EVALUATION OF OXIDATIVE STRESS IN TRANSITION

COWS AND POST PARTUM ENDOMETRIAL CYTOLOGY

By
G. RAVI BABU, B.V.Sc. & A.H.

Thesis Submitted to the

SRI VENKATESWARA VETERINARY UNIVERSITY
In partial fulfilment of the requirements
For the award of the degree of

MASTER OF VETERINARY SCIENCE

In the faculty of veterinary science

DEPARTMENT OF ANIMAL REPRODUCTION, GYNAECOLOGY

AND OBSTETRICS
COLLEGE OF VETERINARY SCIENCE
SRI VENKATESWARA VETERINARY UNIVERSITY
TIRUPATI – 517 502

MARCH, 2013
 CERTIFICATE

Dr. G. RAVI BABU has satisfactorily prosecuted the course of research and

that the thesis entitled “EVALUATION OF OXIDATIVE STRESS IN TRANSITION

COWS AND POST PARTUM ENDOMETRIAL CYTOLOGY” submitted is the result of

original research work and is of sufficiently high standard to warrant its presentation

to the examination. I also certify that the thesis or part thereof has not been previously

submitted by him for a degree of any university.

Date :

Place : Tirupati Chairman of Advisory Committee

(K. MOULI KRISHNA)
 CERTIFICATE
This is to certify that the thesis entitled “EVALUATION OF OXIDATIVE
STRESS IN TRANSITION COWS AND POST PARTUM ENDOMETRIAL CYTOLOGY”
submitted in partial fulfilment of the requirements for the degree of Master of
Veterinary Science of the Sri Venkateswara Veterinary University, Tirupati, is a
record of the bonafide research work carried out by Dr. G. Ravi Babu under my
guidance and supervision. The subject of the thesis has been approved by the
Student’s Advisory Committee.
No part of the thesis has been submitted by the student for any other degree or
diploma. The published part has been fully acknowledged. All assistance and help
received during the course of the investigations have been duly acknowledged by the
author of the thesis.
Chairman of the Advisory
Committee

Thesis approved by the Student’s Advisory Committee

CHAIRMAN : Dr. K. Mouli Krishna _______________________

Professor & Head
Department of Veterinary
Gynaecology and Obstetrics
College of Veterinary Science
Tirupati – 517 502.

MEMBER : Dr. K. Veerabramaiah ______________________

Professor
Department of Veterinary
Gynaecology and Obstetrics
College of Veterinary Science
Tirupati – 517 502.

MEMBER : Dr. K. Padmaja _______________________

Associate Professor
Department of Veterinary Biochemistry
College of Veterinary Science
Tirupati – 517 502.
 LIST OF CONTENTS

Chapter Page
Title
No. No.

I Introduction

II Review of literature

III Materials and methods

IV Results

V Discussion

VI Summary

Literature cited
 LIST OF TABLES

Table Page
Title
No. No.

1 Profiles of plasma MDA (Mean  SE) in

peripartum crossbred cows (µ mol/L).

2 Analysis of variance for MDA.

3 Results of student’s t-test for MDA.

4 Profiles of plasma Vit.E (Mean  SE) in

peripartum crossbred cows (mg/L).

5 Analysis of variance for Vit.E.

6 Results of student’s t-test for Vit.E.

7 Profiles of plasma GSH (Mean  SE) in

peripartum crossbred cows (µ mol/L).

8 Analysis of variance for GSH.

9 Results of student’s t-test for GSH.

10 Catalase activity in erythrocyte hemolysate (Mean

 SE) in peripartum crossbred cows (KU/g Hb).

11 Analysis of variance for Catalase.

12 Results of student’s t-test for Catalase.

13 SOD activity in erythrocyte hemolysate (Mean 

SE) in peripartum crossbred cows (KU/mg
Hb/min).

14 Analysis of variance for SOD.

15 Results of student’s t-test for SOD.

16 Profiles of plasma Glucose (Mean  SE) in

peripartum crossbred cows (mg/dl).

17 Analysis of variance for plasma glucose.

18 Results of student’s t-test for plasma glucose.

19 Profiles of plasma Total proteins (Mean  SE) in

peripartum crossbred cows (g/dl).

20 Analysis of variance for plasma Total proteins.

21 Results of student’s t-test for plasma Total proteins.

22 Profiles of plasma Progesterone (Mean  SE) in

peripartum crossbred cows (ng/ml).

23 Analysis of variance for plasma progesterone.

24 Results of student’s t-test for plasma Progesterone

concentration.
 LIST OF ILLUSTRATIONS

Figure Page
Title
No. No.

1 Cytobrush catheter.

2 Profiles of MDA (µmol/L).

3 Profiles of Vit.E (mg/L).

4 Profiles of GSH (µ mol/L).

5 Profiles of Progesterone concentration (ng/ml).

6 Neutrophils in endometrial cytological smears.

Leishman`s stain x 1000

7 Comparison of endometrial neutrophils counts between

day 25 and CUI.
 ACKNOWLEDGMENTS

I wish to record my sincere and heartfelt regards to Dr.K.Mouli Krishna,

Professor and Head, Major advisor and Chairman, Dr.K.Veerbramaiah, Professor
and Member student`s advisory committee, Department of Veterinary Gynaecology
and Obstetrics, and Dr.K.padmaja Associate Professor , Department of Veterinary
Biochemistry CVSc, Tirupati for their inspiring and affectionate guidance, during the
course of my work.
I owe my thankfulness to Dr.K.Venugopal Naidu formally Professor and
Head and presently controller of examinations SVVU, Tirupati, and Dr.Ch.Srilatha,
Professor and University Head, Department of Pathology, for providing technical
assistance and encouragement and Dr.Vijayalakshmi Assistant Professor,
Department of Veterinary Microbiology. CVSc, Tirupati for technical assistance in
determination of progesterone.
I owe my thankfulness to Dr.Ch.AppaRao professor and Head, Department of
Biochemistry, S.V.University for his valuable suggestions rendered in Biochemical
analyses.
I avail this opportunity to express my hearty thanks to Dr.G.Suman Kumar,
Dr.K.Anusha,Dr.D.Srividya,Dr.K.Jyothi,Dr.sunnyPraveen,Dr.Prithvi, Dr.Ch.Vredd,
Dr.Subba reddy, Dr Lakshmi, Amar for their immense affection and moral support.
I affectionately acknowledge the help and cooperation received from non
teaching staff Smt.V.Syamala Devi, Sri. M. Chandrasekhar, Smt.K.Rajyalakshmi,
Sri. J. Anand, Sri R. Nagulu, Sri. B. Ankaiah and Smt.Dhanamma Department of
Veterinary Gynaecology and Obstetrics CVS, Tirupati.
I am thankful to Director of Animal Husbandry, Hyderabad for granting
deputation and authorities of S.V.Veterinary University, Tirupati for their support.
I am deeply indebted to my parents G.Purushotham, Saroja and wife
G.Sumalatha and my kids Hasini and Nityasree and my in-laws Chinabba and Rani
for their untiring support and seemingly culminated belief in me.
G. Ravi Babu . . .
 DECLARATION

I, Dr. G.Ravi Babu, hereby declare that the thesis entitled “EVALUATION

OF OXIDATIVE STRESS IN TRANSITION COWS AND POST PARTUM

ENDOMETRIAL CYTOLOGY” submitted to Sri Venkateswara Veterinary University,

Tirupati for the degree of Master of Veterinary Science is a result of original research

work done by me. It is further declared that the thesis or any part thereof has not been

published earlier in any manner.

Date :

Place : Tirupati. (G.RAVI BABU)

Name of the Author : G. Ravi Babu
Title of the thesis : EVALUATION OF OXIDATIVE STRESS IN TRANSITION
COWS AND POST PARTUM ENDOMETRIAL
CYTOLOGY
Degree to which it is Submitted : Master of Veterinary Science
Faculty : Faculty of Veterinary Science
Department : Department of Animal Reproduction, Gynaecology and
obstetrics,
College of Veterinary Science, Tirupati - 517502.
Guide : Dr. K. Mouli Krishna
Professor & Head

University : Sri Venkateswara Veterinary University, Tirupati.

Year : March, 2013

ABSTRACT
The transition period is physiologically the most important, highly sensitive

and venerable in the reproductive life of dairy cows. The physiological and metabolic

changes occurring in peripartum period imposes oxidative stress and directs the

subsequent reproductive events in dairy cows. The objective of the study was to

evaluate oxidative stress, antioxidant status and endometrial cytology for predicting

the uterine involution in cows. A total of 14 multiparous crossbred cows maintained

in a commercial farm were assigned to group I (8 Healthy) & II (6 Infected). Blood

samples were collected periodically on day -10, 0, 10, 25 and CUI. Spectrophoto-

metric procedures were adopted to determine concentrations of MDA, Vit.E, GSH,

glucose, total proteins and SOD and Catalase activity, in addition to endometrial

cytological smears on day 25 and CUI. Neutrophils counts were decreasing after day

25 and in infected group II the counts were significantly higher. Values of MDA,
Vit.E, GSH and Catalase activity were significantly different (p≤0.05) between

collections in group I and II while, glucose and total proteins remained within

physiological ranges. Progesterone concentration significantly different (p≤0.05)

between collections in group I and II. The cows in group II had high lipid peroxidese

and low antioxidant status during early part of the experiment and was affected with

uterine infections.

It was concluded that there were significant differences between two groups of

cows investigated. The high MDA profiles recorded on day 0 and low antioxidant

status observed in group II animals might have compromised host`s immune defense

and suffered them from uterine infections. Further, the presence of neutrophils

significantly in high percentages in uterine cytological smears obtained at CUI (after

morphological completion of uterine involution) indicates the persistence of infection

after declaring completion of involution. From the present study, it was suggested that

the routine rectal examination of the tract is insufficient to conclude completion of

involution. It was felt that in routine practice the endometrial cytology may be

adopted in clinical diagnosis of uterine involution.

 LIST OF ABBREVIATIONS

≤ : less than or equal to

% : percentage
µ : micron
mm : millimeter
cm : centimeter
ml : milliliter
min : minutes
viz. : such as
et al. : and others
df : degrees of freedom
Fig. : figure
No. : number
CUI : complete uterine involution
Hb : hemoglobin
KU : katal unit
OD : optical density
mg : milligram
µg : microgram
g : gram
dL : deciliter
L : liter
MDA : melonylaldehyde
SOD : superoxide dismutase
Vit.E : vitamin A
GSH : glutathione
GSSH : oxidized glutathione
IU : intrauterine
Nmol : nanomole
µmol : micromole
mmol : millimole
ng : nanogram
rpm : revolutions per minute
NS : natural service
SE : standard error
ELISA : enzyme linked immunosorbent assay
Wk : week
 CHAPTER - I
1. INTRODUCTION

The transition from the pregnant, non-lactating state to the non-pregnant,

lactating state is too often a disastrous experience for the cows. Dairy cattle are more

susceptible to a variety of metabolic and infectious diseases during the transition

period compared with peak lactation (Sordillo et al., 2007). The defense mechanisms

of the dairy cow is compromised during the transition period because of rapid

differentiation of secretory parenchyma, intense mammary gland growth, and the

onset of copious milk synthesis and secretion are accompanied by a high energy

demand and an increases oxygen requirement. This increased oxygen demand

augments the production of oxygen derived reactants, collectively termed reactive

oxygen species (ROS).

Oxidative stress is simply the elevation of free radicals (i.e., ROS) found in

cells that accumulate to higher than normal levels (Mandelkar, 2008). Several cell

constituents including poly unsaturated fatty acids in cell membranes are damaged

due to oxidative stress resulting in increased production of thiobarbituric acid reactive

substances (TBARS) like Malondialdehyde (MDA) in as their degradation product

(Sharma et al., 2011). Hence, determination of MDA levels in biological fluids

formed the basis for assessing oxidative stress.

Excessive production of free radicals and concomitant damage at cellular and

tissue levels are controlled by cellular antioxidants defense systems. Antioxidants can

be broadly defined as any substance that delays, prevents or removes oxidative

damage to target molecules (Halliwell and Gutteridege et al., 2007). The preventive

body antioxidant defense systems can be accomplished by enzymatic (SOD, CAT)

and non enzymatic (GSH, Vit. E) mechanisms.

 An imbalance between increased production of ROS and reduced availability

of antioxidant defenses near the time of parturition increases oxidative stress and may

contribute to periparturient disorders in dairy cows including delay in uterine

involution. (Waller, 2000; Gitto et al., 2002). The ability to resolve infection depends

on immune functions and antioxidant status of the body. The determination of

products of peroxidative damage and antioxidant substances like Vit. E, glutathione

and enzymes (SOD, CAT.) are useful markers for the oxidative stress and antioxidant

status respectively (Celi et al., 2010) and postpartum cytological examinations throw

light on the prediction of uterine involution.

In infected animals Neutrophils counts were significantly in high percentages

in uterine cytological smears obtained at CUI (after morphological completion of

uterine involution) indicated the persistent of infections after morphological

completion of involution (Yavari et al., 2008). The routine rectal examination of the

tract is insufficient to conclude completion of involution. It was felt that in routine

practice the endometrial cytology technique may be adopted in clinical diagnosis of

uterine involution. Thus, early detection of oxidative stress might give a clue to

predict various insults (uterine involution) caused by free radicals and plan to develop

preventive measures to combat the parturient complications (uterine involution).

In view of this, the present work was designed with the objective of predicting

completion of uterine involution in crossbred cows by cytological examinations.

 CHAPTER-II
2. REVIEW OF LITERATURE

2.1 OXIDATIVE STRESS IN TRANSITION COWS

The transition period between late pregnancy and early lactation is the most

interesting stage of lactation cycle. The transition from pregnant, non-lactating state to

the non-pregnant, lactating state is a disastrous experience for the cows. Periparturient

period is critical for health and subsequent performance of dairy cows.

The cows are subjected to physiological stresses associated with high energy

demand and increased oxygen requirement (Gitto et al., 2002) which leads to

production of ROS. ROS are continuously generated in the body during normal

cellular metabolic processes and have very short half life in milliseconds. They react

with other compounds and generate several thousand free radicals. They include

hydroxyl (OH), superoxide O2   , nitric oxide (NO  ), nitrogen dioxide NO2  ,

peroxyl (ROO  ) and lipid peroxyl (LOO  ). On other hand, hydrogen peroxide

(H2O2), ozone (O3), singlet oxygen (1O2), hypochlorus acid (HOCl), nitrous acid

(HNO2), peroxy nitrite (ONOO-), dinitrogen trioxide (N2O3), lipid peroxide (LOOH)

have not been considered free radicals, but generally called oxidants and involve in

free radical reactions in living organisms (Genestra, 2007).

Excessive production of free radicals and concomitant damages at cellular

levels are controlled by enzymatic and non-enzymatic antioxidant system. Under

physiological conditions normal threshold levels are maintained, where as imbalance

between increased production of ROS and reduced availability of antioxidant defense

near the time of parturition results in oxidative stress and contribute to periparturient

disorders in dairy cows (Bernabucciet et al., 2005; Castillo et al., 2005).

2.1.1 MALONYLALDEHYDE

Lipid per oxidation is a non-enzymatic chain reaction based on oxidation of

unsaturated fatty acids. Damage to these poly unsaturated fatty acids results in

increased production of Thio barbituric acid reactive substances (TBARS) like MDA

as their degradation product. Hence determination of MDA levels in plasma formed

the basis of assessing the oxidative stress in periparturient cows.

Perusal of literature revealed that different workers have studied on the levels

of MDA in calves, periparturient cows, endometritis, repeat breeding cows and

stressed cows (Miller et al., 1993, Brzezinska et al., 1994, Gupta et al., 2004 and

Castillo et al., 2005).

Gupta et al. (2004) reported that mean lipid peroxidation levels in erythrocytes

at 3 weeks and 0 week prepartum in RFM affected animals in (nano mol MDA/mg

Hb) were 2.46 ± 0.07 and 3.67 ± 0.1. Where as in control non-RFM at 3 weeks and 0

week was 2.78 ± 0.06 and 2.76 ± 0.08.

Sivanagapavani (2005) reported that in group I the mean MDA levels in

erythrocyte hemolysate were 2.71 ± 0.08, 2.78 ± 0.07, 2.88 ± 0.08, 2.68 ± 0.09 and

2.62 ± 0.08 , where as in control 2.74 ± 0.06, 2.63 ± 0.06, 2.84 ± 0.09, 2.58 ± 0.06

and 2.52 ± 0.07 nmol/mg Hb at -3, -2, -1, 0 and 1 week peripartum, respectively. The

incidence of RFM in group I and II was 36.36 (4/11) and 45.45% (5/11).

Castillo et al. (2005) reported MDA levels in periparturient dairy cows. They

reported that MDA profiles were 34.57 + 2.40 uM/L in late lactation, 51.03 + 20.42

uM/L one week before calving and 68.99 + 33.64 uM/L one week after calving.

Pintea et al. (2006) reported that maximum level of lipid peroxidation was

observed in the first week after parturition (62µmols/L), comparing with the level of

lipid peroxidation in the cows in late lactation (35µmols/L).

 Kizil et al. (2007) reported that the MDA level in healthy cow is 1.88 ±

0.21µmol/L and in sub clinical mastitis the MDA levels were 2.28 ± 0.15 µmol/L.

Serum MDA profiles in healthy buffaloes were 1.98 + 0.90 mmol/ml and in

endometritis affected buffaloes 5.71 + 0.84 mmol/ml (Hanafi et al., 2008).

Turk et al. (2008) reported that MDA levels were significantly higher in the

dry period compared to postpartum cows. In dry period MDA was 2.43mmol/L and

post partum 1-15 days, 20-30 days and 60-100 was 1.89 mmol/L, 1.92mmol/L and

2.50mmol/L.

Ahmed et al. (2010) reported MDA levels in different conditions in buffaloes.

The MDA levels in normal cyclic, inactive ovaries, delayed puberty, endometritis

repeat breeding, retained placenta and cystic ovaries were 1.94 ± 0.07, 3.90 ± 0.41,

4.18 ± 0.89, 5.47 ± 0.74, 1.96 ± 0.05, 5.51 ± 0.54 and 6.32 ± 1.19 nmol/L

respectively.

Pathan et al. (2010) reported erythrocytic MDA in non pregnant buffaloes as

4.5 ± 0.69 nM/ml and in periparturient buffaloes 30 days,15 days before parturition

was 7.50 ± 0.32 nM/ml, 8.32 ± 0.25 nMml and 0 day, 15 day and 30 day after

parturition was 9.74 ± 0.15 nM/ml, 6.96 ± 0.17 nM/ml, 5.43 ± 0.14 nM/ml. The

concentration of erythrocytic MDA was significantly higher on the day of parturition

as compared to 30 and 15 days before and 15 and 30 days after parturition.

Sharma et al. (2011) reported that lipid peroxidation was significantly higher

in cows during early lactation as compared to the cows in advanced pregnancy. MDA

levels in advanced pregnancy are 7.59 ± 0.72 nmol/g Hb/h and in late lactation was

13.34 ± 2.68 nmol/g Hb/h.

2.2 ANTIOXIDANTS

Production of free radicals and their neutralization by antioxidants has been

the normal bodily process. A biological antioxidant may be defined as a substance

that significantly delays or inhibits oxidation of a substrate. Antioxidants were

classified into preventing antioxidants or enzymatic antioxidants and chain breaking

antioxidants or non enzymatic antioxidants. Enzymatic antioxidants will block the

initial production of free radicals by scavenging both intercellular and extracellular

superoxide radicals. eg: Catalase, superoxide dismutase and Glutathione peroxides

while non enzymatic antioxidants will inhibit propagative phase of lipid peroxidation

eg: α Tochopherol (Vit. E), proteins like albumin, transferin, glutathione (GSH),

(Kizil et al., 2007, Sunil Kumar et al., 2011).

2.2.1 Vitamin. E

In normal healthy cows Vit.E concentration ranges from 2 to 5 mg/L

(Ashutosh and Singh, 2007).

The levels of Vit.E in normal cyclic buffalo heifers were 4.22 + 0.014 µmol/L

and repeat breeder buffalo heifers were 3.35 + 0.22 µmol/L (Nayyar et al., 2002).

Kizil et al. (2007) reported Vit.E levels in sub clinical infections and in control

group as 2.1 + 0.1 and 2.4 + 0.2 µg/L respectively.

Bowstra et al. (2008) supplemented 3000 IU of Vit. E, 60 days prior to

calving. Blood was sampled nine times before calving, on day of calving and twice

after calving. He reported that Vit. E significantly lowered blood MDA concentration

of supplemented heifers in the two weeks after calving, indicating Vit.E has a role in

recovery from parturition related oxidative stress.

Sordillo et al. (2009) reported that Oxidative stress is thought to be a

significant underlying factor leading to dysfunctional host immune and inflammatory

responses during times of metabolic stress. Vit E have impact on inflammation,

uncontrolled inflammation is a dominant factor in metritis and mastitis.

Bowstra et al. (2010) reported that physiological states influence the effect of

Vit. E supplements during the dry period on the level of oxidative stress 2 week ante

partum and the risk of clinical mastitis in early lactation.

Where as in cows vitamin E values were 5.7 + 0.8 and 2.0 + 0.01 µmol /L in

healthy and infected cows, respectively (Kataria et al., 2010). In healthy and infected

she buffaloes the values were 4.20 ± 0.09 and 2.20 ±0.02 µmol /L respectively

(Kataria et al., 2012).

Graugnard et al. (2012) reported that Vit. E in overfed group 14 days

prepartum and 7 days post partum were 1.57 ± 4.78 µg/mL and 1.01 ± 2.75 and in

control group 14 days prepartum and 7 days post partum were 1.35 ± 3.88 and 0.88 ±

2.41 µg/mL, respectively.

2.2.2 GSH

Hanafi et al. (2008) reported that the concentration of reduced Glutathione

was 6.38 ± 0.11and 1.99 ± 0.30 mmol/L in healthy and endometritis affected she

buffaloes, respectively.

Kizil et al. (2007) reported reduced Glutathione levels in sub clinical infected

and healthy control group as 0.27 + 0.01 and 0.24 + 0.01 mmol/L, respectively.

In buffaloes, Ahmed et al. (2010) compared the levels of reduced Glutathione

between endometritis affected and normal cyclic groups. Reduced Glutathione was

2.67 ± 0.24 and 6.94 ± 0.13 mmol/L in endometritis affected and control group,

respectively.

Sharma et al. (2011) reported that GSH was significantly higher in cows

during advance pregnancy as compared to the cows in late lactation. GSH levels were
71.81 ± 11.41 and 28.99 ± 6.58 µg/ml in advanced pregnancy and late lactation,

respectively.

2.2.3 Catalase

The serum Catalase activity in healthy buffaloes and endometritis affected

buffaloes were 2.28 + 0.04 and 1.37 + 0.07U/ml, respectively (Hanafi et al., 2008).

Pintea et al. (2006) recorded the increase in blood Catalase activity from first

to sixth week post partum.

Moghazy et al. (2011) reported that Catalase was significantly higher in

healthy buffaloes compared to parasitic infected buffaloes. Catalase levels in healthy

buffaloes were 2.38 ± 0.05 U/ml and in parasitic infected buffaloes ranged from 0.87

± 0.03 to 0.97 ± 0.36 U/ml.

In cows Sharma et al. (2011) noticed that Catalase activity was significantly

higher in late lactation when compared to advanced pregnancy. Catalase activity in

advanced pregnancy was 42.52 ± 6.98 µmoles/min/mg Hb and in late lactation was

48.33 ± 6.55 µmoles/min/mg Hb.

Kataria et al. (2012) reported that Catalase activity was significantly higher in

infected buffaloes as compared to healthy buffaloes. Catalase activity levels in

infected buffaloes was 132.65 ± 8.56 KU/L and in healthy buffaloes was 68.55 ± 10

KU/L.

2.2.4 SOD

Bernabucci et al. (2005) reported that SOD values in Low Body condition

score, Medium Body condition score , High Body condition score precalving were

1830 ± 39; 1560 ± 112, 1737 ± 69 U/mL of PCV and 1828 ± 54; 1587 ± 78 and

1528 ± 59 U/mL of PCV, respectively.

 Pintea et al. (2006) reported that SOD activity was 1365 U/g Hb in the first

week, 1890 U/g Hb in the second week, 2290 U/g Hb in the third week, 1869 U/g Hb

in the fourth week and 1905 U/g Hb in the sixth week after parturition.

Hanafi et al. (2008) reported that superoxide dismutase values were 338.165

+ 7.11 U/ml in healthy buffaloes and 335.12 + 18.33 U/ml in endometritis buffaloes.

In cows, levels of SOD in erythrocytes hemolysate of control and

inflammatory conditions were 999.2 + 109.4 and 1321.5 + 163.2 U/g Hb, respectively

(Kleczkowski et al., 2008).

Whereas in estrus buffaloes, Megahed et al. (2008) reported that the SOD

levels in winter and summer were 3.80 + 0.16 and 3.17 + 0.13 U/L, respectively.

In cows, Kataria et al. (2010) reported that serum SOD levels in control and

affected with bacterial infections were 127.0 + 12.9 and 218.9 + 12.0 KUL-1 ,

respectively.

Pathan et al. (2010) reported that in Mehsana she buffaloes, erythrocytes

hemolysate SOD activity was 2.13 ± 0.19, 2.33 ± 0.13, 3.03 ± 0.13, 2.47 ± 0.11 and

2.39 ± 0.11 U at day -30, -15, 0, +15 and +30 peripartum, respectively as against

1.95 ± 0.14 U recorded in non pregnant control buffalos.

Sharma et al. (2011) reported that SOD activity was significantly higher in

cows during advance pregnancy as compared to the cows in late lactation. SOD

activity in advanced pregnancy were 6.99 ± 0.45 units/mg Hb and in late lactation

were 6.37 ±0.72 units/mg Hb.

Ghasemian et al. (2011) reported that SOD levels in normal cows were

1668.87 ± 867.19 IU/g Hb and in subclinical mastitis affected cows were 1769.34 ±

755.73 IU/g Hb.

2.3 Glucose

Metabolic profiles have been used to monitor health and nutritional status of

dairy cattle. Many workers suggested that alterations in biochemical constituents

caused fertility problems.

Mandali et al. (2002) reported that the mean glucose concentration was lower

in RFM affected buffaloes (53.42 ± 0.73 mg %) than normal buffaloes (57.73 ± 1.42

mg %).

Castillo et al. (2005), reported that mean glucose values at 10,6,2 and 1 week

before calving were 65 ± 3.59, 70.71 ± 2.85, 73.44 ± 2.18, 74.86 ± 12.95 and 1,2

weeks after calving were 70.87 ± 6.95, 68.31 ± 5.34 and at late lactation were 73.88 ±

1.42 mg/dL.

The serum glucose profile in normally cyclic parous lactating cows of >90

days post partum interval was reported as 58.33 ± 6.22 mg% (Singh et al., 2007).

Magnus and Lali (2009) ascertained the relationship between serum

biochemical profile and postpartum metritis by examining animals with recent

history of calving and subsequent metritis and observed serum glucose levels as 22.3

 2.18 mg% which was below the reference range of 45-75 mg% (Radostitis et al.,

2000).

Turk et al. (2008) reported that glucose levels were significantly higher in the

dry period compared to postpartum cows in early purperium and late purperium. The

level of serum glucose in dry period, during 1-15 days post partum, 20-30 days post

partum and 60-100 days post partum was 3.30, 2.20, 2.30 and 3.30 mmol/L.

Graugnard et al. (2012) reported that overfed 14 days prepartum and 7 days

early postpartum glucose were 1.44 ± 4.24 and 1.25 ± 3.49 mmol/L and in control

were 1.39 ± 4.0 in 14 days prepartum and 1.19 ± 3.30 mmol/L 7 days post partum.
2.4 Total Proteins

Mandali et al. (2002) reported that the mean total protein concentration was

higher in RFM affected buffaloes (6.78 ± 0.05g %) than normal buffaloes (6.64 ± 0.09

g %).

Castillo et al. (2005) reported that mean total protein values at 10, 6, 2 and 1

week before calving were 7.51 ± 0.13, 7.99 ± 0.19, 7.76 ± 0.30 , 6.91 ± 0.40 and 1,2

weeks after calving were 7.32 ± 0.32, 8.33 ± 0.30 g%, respectively.

Sahadev et al. (2007) found that serum total protein concentration in

endometritis affected and in healthy cows was 8.32  0.07g% and 8.49 

0.17g%, respectively.

Magnus et al. (2009) recorded that the serum total protein profile of post

partum metritis affected and normal cows as 6.1 ± 0.51 and 5.7-8.1g/dl (Radostitis et

al. 2000).

2.5 Progesterone

Corah et al. (1974) determined the plasma progesterone concentration during

the first 21 days postpartum period in beef heifers. The plasma progesterone profiles

ranged from 0.7 to 0.9 ng/ml up to day 7 and then declined to 0.16 ± 0.04 ng/ml by

day 13 and thereafter remained stable till day 21 postpartum.

The mean base line progesterone concentration on day 6 and 10 was 0.04 and

0.03 ng/ml in primiparous and multiparous beef cows, respectively throughout the

postpartum anoestrous period (Dimmick et al., 1991)

Saxena et al. (1995) reported the plasma progesterone profiles in crossbred

cow of lactation I and II as < 1 ng/ml up to day 45 postpartum. The progesterone

concentration did not differ between days up to day 30 postpartum. However, the
levels were significantly higher on day 45 in cows that returned to estrum by day 60

than at latter postpartum period.

Srinivas rao, (2004) reported that the overall mean serum progesterone was

0.54 ± 0.03, 0.76 ± 0.11, 0.66 ± 0.11, 0.62 ± 0.09 and 0.80 ± 0.08 ng/ml on day 1, 8,

15, 22 and at complete uterine involution.

Patel et al. (2007) reported the circulatory level of progesterone was lowest on

the day of calving (1 ng/ml) and increased linearly (2-3 ng/ml) by second and third

week postpartum.

Hanafi et al. (2008) reported that serum progesterone level were <0.02 and

1.47 ng/ml in animals having inactive ovaries and persistent corpora lutea in

endometritis. Whereas in healthy the levels in follicular and luteal phase were 0.44 ±

0.13, 2.89 ± 0.17.

Venkatnaidu et al. (2010) reported that serum progesterone levels on day 0, 5,

10, 15, 20, 25, 30, 35, 40, 45 in lactating postpartum Ongole cows were, 0.33 ± 0.04,

0.40 ± 0.55, 0.65 ± 0.22, 1.13 ± 0.25, 1.58 ± 0.32, 1.85 ± 0.52, 1.40 ± 0.25, 0.82 ±

0.04, 0.65 ± 0.16, 0.75 ± 0.05 ng/ml, respectively. The normal range for the above

days were 0.02-0.4, 0.2-0.6, 0.4-1.6, 0.7-2.2, .5-2.5, 0.6-4, 0.6-4, 0.7-1, 0.4-1.1, 0.7-

0.8 ng/ml.

Kafi et al. (2010) reported that cows with progesterone concentration ≥ 1ng/ml

on day 24 after calving were more at risk for prolonged luteal phase compared to

normal.

2.6 Cytological studies

For cytological studies different approaches viz., uterine lavage / aspiration

(Gilbert et al., 1998; Hammon et al., 2006), cytobrush technique (Kasimanickam et

al., 2005; Barlund et al., 2008; Burke et al., 2010: Baranski et al., 2011: Kaewlamun
et al., 2011), cotton swab (Azawi et al., 2007and Yavari et al., 2008) were adopted in

bovine species.

2.6.1 Evaluation of technique

In clinically normal postpartum cows Gilbert et al. (1998), Kasimanickam et

al. (2005) and Barlund et al. (2008) reported the merits and demerits of cytobrush,

uterine lavage and swab method meant for sampling uterine contents.

Though the cytobrush technique required specialized equipment, sampling by

this method was easy, simple, more consistent and precise and produce rapid results,

when compared to swab technique. The technique yielded in-situ samples which

might represent the inflammatory nature of the endometrial compared with the uterine

swab technique, which yielded minimum samples of luminal contents. The technique

resulted in less distortion of cells compared with the lavage and swab method (Studer

and Morrow 1978 and Miller et al. 1980). Kasimanickam et al. (2005) reported that

delay in the cytological evaluation may result in inaccurate total nucleated cell count

and thus later the cytological evaluation.

Kasimanickam et al. (2005) reported that the number of red blood cells in the

sample tended to be higher in the lavage technique compared with cytobrush

technique. But when compared to swab technique number of red blood cells in

cytobrush was more. This might be related to trauma resulting from the manipulations

of the uterus and infusion rod, while attempting to recover the sample. The failure to

recover sufficient samples from uterus indicated that the swab technique was

inconsistent.

2.6.2 Staining Cytological Smears

Scientists have used either Giemsa or Wrights’ Geimsa for staining the

cytological smears (Kasimanickam et al., 2004 and 2005; Gilbert et al., 2005; Ahmadi
et al., 2006; Azawi et al., 2007; Yavari et al., 2008; Santos et al., 2009; and

Chapwanya et al., 2009). Whereas Zebre et al. (1996) adopted Acridin orange stain.

2.6.3 Neutrophils Population

In cytological studies, the threshold number of Neutrophils, the cutoff point at

different postpartum periods to define endometritis was worked out by different

scientists (Kasimanickam et al., 2004; Gilbert et al., 2005; Ahmadi et al., 2006;

Sheldon et al., 2006; Barlund et al., 2008 and Yavari et al., 2008).

It was stated during postpartum period, the Neutrophils population was

varying with increasing postpartum interval. In early postpartum the percentage of

Neutrophils tended to be on higher side and declining with every increase in

postpartum period (Stevenson, 1997 and Bondurant, 1999).

The cutoff point of Neutrophils population for defining prevalence of

endometritis ranged from 5-25% in different studies (Kasimanickam et al., 2004;

Barlund et al., 2008; Kaufmann et al., 2009; Santosh et al., 2009). It was concluded

that the population of Neutrophils over and above the cutoff point was negatively

correlated with reproductive performance of dairy cows.

Rameshbabu et al. (2011) reported that the mean Neutrophils counts in smears

obtained at day 0 by aspiration technique were 4.6 ± 0.64 and 45.69 ± 3.88% in

group- I (control) and group-II (Infertile), respectively. Whereas at day 4 by lavage

technique were 5.6 ± 0.78 and 57.82 ± 3.50 % in group I and group II, respectively.

Sensory et al. (2012) reported that, there was no significant difference in

polymorph nuclear leukocyte percentage between conceived and non-conceived cows

during different weeks post partum. However, first service conception rate in animals

with normal vaginal discharge was significantly low when compared to those of
abnormal discharge during W3 (2.3% vs. 30.3%) and W4 (4.7% vs. 29.7%) post

partum.

2.7 UTERINE INVOLUTION

The uterine involution was considered as complete when the discharge were

stopped, the two uterine horns were felt equally rigid, appeared approximately similar

in size and had returned to its normal intrapelvic location (Francis and Raja 1971; Rao

and Rao 1980). Completion of uterine involution was judged by rectal palpation of

the genital organs. The rectal examination of the cow was done from day 25

postpartum at weekly intervals until completion of uterine involution.

In Nili-Ravi buffaloes Usmani et al. (2001) reported 24 % (7/29) subclinical

uterine infection, which delayed uterine involution over that of control (46.3 vs. 35.8).

Mateus et al. (2002) designed to study the effect of puerperal uterine infection

on uterine involution and on ovarian activity in dairy cows from parturition to six

weeks postpartum. Infection retarded the uterine involution assessed by uterine body

diameter and score of intra uterine fluid volume.

El-wishy et al. (2007) reported that the mean interval to complete uterine

involution varied widely between 19 and 52 days. He reported that uterine involution

does not seem to be a limiting factor for achievement of satisfactory fertility in post

partum buffaloes.

Nechifor et al. (2011) reported that the involution of the uterus in 5% of the

cows started at 14 days postpartum and in 70% of the cows involution reached a peak

at 21 days post partum.

Rolim et al. (2011) evaluated the reproductive characteristics such as uterine

involution, ovarian activity and first estrus after parturition and reported that the
resumption of ovarian activity, verified by visual observation of the first estrus, was

47.06 ± 25.66 days, while the uterine involution was 27.5 ± 7.77 days.

Cengic et al. (2012) monitored uterine involution by ultrasound and reported

that 48% of cows had normal uterine involution, while the remaining 52% had

delayed uterine involution. Cows with normal puerperium have completed involution

in the period from 38-45 days postpartum, while those with abnormal puerperium

needed more time to complete it or required therapy of disturbed uterine status.

 CHAPTER-III
3. MATERIALS AND METHODS

3.1. SELECTION OF ANIMALS

A total of fourteen clinically healthy parous pregnant crossbred cows

maintained stall fed under uniform managemental practices in a commercial dairy

farm were utilized. The cows were averaged 400-450 kg body weight. Cows were

housed and maintained in groups according to their reproductive status. During the

course of study the animals, which suffered from infections, were placed in group II

(6) and the other normal animals were placed in group I (8). Every cow was closely

and regularly watched for signs of ill health if any throughout the duration of the

study.

3.2 COLLECTION OF SAMPLES

Adequate volumes of jugular blood was collected from every cow on day -10,

0, 10, 25 and completion of uterine involution (CUI) into heparinised vacutainers and

transported with in 30minutes to the laboratory under cold chain. The whole blood

was centrifuged at 1500 rpm for 20 minutes and plasma was separated. The plasma

was stored at -20˚C until biochemical analyses were carried out. The erythrocytes

remained thereafter in the tubes were processed to obtain 10% erythrocytes

hemolysate (Cohn, 1970). The erythrocytes were washed thrice in physiological

saline and centrifuged at 3000 rpm at every washing.

3.2.1 Biochemical Analyses

Erythrocytes hemolysate will be obtained by lysing the erythrocytes in 10

parts of ice-cold distilled water. The Catalase and SOD activities were determined in

erythrocyte hemolysate. The concentrations of MDA, Vit. E, GSH, Protein and

Glucose were determined in plasma. The plasma concentrations of MDA (Yagi et al.,

1987), Vit.E (Baker et al., 1951), GSH (Ellman, 1959), SOD (Mishra and Fidrovich,

1972) and Catalase (Aebi, 1984) were determined by spectrophotometric procedures.

While glucose and total proteins were determined by calorimetric procedures. (kit*)

Whereas commercial ELISA (kit**) was adapted to determine plasma progesterone

concentrations.

3.2.2 Harvesting cellular portion

The cytobrush used in medical practice for obtaining pap smears was adopted

in this study to collect uterine secretions as suggested by Barlund et al. (2008) and

Galvao et al. (2011). In the present study for harvesting cellular fraction from uterine

secretions, the metal cytobrush catheter was designed with the locally available

materials (Fig: 1). The normal cytobrush handle was cut approximately to 3cm,

threaded onto a 55cm solid stainless steel rod and placed in a 45cm stainless steel

tube. The tool was protected with a plastic sheath protector during insertion into the

vagina. The catheter was then introduced into the cervix and the uterus by gentle

manipulation of the cervix per rectally. Then the stylet was pushed and cytobrush was

gently pressed against the uterine wall and rolled clock wise approximately one

quarter turn, before it was withdrawn into the catheter. Later the catheter was

removed and the brush was rolled on clean glass slide to make smears.

-------------------------------------------------------------------------------------------------------
* M/s Excel Diagnostics, Pvt. Ltd.
** Pathozyme, Omega Diagnostics, Pvt. Ltd.

3.2.3 Staining

Immediately after obtaining smears the slides were fixed in methanol and

stained with Leishmans stain and examined under oil immersion lens of a microscope

(x 1000). A total of hundred cells were counted on the slide and the percentages of

Neutrophils were determined.

3.3 Uterine involution

The uterine involution was considered as completed when the discharges were

stopped; the two uterine horns were felt equally rigid, appeared approximately similar

in size and had returned to its normal intrapelvic location (Francis and Raja, 1971).

Each cow was evaluated for complete uterine involution by palpation per rectum. The

rectal examination of each cow was done initially on day 25 and there after at 5 days

interval. The values of the last two examinations were averaged and the duration of

uterine involution was completed.

3.4 Statistical Analysis

The statistical analysis of the data was done by adopting computer soft ware

programmed for windows XP (Version 9.0, spss Inc. Munich) and Excel (Version

2003, Microsoft) and also the methods described by Snedecor and Cochran (1967).
 CHAPTER – IV
4. RESULTS

4.1 Oxidative stress

4.1.1 MDA

The profiles of MDA recorded in the present study are presented in Table 1.

The mean blood plasma concentration of MDA was 4.48 ± 0.72, 5.88 ± 1.01, 7.19

± 1.12, 4.63 ± 0.75 and 2.96 ± 0.40; and 4.12 ± 0.48, 7.05 ± 1.42, 7.11 ± 0.96, 2.7

± 0.45 and 2.7 ± 0.5 µmol/L on day -10, 0, 10, 25 and CUI in group I and II,

respectively. There was a significant difference between collection intervals in group I

and II (P ≤ 0.05). In both groups in general the trend were increasing by day 10 post

partum and thereafter continued to decline towards the end of the experiment. In

group I and II the concentration of MDA was significantly higher on day 10 post

partum than other days of collection. Further there was a significant difference in the

mean concentration of MDA between group I and II on day 25 (P ≤ 0.05) and non

significant between groups on other days. Further in group I the MDA concentration

was negatively correlated with Vit.E (r: - 0.36206; P ≤ 0.05) and positively correlated

with Catalase activity (r: 0.3044; P ≤ 0.05) and non significant with GSH

(r: 0.279321) and SOD (r: 0.24847). Where as in II the MDA concentration was

negatively correlated with Vit.E (r: - 0.47241; P ≤ 0.05) and Catalase activity

(r: - 0.39205; P ≤ 0.05); and positively correlated with GSH (r: 0.392004; P ≤ 0.05)

and non significant with SOD (r: 238874).

4.2 Antioxidants

4.2.1 Vit.E

The mean profiles of Vit.E in the present study are presented in Table 4. The

Vit.E concentration was 4.16 ± 0.33, 0.73 ± 0.20, 0.86 ± 0.15, 1.93 ± 0.87 and 4.95 ±

2.36 mg/L in group I and 1.6 ± 0.33 ,1.14 ± 0.32, 1.49 ± 0.27, 2.73 ± 0.48 and 2.5 ±

0.51 mg/L in group II on days -10, 0, 10, 25 and CUI, respectively. There was a

significant difference in Vit.E profiles between collection intervals in group I and II.

In group I the mean Vit. E concentration declined significantly by day 0 and remained

stable up to day 10 and thereafter continued to increase towards the end of the

experiment. In Group II the concentration non significantly fluctuated up to day 10

and thereafter the increase was significant towards the end of the experiment .further

there was significant difference on day -10 (P ≤ 0.05) and on day 10 (P ≤ 0.05) and

non significant on day 0, 25 and CUI between groups. Further in group I the

concentration of Vit E was significantly correlated with MDA (r: - 0.36206; P ≤ 0.05),

but non significant with SOD activity (r: - 0.23703) and GSH (r: - 0.09855) and

Catalase activity(r: 0.07470). Where as in group II the mean Vit.E concentration was

significantly correlated with MDA (r: - 0.47241; P ≤ 0.05) and GSH (r: - 0.34468; P ≤

0.05) but non significant with SOD (r: -0.21715) and Catalase activity (r: 0.208639).

4.2.2 GSH

The profiles of GSH concentration recorded in the present study are presented

in Table 7. The GSH concentration was 0.74± 0.12, 0.53± 0.15, 0.48± 0.15, 0.26 ±

0.05 and 0.11 ± 0.03 µ mol/L in group I; and 0.89± 0.18, 0.46± 0.07, 0.77± 0.24,

0.25± 0.07 and 0.20± 0.03 µ mol/L in group II on days -10, 0, 10, 25 and CUI,

respectively. There was a significant difference between collection intervals in group

I and II (P ≤ 0.05). In general in both groups a similar trend was noticed between day

– 10 and 0 peripartum and after day 10 postpartum. In group I an initial significant

fall in the concentration was found till day 0 (P ≤ 0.05) and continued to decline

further non significantly and later significantly declined (P ≤ 0.05) by day 25

postpartum which continued to decline further non significantly. In group II except

an abrupt raise between day 0 and 10 peripartum, the trend was similar to that of

group I. Further there was no significant difference between groups at different

collection intervals. In group I GSH concentration was non significantly correlated

with MDA (r: 0.27931), Vit.E (r: - 0.09855), Catalase (r: 0.070409) and SOD (r:

0.058115); and in group II it was significantly correlated with MDA (r: 0.392004; P ≤

0.05) and Vit.E (r: - 0.34468 P ≤ 0.05) and non significant with Catalase (r: - 0.23125)

and SOD (r: - 0.03955).

4.2.3 Catalase

The mean values of Catalase activity recorded in the present study are

presented in Table 10. The Catalase activity was 0.056 ± 0.03, 0.008± 0.002, 0.008±

0.003, 0.040± 0.007 and 0.07± 0.01 KU/g Hb in group I and 0.06± 0.04, 0.018±

0.007, 0.007± 0.003, 0.508± 0.332 and 0.47± 0.02 KU/g Hb in group II on day -10,

0, 10, 25 and CUI, respectively. There was a significant difference in the mean values

of Catalase activity in group I and II (P ≤ 0.05). In group I the mean activity of

Catalase non significantly declined until postpartum day 10 and thereafter continued

to increase to a significant peak value at CUI. By and a large a similar trend was

observed in group II, but the rise between day 10 and 25 postpartum after an initial

decline until day 10 postpartum, was significant. There was a significant difference at

CUI (P ≤ 0.05) between groups and non significant at other periods. Further in group I

the Catalase activity was non significantly correlated with MDA (r: - 0.1625), Vit E
(r: 0.0747); SOD (r: - 0.23264) and GSH (r: 0.0704). In group II there was a

significant correlation with MDA (r: - 0.39205; P ≤ 0.05), but non significant with Vit

E (r: 0.2086); SOD (r: - 0.31266) and GSH (r: -0.23125).

4.2.4 SOD

The means of SOD activity recorded in the present study are presented in

Table 13. The SOD activity was 35.12± 2.00, 118.25± 18.3, 122.0± 36.2, 71± 14.5

and 36 ± 1.99 KU/mg Hb/min in group I; and 61± 14.7, 95± 22.2, 98.5± 15.5,

78.6± 16 and 52.6± 15.7 KU/mg Hb/min in group II on days -10, 0, 10, 25 and CUI,

respectively. There was a significant difference between collection intervals in group I

and II (P ≤ 0.05). In general in both groups the trend was increasing to the peak by

day 10 and thereafter declined towards end of the experiment. In group I the mean

SOD activity increased significantly on day 0, non significantly peaked on day 10,

followed by non significant decline on day 25 and thereafter decrease was significant

(P ≤ 0.05). A similar trend was found in group II also. Further there was no significant

difference between groups at different collection intervals. In group I SOD activity

was non significant with MDA (r: 0.248472), Vit.E (r: - 0.23703), Catalase

(r: - 0.23264) and GSH (r: 0.058115); and in group II it was non significant with

MDA (r: 0.238874), Vit.E (r: - 0.21751), Catalase (r: -0.31266) and GSH

(r: - 0.03955).

4.3 Glucose

The profiles of Glucose recorded in the present study are presented in Table

16. The Glucose concentration was 31.67± 3.14,42.05± 8.04, 21.37 ± 3.88, 23.47 ±

5.11, and 35.12± 6.17 mg/dl in group-I and 30.81± 4.17, 42.8 ± 10.1,29.6± 5.8,32.5

± 6.83 and 43.5± 7.61 mg/dl in group II on days -10, 0, 10, 25 and CUI, respectively.

There was significant difference between collection intervals in group I and II. In
general in both groups the trend was increasing to the peak value recorded on day 0

and thereafter continued to decline until day 25 and then started increasing towards

end of the experiment. There was no significant difference between groups at

different collection intervals.

4.4 Total proteins

The profiles of total proteins recorded in the present study are presented in

Table 19. The total proteins concentration was 8.5 ± 0.28, 9.17 ± 0.80, 9.41 ± 0.53,

9.11 ± 0.21 and 9.35 ± 0.24 g/dL in group I and 9.11 ± 0.56, 9.35 ± 0.63, 9.11 ± 0.46,

9.96 ± 0.66 and 9.13 ± 0.64 g/dL on day -10, 0, 10, 25 and CUI in group II,

respectively and did not differ. In general in both groups the trend was increasing to

the peak recorded on day 10 and thereafter continued to remain same towards the end

of the experiment period. There was no significant difference between groups at

different collection intervals.

4.5 Progesterone profiles

The mean plasma Progesterone concentrations recorded in the present study

are presented in Table 22. The progesterone concentration was 1.01 ± 0.142, 0.083 ±

0.040, 0.233 ± 0.021, 0.366 ± 0.108 and 0.383 ± 0.164 ng/ml in group I and 1.6 ±

0.233, 0.516 ± 0.340, 0.466 ± 0.158, 0.116 ± 0.040 and 0.35 ± 0.27 ng/ml in group II

on day -10, 0, 10, 25 and CUI, respectively. There was a significant difference

between collection intervals in group I and II (P ≤ 0.05). In Group I the trend was

significantly high on day -10 than other collection intervals and the lowest was on day

0. In group II comprehensively a similar trend, being highest on day -10 and thereafter

continued to decline significantly until day 10 and later largely remained same. The

trend differed significantly between groups on day -10 and 25 (P ≤ 0.05). Apparently

the levels were higher during early part of the experiment in group II than group I.
4.6 Endometrial cytology

The mean percent of neutrophils recorded in the present study are shown in

Fig: 7. In group I the mean percent of neutrophils in the endometrial cytological

smears were 31.66 and 7.00 on day 25 and CUI, respectively. In group II the mean

percent of neutrophils were 78.33 and 57.16 on day 25 and CUI, respectively and

differed significantly. The counts differed significantly between sampling intervals in

both groups (P ≤ 0.05).

 CHAPTER - V
5. DISCUSSION

5.1 Oxidative Stress

5.1.1 MDA

The mean blood plasma concentration of MDA was 4.48 ± 0.72, 5.88 ±

1.01, 7.19 ± 1.12, 4.63 ± 0.75 and 2.96 ± 0.40; and 4.12 ± 0.48, 7.05 ± 1.42, 7.11 ±

0.96, 2.7 ± 0.45 and 2.7 ± 0.5 µmol/L on day -10, 0, 10, 25 and CUI in group I and

II, respectively. The mean values of MDA recorded in the present study were not in

line with Castillo et al. (2005) and Pathan et al. (2010) whereas, similar to

Sivanagapavani (2005). The changes in the MDA values reported in different studies

were attributed to age, breed, timing and frequency of sampling, nutrition,

environment, analytical procedures and management (Bell, 1995; Castillo et al.,

2005).

In both groups in general the trend was increasing to the peak on day 10 post

partum and thereafter continued to decline towards the end of the experiment. There

was significant difference between collection intervals in group I and II (P ≤ 0.05). In

group I MDA levels increased non significantly on day 10 and later significantly

decreased on day 25 (P ≤ 0.05) post partum and thereafter non significantly decreased

by day 45. In group II MDA significantly increased on day 0, whereas continued to

remain stable until day 10, but significantly declined on day 25 (P ≤ 0.05) and

thereafter no change towards the end. A similar trend was reported by Pathan et al.

(2010). In their study, the mean MDA value significantly increased to peak at 0 day of

peripartum and later steadily declined towards the end of the experiment. The

frequency of sampling was fifteen days in their study, while it was different in the

present study.
 The trend in MDA values observed in group I and II conveyed that towards

the end of the gestation period animals were experiencing the phenomenon of

oxidative stress, which continued until day 10 post partum. In group II the value was

elevated significantly at day 0 against day 10 in group I. It showed that animals in

group II experienced oxidative stress between day -10 and 0. It might be the reason

for uterine infections observed in group II (Sordill, 2005). Based on this observation

it was felt that post partum performance could be predicted by evaluating oxidative

stress prepartum. Further it showed that beyond 10 days postpartum the profiles of

MDA followed statistically a similar trend in both groups. Animals in both groups

experienced nearly a similar phenomenon during the later part of the experiment.

The Reactive Oxygen Species, free radicals are continuously generated in the

body during normal cellular metabolic processes and have very short half life of

milliseconds. They react with other compounds and generate several thousand free

radicals. Under physiological conditions normal threshold levels are maintained by

certain scavenging systems, which are readily available in the internal environment as

antioxidants. Whereas, the imbalance between their production and safe disposal

could lead to oxidative stress in some physiopathological conditions and impair the

performance. Several cell constituents including polyunsaturated fatty acids in the

cell membranes are damaged due to oxidative stress resulting in increased production

of thiobarbituric acid reactive substances (TBARS) like MDA in the system as a

degradation product. Hence, estimation of MDA levels in biological fluids formed

the basis for assessing the oxidative stress.

The peripartum period is metabolically most sensitive and stressful, since

remarkable metabolic changes are known to occur. The metabolic processes are

directed to meet the demands of fetus such as preparation for calving and initiation of
lactation (colostrum and milk). It was stated that the demands of metabolic adaptation

for lactation (colostrum and milk) far exceed that of fetus. Hence, the increased

concentrations observed until day 10 might reflect metabolic adaptation, a normal

eventual physiological phenomenon in response to body demands. Further, after day

10 the animals were relieved from stress and it was associated with a sudden decrease

in the level of lipid peroxides that further declined steadily towards the end of the

experiment (Castillo et al., 2005; Pathan et al., 2010).

The production of free radicals in excess of body’s antioxidant capacity could

be found at occasions resulting in oxidative stress. (Castillo et al., 2005; Ahmed et al.,

2010). Oxidative stress in excess of physiological levels is a contributory factor to

increase susceptibility to diseases (Sordill, 2005). (Sordill, 2009) reported that

oxidative stress could be a significant underlying factor leading to dysfunctional host

immune and inflammatory responses during times of metabolic stress. That could be

the reason why the animals in group II that had high MDA values on day 0 suffered

from uterine infections later.

In both groups there was a negative correlation with Vit. E. levels. It showed

that Vit.E might have been utilized as an antioxidant in preventing free radical

production (Miller et al. 1993). Further it was correlated with Catalase activity in

group I (r: 0.3044; P ≤ 0.05) and group II (r: - 0.39205; P ≤ 0.05). The difference

between two groups was related to the variations in the trend of MDA recorded

between groups. In group I the initial elevation on day 10 followed by subsequent

decline on day 25 was gradual, while in group II the trend was either abrupt raise or

decline as reported by Sharma et al. 2011. Similarly there was no correlation with

GSH levels in group I, but positively correlated in group II. (r: 0.392004; P ≤ 0.05).
The difference between groups was related to variations in the levels of MDA

recorded in the two groups.

5.2 Antioxidants

5.2.1 Vitamin E

The Vit.E concentration was 4.16 ± 0.33, 0.73 ± 0.20, 0.86 ± 0.15, 1.93 ±

0.87 and 4.95 ± 2.36 mg/L in group I and 1.6 ± 0.33 ,1.14 ± 0.32, 1.49 ± 0.27, 2.73 ±

0.48 and 2.5 ± 0.51 mg/L in group II on day -10, 0, 10, 25 and CUI, respectively.

The mean values recorded in the present study were not in agreement with earlier

reports (Kizil et al., 2007; Graugnard et al., 2012). The variations in the mean values

in different studies were attributed to age, breed, timing and frequency of sampling,

nutrition, environment, analytical procedures adopted and management (Bell, 1995;

Castillo et al., 2005).

In the present study the mean values of Vit. E was lower in group II compared

to group I. In general the profiles followed nearly a similar pattern in both groups.

There was a significant difference in Vit.E profiles between collection intervals in

group I and II (P ≤ 0.05). In group I Vit. E concentration declined significantly

(P ≤ 0.05) on day 0 and remained stable up to day 10 and thereafter non significantly

increased towards the end of the experiment. In group II there was no change up to

day 10 and increased significantly (P ≤ 0.05) on day 25 and thereafter non

significantly declined towards the end of the experiment. Further there was significant

difference on day -10 (P ≤ 0.05) and on day 10 (P ≤ 0.05) between groups.

The present trend was in agreement with Boustra et al. (2008), who reported

decrease on the day of calving and increase two weeks postpartum. Similarly Kizil et

al. (2007) reported that the Vit. E concentration decreased in animals affected with

clinical and subclinical infections during early lactation soon after parturition. Plasma
concentrations of Vit. E were reported to be significantly lower during peripartum

period (Weisse et al., 1990).

There were two possible explanations for the lowered concentrations of Vit. E

observed in group II. Primarily first explanation was that, in group II the antioxidant,

Vit. E available in the system might have got exhausted in a transitory effect, due to

increased production of lipid peroxides. If this concept could be true some time later

in the immediate post partum the levels might have returned to that of group I. Second

explanation was that, the animals in group II might have had deficit reserves of Vit. E.

in the body. Animals in group II suffered from oxidative stress at parturition, which

was evident from the higher MDA concentrations recorded on day 0 over that of

group I. Further, Vit. E levels in group II remained lower throughout the course of the

experiment. Probably, in the present study the second concept of deficit reserves of

Vit.E in the body should be correct. This might have led to increased susceptibility to

oxidative stress and compromised the immune regulatory mechanisms (Samanta et

al., 2005). As a result animals in group II were affected with uterine infections.

During the process of infection and inflammation, production of oxygen free

radicals increased leading to oxidative stress. Due to oxidative stress endogenous

antioxidant, Vit. E in the system was utilized (Kizil et al., 2007). There are two forms

of oxidative stress; acute and chronic in nature based on the duration and presence of

high levels of free radicals in the body. Acute oxidative stress is expressed as a

sudden increase in free radicals due to parturition where as in chronic conditions

constant production of free radicals might occur relative to healthy bodies.

Insufficient Vit. E regeneration system and high levels of free radicals persist in the

body in chronic infections as result cell membranes of immune cells might be

damaged and the body becomes susceptible to diseases (Sordillo, 2005;

Boustra, 2010). Further, Vit. E levels was significantly correlated with MDA in group

I (r: - 0.36206; P ≤ 0.05) and II (r: - 0.47241; P ≤ 0.05).

5.2.2 GSH

The GSH concentration was 0.74± 0.12, 0.53± 0.15, 0.48± 0.15, 0.26 ± 0.05

and 0.11 ± 0.03 µ mol/L in group I; and 0.89± 0.18, 0.46± 0.07, 0.77± 0.24, 0.25±

0.07 and 0.20± 0.03 µ mol/L in group II on day -10, 0, 10, 25 and CUI, respectively.

The mean values recorded in the present study were not in line with earlier reports

(Kizil et al., 2007; Ahmed et al., 2010). The variations reported in different studies

were attributed to age, breed, timing and frequency of sampling, nutrition,

environment, analytical procedures adopted to measure and management practices

(Bell, 1995; Castillo et al., 2005).

In the present study though the mean levels of GSH were apparently higher in

group II on day 10, in general the profiles followed nearly a similar pattern in both

groups. There was a significant difference in GSH levels between collection intervals

in group I and II (P ≤ 0.05). In both group I and II the mean GSH concentration

declined significantly (P ≤ 0.05) on day 0 and remained non significant up to day 10,

later significantly declined on day 25 (P ≤ 0.05) and thereafter continued to decrease

non significantly towards the end of the experiment. Further, there was no significant

difference between groups at different collection intervals.

The trend observed in the levels of GSH was similar to (Sharma et al., 2011),

who reported decreased GSH concentration in dairy cows in early lactation compared

to advance pregnant. It was demonstrated that GSH concentration decreased towards

the end of the experiment. It showed that the animals had higher concentration of

GSH before parturition and later gradually declined as uterine involution progressed.

It implied that production of GSH from GSSH (oxidized glutathione) was more prior
to parturition and less after parturition. Generally, whenever cell experiences

oxidative stress, GSSH is transformed to GSH resulting in enhanced concentrations of

the later (Park, 1997). This concept might be true for the present trend observed in

GSH levels. The apparent higher value of GSH observed in group II on day 10 could

be due to uterine infections. Due to infection the cells might have experienced

oxidative stress as a result more and more GSSH could have been converted to GSH

as a function of scavenging antioxidant defense mechanism.

The possibility for the higher concentrations of GSH observed in group II on

day 10 could be due to involvement of GSH dependent enzymes GPx and GR

(Glutathione- reductase) to control oxidative stress caused during infections. The

coupled activity of these two enzymes would be probably enhanced, leading to

intense regeneration of reduced glutathione (GSH) from the oxidized form (GSSH)

obtained after reduction of peroxides into alcohols (Kizil et al., 2007). Further GSH

concentration in group II was significantly correlated with MDA (r: 0.392004;

P ≤ 0.05) and Vit.E levels (r: - 0.34468 P ≤ 0.05), while in group I there was no

correlation between the same. Probably, in group I GSH values might reflect

physiological phenomenon. Whereas in group II uterine infection triggered oxidative

stress might have disturbed the inherent physiological homeostasis (Yavari et al.,

2008).

5.2.3 Catalase

The Catalase activity was 0.056 ± 0.03, 0.008± 0.002, 0.008± 0.003, 0.040±

0.007 and 0.07± 0.01 KU/g Hb in group I and 0.06± 0.04, 0.018± 0.007, 0.007±

0.003, 0.508± 0.332 and 0.47± 0.02 KU/g Hb in group II on day -10, 0, 10, 25 and

CUI, respectively. The mean values recorded in the present study were not in

agreement with earlier reports (Hanafi et al., 2008; Pintea et al., 2006). The variations
in the mean values of Catalase activity in different studies were attributed to age,

breed, timing and frequency of sampling, nutrition, analytical procedures and

management (Bell, 1995; Castillo et al., 2005).

Catalase activity significantly differed between collection intervals in group I

and II (P ≤ 0.05). In Group I Catalase activity was not increased significantly towards

the end of the experiment. Where as in group II the trend was similar to that of group

I until day 10, later significantly increased on day 25 and thereafter apparently

remained stable. Further, there was no significant difference between groups at

different collection intervals. (Sharma et al., 2011) compared the Catalase activities

between advance pregnant and early lactating dairy cows. They reported increased

Catalase activity in early lactating cows (First 4 weeks of lactation) over that of

advance pregnant cows. A similar trend was observed in the present study, where

Catalase activity was increased on day 25.

The present trend observed in Catalase activity was correlated with the values

of MDA in group I (r: - 0.1625) and II (r: - 0.39205; P ≤ 0.05). It showed that up to

day 10 the Catalase activity decreased, since it might have been involved in the

defense mechanism to neutralize the lipid peroxides. Thereafter, an increase in

Catalase activity was observed in both groups and was correlated with decreased

values of MDA recorded at the corresponding periods (Sharma et al., 2011). Further,

in group II Catalase activity increased suddenly at day 25, which might be correlated

with decreased values of MDA recorded during that period.

During late pregnancy and early lactation, increased demand for

micronutrients do not usually fulfill the resulting deficiencies occurring due to natural

protective substances or excess exposure to stimulators of ROS and this might result

in increased lipid peroxidation and decreased levels of antioxidant enzymes. The

Catalase might have important functions in alleviating the toxic effects of ROS (Kale

et al., 1999).

5.2.4 Superoxide Dismutase

The SOD activity was 35.12± 2.00, 118.25± 18.3, 122.0± 36.2, 71± 14.5

and 36 ± 1.99 KU/mg Hb/min in group I; and 61± 14.7, 95± 22.2, 98.5± 15.5, 78.6±

16 and 52.6± 15.7 KU/mg Hb/min in group II on day -10, 0, 10, 25 and CUI,

respectively. The mean values recorded in the present study were not in line with

earlier reports (Pintea et al., 2006; Hanafi et al., 2008). The trend was comparable

with (Pathan et al., 2010; Sharma et al., 2011). The variations in the mean values of

SOD reported in different studies were attributed to age, breed, timing and frequency

of sampling, nutrition, environment, analytical procedures and management (Bell,

1995; Castillo et al., 2005).

Sharma et al. (2011) compared SOD activities between advance pregnant and

early lactating dairy cows. They have reported increased SOD activity in early

lactating cows (First 4 weeks of lactation) over that of advance pregnant cows.

Similar trend was observed in the present study where SOD activity was increased at

day 0 and at day 10. Similarly (Pathan et al., 2010) reported increased SOD activity

on day 0 and on day 15 which correlates with present study. There was significant

difference in profiles recorded between collection intervals in group I and II.

(P ≤ 0.05) In general in both groups the SOD activity increased until day 10

postpartum and there after decreased non-significantly until completion of the

experiment. However, in group I there was a significant raise in SOD activity on day

0, with peak value on day 10, which was non significant and there after gradually

declined until completion of the experiment.

 SOD activity followed a similar pattern both in group I and II, but it was non

significant. From the present study it appeared that animals experienced oxidative

stress up to day 10, as a result SOD activity increased proportionally in both groups to

convert superoxide radicals to hydrogen peroxides. There after the trend was a

gradual non-significant decline towards end of the experiment, showing that animals

were relieved from oxidative stress. Further, SOD activity was correlated positively

with MDA and negatively with Vit. E values and Catalase activity in both groups. The

present results were in agreement with earlier publications (Pathan et al., 2010;

Sharma et al., 2011).

5.3 Glucose

The Glucose concentration was 31.67 ± 3.14, 42.05 ± 8.04, 21.37 ± 3.88,

23.47 ± 5.11, and 35.12± 6.17 mg/dl in group-I and 30.81 ± 4.17, 42.8 ± 10.1, 29.6 ±

5.8, 32.5 ± 6.83 and 43.5 ± 7.61 mg/dl in group II on day -10, 0, 10, 25 and CUI,

respectively . The mean values recorded in the present study were not in line with

Mandali et al. (2002), Castillo et al. (2005) and Turk et al. (2008), who reported

higher values. The variations in the mean values of glucose reported across

publications were attributed to age, breed and nutrition (Bell, 1995; Castillo et al.,

2005).

There was significant difference in profiles recorded between collection

intervals in group I. In group I the trend increased non significantly on day 0, later

significantly declined on day 10 and thereafter non significantly increased. Where as

in group II there was no significant difference between collection intervals. In general

in both groups maximum induction in glucose concentration was observed on day 0.

Further, there was no significant difference between groups at any collection interval.

The levels of glucose recorded during peripartum period might be considered normal
eventual phenomena associated with body`s demands to meet the requirements of

fetus, calving and lactation (Castillo et al., 2005). It was felt that in the absence of

coverage of other parameters like non esterified fatty acids (NEFA), beta hydroxy

butyrate, nutritional components, which were beyond the scope of the present study, it

would be very difficult to draw light based on glucose levels.

5.4 Total proteins

The total proteins concentration was 8.5 ± 0.28, 9.17 ± 0.80, 9.41 ± 0.53, 9.11

± 0.21 and 9.35 ± 0.24 in group I and 9.11 ± 0.56, 9.35 ± 0.63, 9.11 ± 0.46, 9.96 ±

0.66 and 9.13 ± 0.64 in group II, on day -10, 0, 10, 25 and CUI, respectively. The

mean values recorded in the present study were not in line with earlier reports

(Sahadev et al., 2007; Mandali et al., 2002). However, the trend was comparable with

Castillo et al., 2005) The variations in the mean values of total proteins reported in

different studies were attributed to age, breed, timing and frequency of sampling,

nutrition, environment, analytical procedures and management (Bell, 1995; Castillo et

al., 2005).

There was no significant difference either between groups or between

collection intervals. The values might be reflecting physiological concentrations only.

Of course, the fractions of total proteins, which were not determined in the present

study, might have thrown more light. It was felt that determination of simply total

proteins might not provide information relating to oxidative stress.

5.5 Progesterone

The blood plasma progesterone concentration was 1.01 ± 0.142, 0.083 ±

0.040, 0.233 ± 0.021, 0.366 ± 0.108 and 0.383 ± 0.164 ng/ml in group I and 1.6 ±

0.233, 0.516 ± 0.340, 0.466 ± 0.158, 0.116 ± 0.040 and 0.35 ± 0.27 ng/ml in group II

on day -10, 0, 10, 25 and CUI, respectively. The mean values recorded in the present
study were not in line with earlier reports in buffaloes (Srinivasrao, 2004) and cows

(Kafi et al., 2010). However, the trend was comparable with Saxena et al. (1995).

Probably, sample size, timing and frequency of sampling, breed; and the factors

governing uterine involution viz., production capacities, nutrition and management

could have resulted in variations in progesterone concentrations reported in different

studies (Mwaanga et al., 2000).

The determination of progesterone was suggested as a criterion for assessing

ovarian status of postpartum cows and buffaloes (Tiwari et al., 1995). It was

suggested that the progesterone might remain at baseline concentration through most

of the postpartum period, however the concentrations were reported to be fluctuating

possibly due to the presence of short lived dominant luteinized ovarian follicles

(Yelich et al., 1997). Further, this group of scientists reported that the transitory

increase in progesterone concentration during uterine involution prior to the first

postpartum estrus might possibly play a role in the preparation of reproductive tract

for subsequent reproductive functions. The animals are resistant to uterine infections

when progesterone concentrations are basal and susceptible when progesterone

concentrations are increased (Seals et al., 2002). Spontaneous uterine infections in

dairy cows do not usually develop until after formation of the first postpartum corpus

luteum, although bacterial contamination can be sufficient to induce the onset of

puerperal metritis very soon after calving when progesterone concentrations are basal

(Gregory, 2003).

There was significant difference between collection intervals in group I and II

(P ≤ 0.05). In group I there was a significant decline (P ≤ 0.05) on day 0, there after

non significant increase was noted until completion of the experiment. In group II,

there was a significant decline on day 0 (P ≤ 0.05) and thereafter non significantly
declined. The trend differed significantly between groups on day -10 and day 25

(P ≤ 0.05). Apparently the progesterone concentrations were higher during early part

of the experiment in group II than I and it was group II suffered from uterine

infections. It was group II that had higher MDA levels and lower antioxidant status

during early part of the experiment. The high progesterone levels in group II on day -

10 and 0 was correlated with high levels of lipid peroxides and decreased levels of

antioxidants. Elsewhere it was stated that progesterone down regulates the host’s

immune defense (Lewis, 2003). Though the available information was insufficient to

draw such a conclusion, its role could not be undermined. Significantly low levels of

progesterone observed on day 25 in group II could be just a chance occurrence,

because in 40% (2/6) of animals detectable levels were not observed.

5.6 Uterine involution

The mean days of uterine involution in group I and II was 36.5 ± 0.653 and

36.16 ± 0.94. Uterine involution is affected clinically by parity, retained placenta,

puerperal endometritis and ovarian cysts which develop during involution process

(Jaroslav et al., 2005). There was no significant difference in the mean days of uterine

involution between group I and II. The present values were not in line with Usmani et

al. (2001), Nechifor et al. (2011), however comparable with Cengic et al. (2012), who

reported that cows with normal puerperium have completed uterine involution during

38-45 days post partum. In the present study though the group II animals suffered

from uterine infections postpartum, the uterine involution as examined clinically by

rectal examination was completed in 36.16 days. But cytological examination of

endometrial smears revealed presence of neutrophils significantly in higher side,

conforming presence of uterine infection even after declaring completion of uterine

involution grossly by rectal examination. It was felt that routine practice of defining
completion of uterine involution through rectal examination might be inadequate and

the laboratory diagnostic tool, the endometrial cytology might be more useful to

appropriately declare completion of uterine involution.

5.7 Endometrial cytology

The mean count of Neutrophils in endometrial cytological smears was 31.66

and 7.00% in group I and 78.33 and 57.16% on day 25 and CUI, respectively and

differed significantly between sampling intervals and between groups (P ≤ 0.05).

In the earlier reports the Neutrophils count of 25% was reported during

different postpartum intervals. In those studies the postpartum period was not well

defined (Kasimanickam et al., 2004; Gilbert et al., 2005; Sheldon et al., 2006;

Kaufmann et al., 2009). And the samples were obtained at a wide range of postpartum

intervals as against on a specific day in the present study. However, Barlund et al.

(2008) reported 8% Neutrophils count on day 28-41postpartum. Of course the present

findings were in line with Santos et al. (2009), who reported 5.5% Neutrophils at 7

weeks postpartum. Further in those reports the methodology of sampling was lavage

technique as against cytobrush technique in the present study. However, in the

literature neutrophils counts were decreasing with increase in postpartum interval and

the present observations were in agreement with those reports.

In general in both groups there was a decline in the Neutrophils counts from

day 25 to CUI. In group II uterine infection was persisting on CUI as was evident

from the high Neutrophils count of 57.16%. While in group I the count was 7% on

CUI. During the early period of the experiment itself the animals in group II had high

profiles of lipid peroxides and low antioxidant status and later affected with uterine

infections, which persisted on CUI also. It implied the routine clinical diagnosis of

uterine involution through rectal examination was not sufficient enough to declare
completion of involution in its true sense. Based on the present findings it was felt

that examination of uterine cytological smears is to be made a routine for judging

completion of uterine involution.

It was concluded that there were significant differences between two groups of

cows investigated. The high MDA profiles recorded on day 0 and low antioxidant

status observed in group II animals might have compromised host`s immune defense

and suffered them from uterine infections. Further observed that the presence of

Neutrophils significantly in high percentages in uterine cytological smears obtained at

CUI (after morphological completion of uterine involution) indicated the persistent of

infections after morphological completion of involution. From the present study it was

suggested that the routine rectal examination of the tract is insufficient to conclude

completion of involution. It was felt that in routine practice the endometrial cytology

technique may be adopted in clinical diagnosis of uterine involution. Future studies

should design a well defined research on a large population nullifying the influence of

parity, season and managemental practices. Such a refined experiment might throw

more light to develop a system that may help to identify the markers to predict the

periparturient complications in dairy cattle.

 CHAPTER VI
6. SUMMARY

The transition period is, physiologically, the most important, highly sensitive

and venerable in the reproductive life of dairy cows. The physiological and metabolic

changes occurring in peripartum period imposes oxidative and directs the subsequent

reproductive events in dairy cows. The present study titled “Evaluation of oxidative

stress in transition cows and post partum endometrial cytology” was carried out at

College of Veterinary Science, Tirupati. The objective of the study was to evaluate

oxidative stress, antioxidant status and endometrial cytology for predicting the uterine

involution in cows.

A total of 14 multiparous crossbred cows maintained in a commercial farm

were assigned to group I (8 Healthy) & II (6 Infected). Blood samples were collected

periodically on day -10, 0, 10, 25 and CUI. Spectrophotometric procedures were

adopted to determine concentrations of MDA, Vit.E, GSH, SOD, Catalase, glucose

and total proteins in addition to endometrial cytological smears on day 25 and

CUI and ELISA kit for progesterone determination.

The mean blood plasma concentration of MDA was 4.48 ± 0.72, 5.88 ± 1.01,

7.19 ± 1.12, 4.63 ± 0.75 and 2.96 ± 0.40; and 4.12 ± 0.48, 7.05 ± 1.42, 7.11 ±

0.96, 2.7 ± 0.45 and 2.7 ± 0.5 µmol/L on day -10, 0, 10, 25 and CUI in group I and II,

respectively . There was a significant difference between collection intervals in group

I and II (P ≤ 0.05). In both groups in general the trend was increasing to the peak by

day 10 post partum and thereafter continued to decline towards the end of the

experiment. Further in group I the MDA concentration was negatively correlated

with Vit.E and positively correlated with Catalase activity. Where as in group II the
MDA concentration was negatively correlated with Vit.E and Catalase activity and

positively correlated with GSH.

The Vit.E concentration was 4.16 ± 0.33, 0.73 ± 0.20, 0.86 ± 0.15, 1.93 ±

0.87 and 4.95 ± 2.36 mg/L in group I and 1.6 ± 0.33 ,1.14 ± 0.32, 1.49 ± 0.27, 2.73 ±

0.48 and 2.5 ± 0.51 mg/L in group II on day -10, 0, 10, 25 and CUI, respectively.

There was a significant difference in Vit.E profiles between collection intervals in

group I and II. In group I the mean Vit. E concentration declined significantly by day

0 and remained stable up to day 10 and thereafter continued to increase towards the

end of the experiment. In group II the concentration non significantly fluctuated up to

day 10 and thereafter the increase was significant towards the end of the experiment.

Further there was significant difference on day -10 (P ≤ 0.05) and on day 10 (P ≤

0.05) and non significant on 0, 25 and CUI between groups.

The GSH concentration was 0.74± 0.12, 0.53± 0.15, 0.48± 0.15, 0.26 ± 0.05

and 0.11 ± 0.03 µ mol/L in group I; and 0.89± 0.18, 0.46± 0.07, 0.77± 0.24, 0.25±

0.07 and 0.20± 0.03 µ mol/L in group II on day -10, 0, 10, 25 and CUI, respectively.

There was a significant difference between collection intervals in group I and II (P ≤

0.05). In general in both groups a similar trend was noticed between day – 10 and 0

peripartum and after day 10 postpartum. In group I an initial significant fall in the

concentration was found till day 0 (P ≤ 0.05) and continued to decline further non

significantly and later significantly declined (P ≤ 0.05) by day 25 postpartum which

continued to decline further non significantly. In group II except an abrupt raise

between day 0 and 10 peripartum, the trend was similar to that of group I. Further

there was no significant difference between groups at different collection intervals.

The Catalase activity was 0.056 ± 0.03, 0.008± 0.002, 0.008± 0.003, 0.040±

0.007 and 0.07± 0.01 KU/g Hb in group I and 0.06± 0.04, 0.018± 0.007, 0.007±
0.003, 0.508± 0.332 and 0.47± 0.02 KU/g Hb in group II on day -10, 0, 10, 25 and

CUI, respectively. There was a significant difference in the mean values of Catalase

activity in group I and II (P ≤ 0.05). In group I the mean activity of Catalase non

significantly declined till postpartum day 10 and thereafter continued to increase

reaching significant peak value at CUI 45 postpartum. By and a large a similar trend

was observed in group II, but the rise between day 10 and 25 postpartum after an

initial decline till day 10 postpartum, was significant.

. The SOD activity was 35.12± 2.00, 118.25± 18.3, 122.0± 36.2, 71± 14.5

and 36 ± 1.99 KU/mg Hb/min in group I; and 61± 14.7, 95± 22.2, 98.5± 15.5, 78.6±

16 and 52.6± 15.7 KU/mg Hb/min in group II on day -10, 0, 10, 25 and CUI,

respectively . There was significant difference in profiles recorded between collection

intervals in group I and II. (P ≤ 0.05) In general in both groups the trend was

increasing to the peak by day 10 and thereafter declined towards end of the

experiment period. Further there was no significant difference between groups at

different collection intervals.

The Plasma Glucose concentration was 31.67± 3.14, 42.05± 8.04, 21.37 ±

3.88, 23.4 ± 5.11 and 35.12± 6.17 mg/dl in group I and 30.81± 4.17, 42.8 ±

10.1,29.6± 5.8, 32.5 ± 6.83 and 43.5± 7.61 mg/dl in group II on day -10, 0, 10, 25

and CUI, respectively. There was significant difference in profiles recorded between

collection intervals in group I and II. In general in both groups the trend was

increasing to the peak value recorded at day 0 and thereafter continued to decline up

to day 25 and then started increasing towards end of the experiment period. There

was no significant difference between groups at different collection intervals.

The total proteins concentration was 8.5 ± 0.28, 9.17 ± 0.80, 9.41 ± 0.53,

9.11 ± 0.21 and 9.35 ± 0.24 g/dL in group I and 9.11 ± 0.56, 9.35 ± 0.63, 9.11 ±
0.46, 9.96 ± 0.66 and 9.13 ± 0.64 g/dL on day -10, 0, 10, 25 and CUI in group II,

respectively and did not differ. In general in both groups the trend was increasing to

the peak by day 10 and thereafter continued to remain same towards the end of the

experiment period. There was no significant difference between groups at different

collection intervals.

The blood plasma progesterone concentration was 1.01 ± 0.142, 0.083 ±

0.040, 0.233 ± 0.021, 0.366 ± 0.108 and 0.383 ± 0.164 ng/ml in group I and 1.6 ±

0.233, 0.516 ± 0.340, 0.466 ± 0.158, 0.116 ± 0.040 and 0.35 ± 0.27 ng/ml in group II

on day -10, 0, 10, 25 and CUI, respectively. There was significant difference between

collection intervals in group I and II (P ≤ 0.05). In group I there was a significant

decline (P ≤ 0.05) on day 0, thereafter non significant increase was noted until

completion of the experiment. In group II, there was a significant decline on day 0 (P

≤ 0.05) and thereafter non significantly declined. The trend differed significantly

between groups on day -10 and 25 (P ≤ 0.05).

In group I the mean percent of Neutrophils in the endometrial cytological

smears were 31.66 and 7.00 at day 25 and CUI postpartum. In group II the mean

percent of Neutrophils were 78.33 and 57.16 and differed significantly between

collection intervals. The counts differed significantly between sampling intervals in

both groups (P ≤ 0.05).

It was concluded that there were significant differences between two groups of

cows investigated. The high MDA profiles recorded on day 0 and low antioxidant

status observed in group II animals might have compromised host`s immune defense

and suffered them from uterine infections. Further observed the presence of

Neutrophils significantly in high percentages in uterine cytological smears obtained at

CUI (after morphological completion of uterine involution) indicated the persistent of

infections after morphological completion of involution. It was felt that in routine

practice the endometrial cytology technique may be adopted in clinical diagnosis of

uterine involution
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Fig 2: Profiles of MDA (µ mol/L).

7
Concentration of MDA (µmol/L)

4
group I
3 group II

0
-20 -10 0 10 20 30 40
Peripartum period (days)
Table 1: Profiles of plasma MDA (Mean ± SE) in peripartum crossbred cows (µ mol/L).

Peripartum period (days)

Group -10 0 10 25 CUI

I (8) 4.48 ± 0.72bc 5.88 ± 1.01ab 7.19 ± 1.12a 4.63 ± 0.75bc 2.96 ± 0.40c

II (6) 4.12 ± 0.48b 7.05 ± 1.42a 7.11 ± 0.96 a 2.7 ± 0.45b 2.7 ± 0.5b

Figures in parentheses are the number of observations.

CUI: Completion of Uterine involution.

Means bearing at least one common alphabet as superscript in rows did not differ

(P ≤ 0.05).

Table 2: Analysis of variance for MDA.

Source of
Group SS df MS F
Variation
Between
81.00775 4 20.25194 3.568803*
Groups
I Within
198.615 35 5.674714
Groups
Total 279.6228 39
Between
118.8128 4 29.70321 6.627468*
Groups
II Within
112.0458 25 4.481833
Groups
Total 230.8587 29

*(P ≤ 0.05)
Table 3: Results of student`s t-test for MDA.

Peripartum Group-I Group-II

period t-stat
(days) (8) (6)

-10 4.48 4.12 0.408NS

0 5.88 7.07 0.679 NS

10 7.11 7.19 0.052 NS

25 4.63 2.7 2.187*

CUI 2.96 2.7 0.383 NS

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

*(P ≤ 0.05)

NS: Non-significant.
Fig 3: Profiles of Vit.E (mg/L).

5
Concentration of Vit.E (mg/L)

3
group I
group II
2

0
-20 -10 0 10 20 30 40
Peripartum period (days)
 Table 4: Profiles of plasma Vit.E (Mean ± SE) in peripartum crossbred cows (mg/L).

Peripartum period (days)

Group
-10 0 10 25 CUI

I (8) 4.16 ± 0.33a 0.73 ± 0.20b 0.86 ± 0.15b 1.93 ± 0.87ab 4.95 ± 2.36a

II (6) 1.6 ± 0.33ab 1.14 ± 0.32b 1.49 ± 0.27b 2.73 ± 0.48a 2.5 ± 0.51ab

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

Means bearing at least one common alphabet as superscript in rows did not differ

(P ≤ 0.05).

Table 5: Analysis of variance for Vit.E.

Source of
Group SS df MS F
Variation
Between
119.22 4 29.80506 2.851799*
Groups
I Within
365.79 35 10.45132
Groups
Total 485.01 39
Between
11.31617 4 2.829042 2.965763*
Groups
II Within
23.8475 25 0.9539
Groups
Total 35.16367 29

*(P ≤ 0.05)
Table 6: Results of student`s t-test for Vit.E.

Peripartum Group-I Group-II

period t-stat
(days) (8) (6)

-10 4.16 1.6 5.403*

0 0.73 1.14 1.047 NS

10 0.86 1.49 2.029*

25 1.93 2.73 0.797 NS

CUI 4.95 2.5 1.012 NS

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

*(P ≤ 0.05)

NS: Non-significant.
Fig 4: Profiles of GSH (µ mol/L).

0.9

0.8
Concentration of GSH (µmol/L)

0.7

0.6

0.5
group I
0.4 group II

0.3

0.2

0.1

0
-20 -10 0 10 20 30 40
Peripartum period (days)
 Table 7: Profiles of plasma GSH (Mean ± SE) in peripartum crossbred cows (µ mol/L).

Peripartum period (days)

Group
-10 0 10 25 CUI

I (8) 0.74 ± 0.12a 0.53 ± 0.15b 0.48 ± 0.15b 0.26 ± 0.05c 0.11 ± 0.03 c

II (6) 0.89 ± 0.18 a 0.46 ± 0.07bc 0.77 ± 0.24ac 0.25 ± 0.07b 0.20 ± 0.03b

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

Means bearing at least one common alphabet as superscript in rows did not differ

(P ≤ 0.05).

Table 8: Analysis of variance for GSH.

Source of
Group SS df MS F
Variation
Between
1.920607 4 0.480152 4.491388*
Groups
I Within Groups 3.741675 35 0.106905
Total 5.662282 39
Between
2.309386 4 0.577347 4.627622*
Groups
II Within Groups 3.119025 25 0.124761
Total 5.428411 29

*(P ≤ 0.05)
Table 9: Results of student`s t-test for GSH.

Group-I Group-II
Peripartum
t-stat
period (days)
(8) (6)

-10 0.7472 0.8945 0.6682NS

0 0.539 0.466 0.436NS

10 0.485 0.779 1.0273NS

25 0.266 0.250 0.167NS

CUI 0.1173 0.2008 1.652NS

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

NS: Non-significant.

Table 10: Catalase activity in erythrocyte hemolysate (Mean ± SE) in peripartum crossbred
cows (KU/g Hb).
 Peripartum period (days)
Group
-10 0 10 25 CUI

I (8) 0.056 ± 0.03ab 0.008 ± 0.002b 0.008 ± 0.003b 0.040 ± 0.007ab 0.07 ± 0.01a

II (6) 0.06 ± 0.04b 0.018 ± 0.007b 0.007 ± 0.003b 0.508 ± 0.332a 0.47 ± 0.02a

Figures in parenthesis indicate the number of observations.

CUI: Completion Uterine involution.

Means bearing at least one common alphabet as superscript in rows did not differ

(P ≤ 0.05).

Table 11: Analysis of variance for Catalase.

Source of
Group Variation SS df MS F
Between
0.027423 4 0.006856 2.708642*
Groups
I
Within Groups 0.088588 35 0.002531
Total 0.11601 39
Between
1.555065 4 0.388766 2.872875*
Groups

II Within Groups 3.383077 25 0.135323

Total 4.938143 29

*(P ≤ 0.05)
Table 12: Results of student`s t-test for Catalase.

Peripartum Group-I Group-II

period t-stat
(days) (8) (6)

-10 0.0556 0.0648 0.1772NS

0 0.0089 0.0186 1.2111NS

10 0.0089 0.0079 0.1937NS

25 0.0408 0.508 1.407NS

CUI 0.0757 0.4775 12.638*

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

*(P ≤ 0.05)

NS: Non-significant
Table 13: SOD activity in erythrocyte hemolysate (Mean ± SE) in peripartum crossbred cows
(KU/mg Hb/min).

Peripartum period (days)

Group CUI
-10 0 10 25

I (8) 35.12 ± 2.0b 118.25 ± 18.3a 122.0 ± 36.2a 71 ± 14.5ab 36 ± 1.99b

II (6) 61 ± 14.7 95 ± 22.2 98.5 ± 15.5 78.6 ± 16.8 52.6 ± 15.7

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

Means bearing at least one common alphabet as superscript in rows did not differ

(P ≤ 0.05).

Table 14: Analysis of variance for Superoxide dismutase.

Source of
Group SS df MS F
Variation
Between
57565.6 4 14391.4 4.803429*
Groups
I
Within Groups 104862.4 35 2996.068
Total 162428 39
Between
9822 4 2455.5 1.377261NS
Groups
II
Within Groups 44572.17 25 1782.887
Total 54394.17 29

*(P ≤ 0.05)

NS: Non-significant.
Table 15: Results of student`s t-test for Superoxide dismutase

Group-I Group-II
Peripartum
t-stat
period (days)
(8) (6)

-10 35.12 61 1.734NS

0 118.25 95 0.805 NS

10 122 98.5 0.5959 NS

25 71 78.66 0.343NS

CUI 36 52.66 1.0514NS

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

NS: Non-significant

Table 16: Profiles of plasma Glucose (Mean ± SE) in peripartum crossbred cows (mg/dl).
 Peripartum period (days)

Group -10 0 10 25 CUI

I (8) 31.67 ± 3.14a 42.05 ± 8.04a 21.37 ± 3.88b 23.47 ± 5.11b 35.12 ± 6.17ab

II (6) 30.81 ± 4.17 42.8 ± 10.1 29.6 ± 5.8 32.5 ± 6.83 43.5 ± 7.61

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

Means bearing at least one common alphabet as superscript in rows did not differ

(P ≤ 0.05).

Table 17: Analysis of variance for plasma glucose.

Source of
Group SS df MS F
Variation
Between
2308.016 4 577.004 2.343356NS
Groups
I Within
8618.04 35 246.2297
Groups
Total 10926.06 39
Between
1101.58 4 275.3964 0.886551NS
Groups
II Within
7765.945 25 310.6378
Groups
Total 8867.53 29

NS: Non-significant
Table 18: Results of student`s t-test for plasma glucose.

Peripartum Group-I Group-II t-stat

period
(days) (8) (6)

-10 31.6 30.81 0.164 NS

0 42.05 42.8 0.0605NS

10 21.3 29.6 0.788NS

25 23.4 32.5 1.416NS

CUI 35.1 43.5 0.859NS

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

NS: Non-significant
Table 19: Profiles of plasma Total proteins (Mean ± SE) in crossbred cows (g/dl).

Peripartum period (days)

Group -10 0 10 25 CUI

I (8) 8.5 ± 0.28 9.17 ± 0.80 9.41 ± 0.53 9.11 ± 0.21 9.35 ± 0.24

II (6) 9.11 ± 0.56 9.35 ± 0.63 9.11 ± 0.46 9.96 ± 0.66 9.13 ± 0.64

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

Table 20: Analysis of variance for plasma Total proteins.

Source of
Group SS df MS F
Variation
Between
4.2035 4 1.050875 0.581857NS
Groups

I Within Groups 63.2125 35 1.806071

Total 67.416 39

Between
3.161147 4 0.790287 0.368048NS
Groups

II Within Groups 53.681 25 2.14724

Total 56.84215 29

NS: Non-significant
Table 21: Results of student`s t-test for plasma Total proteins.

Group-I Group-II
Peripartum
t-stat
period (days)
(8) (6)

-10 8.5 9.11 0.9767NS

0 9.17 9.35 0.1704NS

10 9.41 9.11 0.420 NS

25 9.11 9.96 1.2079NS

CUI 9.35 9.13 0.3134 NS

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

NS: Non-significant
Fig 5: Profiles of Progesterone concentration (ng/ml).

1.8

1.6
Concentration of progestrone (ng/ml)

1.4

1.2

0.8 group I
group II
0.6

0.4

0.2

0
-20 -10 0 10 20 30 40
Peripartum period (days)
 Table 22: Profiles of plasma Progesterone (Mean ± SE) in peripartum crossbred cows
(ng/ml).

Peripartum period (days)

Group
-10 0 10 25 CUI

I (8) 1.01 ± 0.142a 0.083 ± 0.040b 0.233 ± 0.021b 0.366 ± 0.108b 0.383 ± 0.164b

II (6) 1.6 ± 0.233a 0.516 ± 0.340b 0.466 ± 0.158c 0.116 ± 0.040c 0.35 ± 0.272c

Figures in parentheses are the number of observations.

CUI: Completion of Uterine involution.

Means bearing at least one common alphabet as superscript in rows did not differ

(P ≤ 0.05).

Table 23: Analysis of variance for plasma Progesterone.

Source of SS df MS F
Group
Variation
Between
3.05 4 0.7625 10.407*
Groups

I Within Groups 1.831 25 0.0732

Total 4.881 29
Between
7.922 4 1.908 6.07*
Groups

II Within Groups 8.14 25 0.325

Total 16.067 29

*(P ≤ 0.05)
Table 24: Results of student`s t-test for Plasma Progesterone concentration.

Peripartum Group-I Group-II

period t-stat
(days) (8) (6)

-10 1.01 1.6 2.13*

0 0.083 0.516 1.265NS

10 0.233 0.466 1.459NS

25 0.366 0.116 2.160*

CUI 0.383 0.35 0.104NS

Figures in parenthesis indicate the number of observations.

CUI: Completion of Uterine involution.

*(P ≤ 0.05)

NS: Non-significant
Fig 7: Comparison of endometrial neutrophils counts between day 25 and CUI.

90

80

70

60
%Neutrophils

50

group I
40
group II
30

20

10

0
25 CUI
Postpartum(days)