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Study on Persistent Uterine Infection, Ovarian Activity and Reproduction in

Postpartum in Murrah Buffaloes B.U. Wakayo

1, P.S. Brar

1, S. Prabhakar

2, S.P.S. Ghuman

3 and A.K. Arora

4

College of Veterinary Medicine-Jigjiga University, P.O.Box 1020, Jigjiga, (Ethiopian Somali

Regional State)

1,2Department of Veterinary Gynecology and Obstetrics,

3,4Department of Veterinary Microbiology, Guru

Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab-India

*Corresponding author: fikeurga@gmail.com

Rec. Date: Aug 19, 2014 01:55

Accept Date: Sep 10, 2014 17:24

Published Online: October 16, 2014

DOI 10.5455/ijlr.20140910052449

Abstract

Study was conducted on 17 (8 normal and 9 assisted, calving) Murrah buffaloes to evaluate persistent

uterine infections, ovarian activity and reproductive performance. Uterine status was monitored until 35

days in milk (DIM) based on vaginal inspection, rectal examination and evaluation of uterine content.

Ovarian activity was assessed by rectal examination and plasma progesterone estimation. Fetid

muco/purulent vaginal content decreased from 5 (29.4 %) at 21 DIM to 3 (17.6 %) at 35 DIM.

Meanwhile, 10 (58.8 %) of the apparently healthy buffaloes exhibited cytological uterine inflammation at

35 DIM. Fetid purulent uterine contents (Score ≥ 2) was frequently accompanied by muco/purulent

vaginal discharge and exhibited higher polymorphoneuclear leukocyte (PMNLs) % and exclusive

involvement of recognized uterine pathogens (p<0.05). Persistent uterine affections were associated with

reduced ovarian activity and poor reproductive performance. Inspection of vaginal content can provide a

good indication as to uterine health status as well as future reproductive prospects.

Key words: Murrah Buffalo, Uterine infection, Ovarian activity, Reproductive performance

Introduction

The buffalo uterus is subjected to bacterial contamination at calving followed by cyclic clearance and

recontamination for over 5 weeks postpartum. Animals with heavy bacterial contamination and/or poor

immune competence develop persistent uterine infections (Azawi 2010). Infections disrupt structural and

functional integrity of uterus and prevent embryonic development (Azawi et al 2008). Moreover, uterine

bacterial and inflammatory products suppress; hypothalmo-pituitary function, ovarian follicular

development, ovulation and luteal function (Sheldon et al 2002; Ahmed et al 2007; Williams et al 2007;

Hanafi et al 2008). Ultimately, buffaloes with persistent uterine infection tend to have prolonged

acyclicity and reduced fertility on service which extends the calving interval. This study tried to evaluate

different uterine health indicators and assess their implications on ovarian dynamics and reproductive

performance in postpartum Murrah buffaloes.

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Material and Methods

Animals and management

Parturient Murrah buffaloes (8 normal and 9 assisted, calving) from 8 dairy holdings in Ludhian district,

India, were used for the study. Animals were housed under tie -tall barns and loose housing systems and

fed on mixed ration (chaffed forage, hay/silage and concentrates). Mineral block and water were freely

accessible. Buffaloes were monitored at calving and dystocia cases were given obstetrical assistance.

Ceftiofur Sodium (1.5mg/Kg, intramuscular (im), once daily, for 5 days from calving day) was given to

all animals with supportive therapy only for those receiving assistance.

Examination

Timing to expulsion of fetal membranes was noted (< or ≥ 12 hrs) to determine retention of fetal

membranes (RFM). Uterine and ovarian status was monitored weekly until 35 days in milk (DIM) by;

general inspection, inspection of vaginal contents and rectal examination of the reproductive tract.

Behavioral estrus and breeding record of study animals was monitored until 120 DIM.

Sampling and physical evaluation

Ten ml jugular blood was aseptically collected in heparinized centrifuge tubes on 21 and 35 DIM. Low

volume (≥ 5 ml.) uterine flushing samples were collected aseptically on 21 and 35 DIM according to the

procedure outlined by Galvao et al (2011). The physical quality (color, consistency and odor) of uterine

contents was assigned increasing subjective scores and subsequently used for laboratory analysis.

Cytological analysis of uterine contents

Plain uterine samples were homogenized and smeared on microscope slides (duplicate). Smears were

stained with Leishman’s stain solution (s.d. fine-chem limited, Mumbi- India) for 2 minute, stain was

subsequently diluted with normal saline for 8 minutes and smears were finally washed under running

water and air dried. Microscopic examination (x100) and differential cell count (200 cells excluding

erythrocytes) was performed to estimate polymorphonuclear leukocytes/neutrophils % (PMNLs %).

Bacteriological analysis of uterine contents

Uterine flushing sample kept in carry blair transport media were inoculated on to Blood agar (BA) and

Wilkins Chalgreen agar (WC) media (HiMedia Laboratories Pvt. Ltd. India), and incubated at 37o

C for 24

to 72 hrs under aerobic and anaerobic conditions, respectively. Specification of bacteria was done as

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outlined by Quinn et al (1999) and magnitude of bacterial contamination was estimated as outlined in

later parts.

Plasma progesterone estimation

Plasma was separated by centrifugation of whole blood samples and stored under deep freeze condition.

Thawed plasma progesterone level estimated by liquid phase Radioimmunoassay (RIA) using polyclonal

progesterone antisera raised in the RIA lab at department of veterinary gynaecology and obstetrics-Guru

Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana (Ghuman et al 2009).

Working definitions

Clinical uterine disorders were classified according to vaginal inspection findings (fetid

purulent/muco-purulent material) as outlined by Sheldon et al (2006) and Azawi (2013).

Progress of uterine involution was classified at 35 DIM according to criteria described by

Raizada and Pandey (1982).

The physical quality of uterine content was subjectively scored on a scale of 0 - 3 as outlined

below;

(0) Clear to light brown watery fluid without off smell

(1) Clear to yellowish white mucoid fluid with some off smell

(2) Thick white puss with pronounced foul smell

(3) Dirty brownish watery to purulent with pronounced foul smell.

In the absence of abnormal vaginal content, uterine PMNLs content > 18 % and 10 % indicated

sub-clinical endometritis (SCE) at 21 and 35 DIM, respectively (Sheldone et al 2006).

The bacteriological quality of uterine contents was characterized by two criteria i.e. Bacterial

isolation rate reflecting number of bacterial isolates per sample and Semi-quantitative bacterial

load indicating number of distinct primary colonies scored on a 0 - 5 scale as described by

Williams et al (2005).

Rectally ovarian status classified as inactive (small, with flat and smooth surface) or active

(larger size, containing antral follicles (AF) and /or corpus luteum (CL)). Plasma progesterone

levels ≥ 1 ng/µl indicated luteal activity (Royal et al 2000).

Buffaloes not observed in estrus until 90 DIM were considered to have extended anestrous and

those not conceiving until 120 DIM had prolonged open period (Prasad and Neeraj 2010).

Data Analysis

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Data was analyzed on SPSS 16 (Version 16.0, SPSS Inc., Munich, Germany). Descriptive statistics

(Count (N), Percentages (%) and Mean ± SE and %) were used to summarize findings. Association

between categorical variables was tested using SPSS cross tab and Fisher’s exact procedures. Comparison

of numeric variables among animals with different calving and puerperal traits was conducted using one

way analysis of variance. Statistical significance was determined at p ≤ 0.05.

Results and Discussion

Obstetrical Assistance and Uterine Health

The current findings were in agreement with similar observations indicating that assistance at calving

predisposed buffaloes to uterine infection (Prabhakar et al 2011). Indication of clinical uterine pathologies

declined with time whereas cytological evidence of active inflammation persisted in majority of animals

(Table 1). The former trend could reflect progressive clearance of pathogenic bacterial invaders whereas

the latter could indicate carryover effect of sever prior assaults or a continued low-grade infection.

Table 1: Uterine health indicators according to calving condition (N (%))

Parameters of uterine and ovarian status Overall Calving p Value

Normal (8) Assisted (9)

RFM 3(17.6) 0 3 (33.3) 0.072

Metritis < 14 DIM 7(41.2) 1 (12.5) 6(66.7) 0.024

Clinical endometritis (CE)-21 DIM 5(29.4) 0 5(55.6) 0.018

Sub-Clinical endometritis (SCE)-21 DIM 9(52.9) 7(87.5) 2(22.2)

Clinical endometritis (CE)-35 DIM 3(17.6) 0 3 (33.3) 0.138

Sub-Clinical endometritis (SCE)-35 DIM 10(58.8) 5(62.5) 5(55.6)

Delayed uterine involution-35 DIM 5(29.4) 0 5(55.6) 0.012

Characterization of Uterine Contents

There was strong association (p=0.012) between the quality (subjective score) of uterine contents at 21

and 35 DIM (Table 2). Average uterine PMNLs % declined from 36 ± 4.1 at 21 DIM to 24.8 ± 3.8 at 35

DIM (p=0.000). Bacteria negative samples increased from 2 (11.8 %) at 21 DIM to 10 (58.8 %) at 35

DIM, particularly in samples with subjective score of 0. Local immune mechanisms gradually reduce the

incidence and diversity of bacterial invaders returning uterine cavity to a sterile state after 28-35 days

postpartum (Das et al 2013).

Uterine contents with subjective score of ≥ 2 exhibited higher PMNLs % and higher bacterial isolation

with exclusive involvement of recognized uterine pathogens (A. pyogenes and Bacteroides spp.) (Table

2/3). Purulent or fetid genital discharge was associated with growth density of pathogenic bacteria (A.

pyogenes, F. necrophorum, Proteus spp., M. heamolytica and E. coli) but not opportunist contaminants

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(Williams et al 2005). Moreover, uterine content scores of ≥ 2 exhibited strong associations with

simultaneous observations of muco/purulent vaginal content and delayed progress of uterine involution

(Table 4). The latter are considered reliable clinical indicators of persistent uterine infection (Noakes et al

2009; Azawi 2013). Therefore, uterine content with subjective score of ≥ 2 was considered indicative of

clinical pathological condition.

Table 2: Physical quality and PMNLs content (Mean ± SE) of uterine flushing contents

Uterine flushing 21 DIM 35 DIM

Score Characteristics N (%) PMNLs% N (%) PMNLs%

0 Clear to light brown watery fluid without off

smell

9(52.9) 42.8 ± 18 10(58.8) 14.7 ± 3.1

1 Clear to yellowish white mucoid fluid with

some off smell

3(17.6) 45.4 ± 2.75 - -

2 Thick white puss with pronounced foul smell 4(23.5) 54.3 ± 4.9 4(23.5) 41.5 ± 4.5

3 Dirty brownish watery to purulent with

pronounced foul smell.

1(5.9) 65 3(17.6) 36.1 ± 7.3

p Value 0.958 0.001

Table 3: Bacterial profile of uterine content according to subjective physical quality score

Quality of uterine

flushing content

Bacterial isolation

rate (Mean ± SE)

Semi-quantitative

Bacterial load score

(Mean ± SE)

Bacterial isolates

21 DIM 35 DIM 21 DIM 35 DIM

(0)Clear to light brown

watery without off

smell

0.9 ± 0.2 0.4±0.3 2.3±0.6 0.9±0.5 Negative (10),E. coli (3), S. aureus

(2), P. aeruginosa (2), Anthracoides

(1), Citrobacter spp. (1), Enterobacter

spp. (1), Staphylococcus spp. (1),

Seratia spp. (1)

(1) Clear to yellowish

white mucoid fluid with

some off smell

1.7± 0.7 - 3.7±0.7 - P. aeruginosa (1), Staphylococcus

spp. (1), Streptococcsu spp. (1),

Bacillus spp. (1),

(2)Thick white puss

with pronounced foul

smell

1.75 ±

0.25

1.25±0.6 5±0.7 2.25±1.3 Negative (1 ), A. pyogenes (3),

Bacteroides spp (3), P. aeruginosa

(3), S. aureus (1), E. coli (1),

Staphylococcus spp. (1), Seratia spp.

(1)

(3)Dirty brownish

watery purulent with

pronounced foul smell.

4 1.3±0.9 8 4.3±3 Negative (1 ), A. pyogenes (1),

Bacteroides spp (1), P. aeruginosa

(2), S. aureus (2), Klebsiella spp (1)

p Value 0.006 0.310 0.014 0.224

Impact of Persistent Uterine Infections on Ovarian Activity and Reproductive Performance

Most normal buffaloes initiate follicular dynamics shortly after calving with first dominant follicle (>

8mm) detectable by 20-24 days and first ovulation between 24-55 days postpartum (El-Wishy 2007;

Usmani et al 1985). In this study, rectal ovarian follicular activity increased with increasing postpartum

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interval. Plasma progesterone s at 21 and 35 DIM ranged between 0 - 2 ng/µl and 0.2 - 1.6 ng/µl,

respectively. Majority of buffaloes lacked plasma luteal activity until 35 DIM which could be attributed to

poor follicular development and blockage of ovulation.

Table 4: Contrast of Clinical Uterine Disease Indicators

Uterine flushing content Foul muco/purulent vaginal

content (N (%))

Delayed uterine

involution

35 DIM (N (%)) 21 DIM 35 DIM

0)Clear to light brown watery without off smell 0 0 0

(1) Clear to yellowish white mucoid fluid with some

off smell

1(33.3) - -

(2)Thick white puss with pronounced foul smell 3(75) 1(25) 3(75)

(3)Dirty brownish watery purulent with pronounced

foul smell.

1(100) 2(66.7) 2(66.7)

p Value 0.017 0.027 0.006

Persistent uterine infections contributed to prolonged acyclicity in Murrah buffaloes. Compared to

apparently healthy buffaloes those with fetid-purulent/muco-purulent vaginal content had higher

frequency of rectally inactive ovaries at 21 (50 % vs. 80 %, p=0.252) and 35 (28.6 % vs. 100 %,

p=0.023), DIM respectively. Similarly, 5 (100 %) buffaloes with normal (Score < 2) and 5 (41.7 %) with

abnormal (Score ≥ 2) uterine content at 21 DIM lacked rectal ovarian activity (p=0.026). At, 35 DIM,

proportion of respective groups lacking ovarian activity was 3 (20 %) and 5 (71.4 %), respectively

(p=0.034). Buffaloes with atypical uterine content (Score ≥ 2) exhibited limited development of

prominent AF (Table 5) and lacked plasma luteal activity until 35 DIM. Current findings affirm previous

observation that pathogenic bacterial infection of the uterus reduced; development of ovarian follicles,

follicular steroidogenesis, follicular ovulatory prospects well as luteal development and progesterone

secretion (Sheldon et al 2002; Ahmed et al 2007; Williams et al 2007; Hanafi et al 2008).

Overall, 13 (76.5 %) buffaloes were observed in estrus at least once before 90 DIM of which 12 (92.3 %)

were submitted to AI prior to 120 DIM and only 8 (47.1 %) succeeded in establishing pregnancy. The

findings indicate that majority of buffaloes failed to maintain the recommended optimum calving interval

of 13-15 months (Prasad and Neeraj 2010) due to; extended anestrous 4 (44.4 %), prolonged voluntary

period 1(11.1 %) and failure to conceive on service 4 (44.4 %). All buffaloes with prolonged open period

(> 120 DIM) had fetid muco/purulent vaginal content and pathological uterine content (Score ≥ 2) at 35

DIM (p<0.05).

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Table 5: Ovarian findings according to quality of uterine flushing content (N (%))

Quality of uterine flushing content 21 DIM 35 DIM

Inactive Small

Follicle

Large

Follicle

Inactive Small

Follicle

Large

Follicle

CL

(0)Clear to light brown watery no off-

smell

4(44.4) 2(22.2) 3(33.3) 2(20)) 3(30) 4(40) 1(10)

(1)Yellowish-white mucoid with faint

off smell

1(33.3) 2(66.7) 0 - - - -

(2)Thick white puss with pronounced

foul smell

4(100) 0 0 2(50) 1(25) 1(25) 0

(3)Dirty brownish watery purulent

with pronounced foul smell.

1(100) 0 0 3(100) 0 0 0

In conclusion, uterine bacterial contamination was very common after calving. Obstetrical assistance

predisposed buffaloes to persistent clinical uterine affections. The latter were typically associated with a

fetid uterine content contaminated by pathogenic bacteria frequently accompanied with simultaneous

purulent/muco-purulent vaginal content. Buffalo with persistent fetid purulent/sanguine-purulent uterine

and vaginal content tend to have delayed initiation of ovarian follicular dynamics and ovulation as well as

reduced chance of having successful breeding before 120 DIM.

Acknowledgment

The authors thank the Guru Anagd Dev Veterinary and Animal Sciences University (GADVASU) for

providing the necessary facilities and inputs required for the study and respective dairy holding for

allowing use of study animals.

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