study deduced that

nutrients intake by experimental heifers in terms of crude protein(CP), digestible crude

protein (DCP) and total digestible nutrients intake (TDN) did not differ significantly

(P>0.05) during the study.

The present finding also corroborated with the finding of Mondal et al. (2008) who

concluded that digestibility of DM, CP, CF showed no significant difference (P>0.05) among

the minerals supplemented groups. Garg et.al.,(2008) reported that intake of dry matter (DM),

organic matter (OM), crude protein (CP), digestible CP and total digestible nutrients and

digestibility of DM, OM, CP, ether extract, neutral detergent fibre and hemicellulose were

comparable (P>0.05) among the three groups.

Our findings are not in agreement with the results of Zhang et al. (2013) who

concluded that 0.1% chelate Cu and Zn supplementation improved nutrient digestibility in

weanling pigs. Also the results of present study is not supported by Bhoot et al 1981 who

suggested that due to supplementation of chelated minerals in ruminant results in better

fermentability and utilization of organic matter.

5.6 Water Intake

Water intake did not differ significantly (P>0.05) between the treatment groups

during various stages of experimental trial.

Nutrient Intake

The results of the present study deduced that nutrients intake by experimental heifers

in terms of crude protein (CP) , digestible crude protein (DCP) and total digestible nutrients

43

intake (TDN) did not differ significantly (P>0.05). The present finding also corroborated with

the finding of Garg et al.(2008) the intake of dry matter (DM), organic matter (OM), crude

protein (CP), digestible CP and total digestible nutrients and digestibility of DM, OM, CP,

ether extract, neutral detergent fibre and hemicellulose were comparable (P>0.05) among the

three treatment groups.

5.7 Serum mineral concentration

The present study revealed that serum concentrations of Ca and P in the treatment

groups did not differ significantly (P>0.05) throughout the study period although, there was

an increasing trend of serum mineral concentration with the supplementation of minerals in

the diet of all the treatment groups .

It was further revealed that initially there was no significant effect (P>0.05) of

supplemental trace minerals on serum concentration of Cu, Fe, Mn ,Co and Zn. However, day

30 onwards serum copper, zinc and iron increased significantly (P<0.05) in the T3 groups fed

100% chelated mineral mixture. After day 60, T3 exhibited high (P<0.05) serum

concentrations of Manganese, as compared to control group T1 fed inorganic mineral mixture

and 120 day onwards cobalt concentration in serum became significantly (p<0.05) higher in

treatment group T3 than T1 group. Present study revealed serum minerals concentration of

heifers increased linearly with the increase of days due to mineral supplementation and the

effect was more pronounced in group supplemented with 100% chelated minerals that was in

treatment group T3 than T1 . T2 group supplemented with 50% organic and 50% inorganic

minerals showed significantly (P<0.05) higher serum minerals concentration as compared to

T1 but significantly (P>0.05) lower when compared with T3 group. Similar observation was

recorded by Bhanderi et al. (2010) who reported high (P<0.05) serum concentrations of Cu,

Zn and Mn in male calves fed MBOTMs as compared to control group fed inorganic

minerals. Mondal et al. (2008) also found serum mineral concentration of zinc, copper,

manganese and iron increased linearly (P<0.05) with the increase of days due to mineral

supplementation particularly in organic mineral (T3 and T4) supplemented group.

In another study Engle et al. (2000) who reported higher (P<0.05) serum Cu

concentration on 84 days due to CuSO4 and Cu-lysine supplementation. Kegley and Spears

(1994) also found enhanced serum Cu level from both sources of CuSO4 and Cu-lysine like the

present findings. However in contrary to the present findings Tambe et al. (1998) did not find

any improved trace mineral profile on chelated and non-chelated mineral supplementation in

calves.

Higher serum concentration of trace mineral with organic mineral supplementation

probably due to higher absorption and retention in tissue level (Boland, 2003). On the other

hand, the inorganic forms of trace minerals make the intestinal pH more alkaline leading to

precipitation due to formation of inorganic chelates in the gut. These may be the possible

44

reason for the higher concentration of macro and micro elements in the serum of the calves

supplemented with the organic form of minerals (Duz et al., 1996).

5.8 Reproductive parameters

The results of the present investigation revealed that reproductive performance in terms of age

at first heat, age at first conception and A.I per conception were not affected due to 100%

supplementation of chelated minerals in place of inorganic. However,the heifers fed 50%

chelated minerals replacing inorganic attained early pubertal age as compared to control

group. Similar findings reported by Yasui et al (2014) that organic Mn,Zn,Cu

supplementation in cows did not improved uterine health.

In contrary to our findings Stanton et al (2000) found that feeding beef cows with organic

mineral resulted in increased pregnancy rates during the subsequent breeding season.

Similarly, Manspeaker et al. (1987) fed 40 first-calf Holstein heifers a control diet or the

control diet plus an amino acid chelated mineral supplement. Mineral content of the control

diet was not reported. The amino acid chelated supplement supplied additional iron,

manganese, copper and zinc in addition to potassium and magnesium. The study was

conducted from approximately 30 days prepartum until heifers were confirmed pregnant by

rectal palpation. Incidence of periglandular fibrosis (a pathologic response in which

endometrial tissue does not regenerate properly after parturition) was significantly lower (10

vs. 58%)in heifers given chelated minerals. Although not statistically significant, ovarian

activity tended to be higher and embryonic mortality lower for heifers fed the chelated

mineral supplement.

5.9 Cost of feeding

The total cost of feeding for a period of 120 days was Rs.10,392 ,Rs.10,899 and Rs.

11,406 in treatments T1, T2 and T3, respectively has been presented in Table 4.19.

The corresponding values per kg body weight gain were Rs. 165.50, 163.65 and

184.56 in treatments T1, T2 and T3, respectively, which is lowest in T2 treatment i.e. it was

most economical when expressed in term of cost per unit weight gain. In the present

investigations the cost of feeding per unit live weight gain was lower in T2 treatment

supplemented with 50% inorganic and 50% chelated minerals. Though treatment T2 seems to

be better than other two groups, but statistically not significant.

45

CCHHAAPPTTEERR--VVII

SSUUMMMMAARRYY AANNDD CCOONNCCLLUUSSIIOONN

The present study was conducted from January to April month on fifteen apparently

healthy buffalo heifers, between 22 to 28 months of age. The animals were maintained at

Buffalo Farm, Department of Livestock Production Management, College of Veterinary

Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar.

The experiment was conducted for a period of 120 days.

The fifteen buffalo heifers were randomly distributed into three treatment groups each

having five buffalo heifers in such a manner that average body weight and age of each

experimental group was statistically similar. The heifers of dietary treatment T1 (control

group) were fed with seasonal green fodder, wheat straw and conventional concentrate

mixture having 2% inorganic mineral mixture to meet out the nutrients requirements as per

feeding standards (Ranjhan, 1998) . While heifers under treatment groups T2 and T3 were fed

as like T1 but with concentrate mixture having 1% inorganic + 1% chelated minerals

(replacing 50% inorganic mineral mixture with chelated minerals in concentrate mixture of

control group) and 2% chelated minerals + no inorganic mineral mixture (replacing 100%

inorganic mineral mixture with chelated minerals in concentrate mixture of control group),

respectively. The amount of concentrate mixture was given to each group in such a way that

the experimental ration remains iso-proteinaceous. The quantity of different feeds given to

each group was adjusted at fortnightly intervals so that the overall DCP requirements of

buffalo heifers were met according to the change in body weight. Animals were given ad-lib

fresh water throughout the experimental period.

All the experimental animals were weighed before start of the experiment, and

thereafter at fortnightly interval using standard platform weighing balance. The weights were

recorded in the morning for two consecutive days, before feeding and watering of the animals.

The body weights were utilized to calculate metabolic body weight and growth rate or weight

gain per day at fortnightly intervals using standard formula. Daily feed intake during the

experimental period by individual buffalo heifers was determined on the basis of feeds and

fodder offered and weigh back. On the basis of feed and fodder consumption, dry matter

consumed per day, per 100 kg body weight and per kg of metabolic body weight by the

animals at fortnightly intervals were estimated. The DM consumed and body weight gain

were used to calculate the FCR i.e. the amount of dry matter intake in kilogram (Kg) required

for per Kg weight gain and FCE i.e. body weight gain (g) per kg of DM intake at fortnightly

intervals by animals during the experimental period for each treatment.

46

A digestion trial of 7 days was conducted at the end of experiment to know the effect

of treatments on digestibility of feed and fodder.

Blood samples were collected at the beginning of the experiments and thereafter, at

monthly interval before feeding and watering of the experimental animals. About ten milliliter

(ml) of blood was collected by jugular vein-puncture into a set of sterile plastic tubes without

anti-coagulant for serum minerals tests. Blood collected without anti-coagulant were

centrifuged at 2500 to 3000 rpm for 25 minutes and plasma was separated and used for

estimation of biochemical parameters in serum viz. plasma calcium (mg/dl) and phosphorus

(mg/dl) using kits procured from M/S Transasia Biomedical Limited with fully automated

Random Access Clinical Chemistry Analyzer (EM 200TM Erba Mannheim – Germany). The

serum samples were digested in digestion mixture consisting of nitric acid and perchloric acid

for estimation of minerals copper, zinc, manganese, iron and cobalt using by atomic

absorption spectrometer- model Pinaacle 900T, S/N PTAS13050201 of PerkinElmer

Company.

Reproductive parameters viz. Age at 1st heat, age at 1st conception and A.I per

conception were recorded during the experimental period to know the effect of chelated

minerals on the above indices.

The total cost of feeding by each buffalo heifer during the whole period was

calculated. The information about the prices of all feed ingredient including price of greens

prevailing at the time of purchase were obtained from the Department of Animal Nutrition of

the University. Cost of feeding per kg gain was also computed for different treatment groups.

Body weight and average metabolic body weight of experimental buffalo heifers at

fortnightly intervals under different treatment groups showed no significant (P>0.05)

difference during 120 days of experimental period. The average daily body weight gain

during the whole experimental period was 523.3, 555 and 515g/d in T1, T2 and T3 groups,

respectively The results of the study revealed that total weight gain and gain per day did not

differ significantly (P>0.05) among different dietary treatment groups throughout the

experimental trial.

Average increase in body length and height, total gain in heart girth and abdominal

girth were comparable (P>0.05) in different treatment groups.

Dry matter intake per 100 kg body weight and per kg metabolic body weight of

heifers at various intervals under different dietary treatments did not differ significantly

(P>0.05). The digestibility coefficients of crude fiber and NFE; and nutrients intake in terms

of CP, DCP and TDN did not differ significantly (P>0.05) among three treatment groups.

The feed conversion ratio as well as feed conversion efficiency of heifers were also

non- significant (P>0.05).

47

It was observed that the serum calcium, phosphorus concentration did not differ

significantly (P>0.05) among the treatment groups but copper, zinc, manganese, iron, cobalt

concentrations were significantly higher (P<0.05) in group T3 as compared to T1 and T2 .

The increased level of Cu, Zn, Mn, Co and Fe in the serum of the buffalo heifers

supplemented with chelated minerals might be due to the higher bio-availability of these

elements from chelated as compared to inorganic mineral mixture. Unlike the inorganic

form, chelated minerals do not participate in interaction and antagonism between the

minerals, leading to greater absorption from the gastro-intestinal tract.

It can be concluded that reproductive performance in terms of age at first heat, age at first

conception and conception were not affected due to 100% supplementation of chelated

minerals in place of inorganic. However, the heifers fed 50% chelated minerals replacing

inorganic attained early pubertal age as compared to control group.

The results of the present study revealed that mean value for feed cost per unit body

weight gain was marginally lower Rs. 163.65 for T2 as compared to T1 165.50 but the same

value for T3 was quite high owing to the high cost of chelated minerals.

Although the serum micro minerals concentration was found to be significantly

higher in T2 group (50% chelated and 50% inorganic minerals) and T3 group (100% chelated

minerals) but it is not able to justify the additional cost of chelated minerals. However,

numerically T2 group showed higher values for body weight, average daily weight gain, body

measurements and was also cost effective thus, more economical to the dairy farmers but

statistically non-significant. Thus, increasing the organic minerals in the diet of animals

simply increases the cost, which may not be required, as the inorganic mineral is meeting out

the need of animal. But 50% replacement of organic minerals with organic one can be

practised for marginal improvement in cost of production.

In our experiment there was no deficiency of any of the minerals in any of the

treatment group diet throughout the trial. Majority part of the present experiment was

conducted in the winter season, when sufficient green fodder was available to meet out the

bodily demand for minerals. The serum mineral concentration of the treatment groups for all

the animals were in normal level, and that was sufficient to meet out the growth requirement

of animals. The serum micro mineral concentration though found to be higher for organic

mineral fed group but was within normal range, which could not affect the growth, nutrient

utilization like inorganic fed group.

On the basis of the results obtained in the present study it may be inferred that

supplementation of different levels of chelated minerals (50% or 100%) in ration of Murrah

buffalo heifers of 22 to 28 months age during january to april month of experimental period

has no favorable effect on growth performance, body measurements, nutrient digestibility,

nutrient intake and nutritive value of diet along with feed conversion ratio and feed

48

conversion efficiency and reproductive performance.

FURTHER RESEARCH

• Define the optimal level of Chelated minerals added to the diet.

• Better define conditions where performance responses may be seen.

• More closely define the mode of action of metal proteinates to improve performance

in ruminant animals.

Plate 1: Buffalo heifers kept chained and fed individually

Plate 2: Body length measurement of buffalo heifer

Plate 1: Buffalo heifers kept chained and fed individually

Plate 2: Body length measurement of buffalo heifer

Plate 1: Buffalo heifers kept chained and fed individually

Plate 2: Body length measurement of buffalo heifer

Plate 3 : Heart girth measurement of buffalo heifer

Plate 4: Water being offered to buffalo heifers in measurable bucket separately

Plate 3 : Heart girth measurement of buffalo heifer

Plate 4: Water being offered to buffalo heifers in measurable bucket separately

Plate 3 : Heart girth measurement of buffalo heifer

Plate 4: Water being offered to buffalo heifers in measurable bucket separately

Plate 5: Blood collection from buffalo heifer

Plate 6: Weighing concentrate mixture Plate 7: Weighing green roughage

Plate 8: Weighing faecal matter during digestion trial of 7 days

Plate 9: Automated Random Access Clinical Chemistry Analyzer

i

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ABSTRACT

Title of Thesis : Effect of Chelated Minerals Supplementation onGrowth and Reproductive Performance of MurrahBuffalo Heifers

Full Name of the Degree Holder : Anuradha VermaAdmission Number : 2015V13MTitle of the Degree : Master of Veterinary Science in Livestock Production

ManagementName of Discipline : Livestock Production ManagementName and Address of Major Advisor : Dr.S.K.Chikkara,

Principal Scientist,Deptt. of, Livestock Production ManagementLUVAS, Hisar, Haryana (India).125004

Degree Awarding University LUVAS, HisarYear of Award of Degree : 2017Major Subject : Livestock Production ManagementTotal Number of Pages in Thesis : 48 + viiNumber of Words in Abstract : 337Key words: Chelated minerals, buffalo heifers, growth and reproductive performance.

The objective of the present study was to analyze the effect of chelated minerals supplementation on

growth and reproductive performance of Murrah buffalo heifers. Fifteen apparently healthy heifers (22 to 28

months age) were selected randomly and stratified into three treatment groups each having five animals basing on

their average body weight and age. In treatment T1 (control), animals were fed with seasonal green fodder, wheat

straw and conventional concentrate mixture having 2% mineral mixture while for treatment T2 and T3, animals

were fed similar to T1 but, inorganic mineral in concentrate mixture was replaced with 50% and 100% chelated

mineral, respectively. Feeding trial was conducted for a period of 120 days. Body weight of experimental buffalo

heifers at fortnightly intervals under different treatment showed no significant difference (P>0.05) throughout the

experimental period. Metabolic body weight, average daily gain, body measurements (body length, height, heart

girth and abdominal girth) were also followed the same trend. However, each parameter was marginally higher for

T2 group but statistically remained non-significant. Macro minerals (Calcium and Phosphorus) profile in blood

serum was non-significant among the treatment groups. However, serum micro minerals ( Cu,Zn,Mn,Fe and Co)

was found to be significantly (P<0.05) higher for T2 and T3 groups as compared to T1. Digestibility coefficients of

dry matter (DM), crude protein (CP), crude fiber (CF), ether extract (EE), nitrogen free extract (NFE), neutral

detergent fiber (NDF) and acid detergent fiber (ADF) were also non-significant (P>0.05) among treatment groups.

Dry matter intake, crude protein intake, digestible crude protein intake and total digestible nutrient intake was also

comparable (P>0.05) among treatment groups. Reproductive performance in terms of age at first heat, age at first

conception and conception were not affected due to 100% supplementation of chelated minerals in place of

inorganic. However, the heifers fed 50% chelated minerals replacing inorganic attained early pubertal age as

compared to control group. Thus, it can be concluded that replacement of inorganic mineral with chelated mineral

has no beneficial effect on growth and reproductive performance of Murrah buffalo heifers.

MAJOR ADVISOR STUDENT

HEAD OF DEPARTMENT

CURRICULUM VITAE

1. Name : Anuradha Verma

2. Date of birth : 17-11-1991

3. Place of birth : Narnaul

4. Mother’s name : Banarasi Verma

5. Father’s name : Amar Singh Verma

6. Permanent address : V.P.O.-Dharson,

Teh.-Narnaul,

Distt.-Mohindergarh, Haryana, 123001

7. Telephone : –

8. Mobile : 9996811657

9. E-Mail : anuraworld2020@gmail.com10. Academic qualification:

Degree/Examination

College/School

University/Board

Year ofPassing

Percentageof marks

Subjects

M.V.Sc.(LivestockProduction

Management)

College of Vety. &Animal Science

LUVAS 2017 80.5 Major subject:LPMMinor subject: ANN

Supportivesubject:AGB

B.V.Sc. & A.H. College of Vety. &Animal Science

LUVAS 2015 62.8 As per VCI curriculum

12th Saraswati Sr. Sec.School,

Nasibpur,Narnaul

CBSE 2009 72.2 Physics, Chemistry,Biology, English,

Physical Education10th K.V No.1

AFS,Kalaikunda,Midnapure,West Bengal

CBSE 2007 82.2 Mathematics, Science,Social Science,English, Hindi

11. List of publications (Related To Thesis work):Verma, A., Sahu, S., Sihag, S., Chikkara, S. K., Archana. (2017). Effect of chelated minerals

supplementation on growth performance of Murrah buffalo heifers. Submitted to HaryanaVeterinarian.

12. Co-curricular activities :

NCC – ‘B’ and ‘C’ Certificates 2011,2012 Gold Medalist in Inter College Youth festival 2012, 2013, 2016 (On the spot Rangoli and

Painting Competition) CCS HAU Hisar. Gold Medalist in National Science Day 2017 (On the spot Poster Making) CCS HAU

Hisar.

UNDERTAKING OF COPY RIGHT

“I, Anuradha Verma, Admission No. 2015V13M undertake that I give copy right

to the LLRUVAS, Hisar of my thesis entitled “Effect of Chelated Minerals

Supplementation on Growth and Reproductive Performance of Murrah Buffalo

Heifers”. I also undertake that patent, if any, arising out of the research work conducted

during this programme shall be filed by me only with due permission of the competent

authority of LUVAS, Hisar.

Signature of the student(ANURADHA VERMA)