






































Type of the Paper (Article


 

  

 

 
Cluj Vet J 2024, 29, 3 http://clujveterinaryjournal.ro 

Article 

Study on subclinical endometritis in cattle of Kathmandu Valley by using WST 
(WST) 
Praphulla Panta1, Umesh Prasad Mandal2  

1 Himalayan College of Agricultural Science and Technology; vetdr.praphulla@gmail.com 
2 Himalayan College of Agricultural Science and Technology; Faculty of Dairy Technology; 
 mandal4you@gmail.com 
* Praphulla Panta: vetdr.praphulla@gmail.com; mob.no.:977-9860559244 

Abstract:  

The current investigation aimed to identify the different levels of nonspecific bacterial infection in the genitalia of repeat-breeding 
cattle of Kathmandu District using White Side Test (WST). A total of 307 crossbred Holstein Friesian cows were considered for this 
purpose, out of which 246 repeat-breeding cattle were under treatment and 61 cattle with normal cycle (control group) were artifi-
cially inseminated at their first service. Cervical mucus samples were collected 8 to 12 hours after the first signs of behavioral estrus, 
and subjected to a WST (WST) and bacteriological examination. From the results of WST, it has been inferred that only 9(14.75%) 
animals in the control group were positive and the remaining 52(85.24%) were negative. However, the majority of repeat-breeding 
animals were positive 190 (77.22%)) and only 56 (22.76%) of animals were found negative. Microbiological examination of 190 sam-
ples revealed E. coli 54(28.42%), Staphylococcus spp. 39(20.52%), Streptococcus spp. 26(13.68%), Corynebacterium spp. 10(5.263%), Pseudo-
monas spp. 7(3.68%), Klebsiella spp. 9(4.73%), and mix bacterial growth 44(23.15%). The remaining 56 repeat-breeding cattle were neg-
ative with WST, possibly due to the absence of bacteria. Other causes may be viruses, placental retention, ovarian cysts, dystocia, etc. 
The findings of the present study divulge that WST may be utilized in the field to detect subclinical endometritis caused by bacteria.  

Keywords: WST, Subclinical endometritis, estrus cervical mucus, Repeat Breeding  
. 

1. Introduction 
Uterine inflammation is a serious problem in Nepal's breeding dairy cows, causing 

considerable financial losses [1]. Uterine infections include endometritis, metritis, muco-
metra, and pyometra. Among this endometritis is one of the major gynecology problems 
affecting reproductive efficacy and the economy of milk production in dairy animals [1].  

Among different types of endometritis, subclinical or occult endometritis poses a se-
rious threat to fertility since it is extremely difficult to identify by standard clinical-gyne-
cological examination. Subclinical endometritis is currently being considered as a signifi-
cant factor in dairy cows' lower conception rates [2]. Endometrial inflammation modifies 
the uterine environment, impairing the ability to conceive or sustain an embryo. In Nepal, 
subclinical endometritis has been identified as the most frequent reason why bovines are 
unable to conceive. The occurrence of endometritis has been associated with a weak uter-
ine defense mechanism (UDM) in females [3] mainly due to inadequate husbandry and 
sanitation practices and exposed of genital organs to microbial invasion either at parturi-
tion or during estrus. 

Bovine genital tract infections can be specific or non-specific, with the latter being the 
most important in causing endometritis. Some of the specific pathogens such as Campylo-
bactor fetus and Trichomonas fetus develop infection without any predisposing cause, 
whereas non-specific pathogens residing in the genital tract as saprophytes and set infec-
tion under favorable conditions. The postpartum bovine uterus contains wide range of 
bacteria, both Gram-positive and Gram-negative, aerobes and anaerobes [4] Numerous 
bacteria have also been found and grown from the uterus of cattle with endometritis, in-
cluding Streptococci, Staphylococci, Corynebacterium spp., Klebsiella spp., and E. coli. 

Received: 21.05.2024 

Accepted: 10.08.2024 

Published: 12.09.2024 

DOI: 10.52331/t4syss17 

 

 

 

Copyright: © 2024 by the authors. 

Submitted for possible open access 

publication under the terms and 

conditions of the Creative Commons 

Attribution (CC BY) license 

(http://creativecommons.org/licenses

/by/4.0/). 

https://doi.org/10.52331/t4syss17


Cluj Vet J 2024, 29, 3 21 of 30 
 

 

One of the emerging businesses in Nepal that offers a reliable source of income is 
livestock farming. Farmers favor more productive cattle over less productive cattle due to 
the market's rising need for milk, particularly cross Holstein Friesian (with good blood 
levels) livestock since they produce milk in greater quantities. From India to Nepal, a siz-
able number of crossbred cattle are imported. Cattle with various diseases and reproduc-
tive disorders have been reported to be imported as a result of improper inspection during 
quarantine or illegally importing cattle from open borders. Some of the most significant 
issues farmers today are dealing with include the retention of the placenta, repeat breed-
ing, extended calving intervals, infertility, prolapse, dystocia, anestrus, cervicitis, cystic 
ovary, and endometritis. This research was undertaken with the intention of improvising 
these issues by addressing all of the aforementioned challenges. Early detection of metritis 
or endometritis will help to reduce future treatment costs and complications brought on 
by this condition.  

WST has been described as a test for the diagnosis of subclinical endometritis [5]. The 
normal oestrus discharge has too low number of leucocytes to bring about a positive color 
change on doing WST [5]. The subclinical discharges have moderate number of leucocytes 
causing a moderate color change compared to the clinical discharge having very high 
number causing intense color reaction. The relative efficacy of Whiteside test is 77.5% [6]. 

Therefore, it was intended for the current study to assess the validity of the WST in 
detecting genital infection in repeat-breeding cattle raised in the Kathmandu District. Re-
peat breeders are cows with apparently healthy genitalia, normal estrous, has no abnor-
mal vaginal discharge but failed to conceive in three or more consecutive inseminations 
[7]. To further demonstrate that the results of the WST for endometritis are accurate under 
field conditions, bacterial culture was performed. 

WST is use for diagnosis of subclinical uterine infection in repeat breeding cows [8]. 
The investigators classify the positive samples as slight, moderate, and intense subclinical 
infections based on the color reaction, such as light yellow, yellow, and dark yellow. The 
test revealed 92.85%of cervical mucus positive from repeat breeding cows indicating sub-
clinical uterine infection. Out of the total, 44.75% were slightly positive, 42.50% were mod-
erate, whereas, 15-38 % were detected to have an intense uterine infection. 

 WST is interpreted as negative, mild, moderate, and severe as recorded 16.67, 66.67, 
09.26, and 7.41 percent color pattern of no color, light yellow, yellow, and dark yellow 
type, respectively in repeat breeder crossbred Jersey cows. On culture 60 (75.00 %) of the 
80 repeat breeding animals were found positive and 20 (25.00 %) were free of bacteria. The 
different bacterial isolates from repeat breeding cows were Staphylococcus spp. 16 (21.05%), 
E. coli 14 (18.42%), Bacillus spp. 10 (13.16%), Corynebacterium spp. 10 (13.16%), Pseudo-
monas spp. 8 (10.53%), Proteus spp. 8 (10.53%), Klebsiella spp. 6 (7.89%), and Streptococcus 
spp. 4 (5.26%). [9] 

2. Materials and Methods 

2.1 Site of Study 

The study was conducted in a Kathmandu district located in Kathmandu Valley, Bagmati Province of Nepal. 
It covers an area of 413.69 km2 (159.73 sq mi) with 11 municipalities [10]. Total Population of cattle in Kath-
mandu District is about 52,470 [11]. 

The Kathmandu district is surrounded by: 

East: Bhaktapur District and Kavrepalanchok District 

West: Dhading District and Nuwakot District 

North: Nuwakot District and Sindhupalchok District 

South: Lalitpur District and Makwanpur District  



Cluj Vet J 2024, 29, 3 22 of 30 
 

 

2.2 Sample size 

For estimating the minimum sample size for an unknown population, the formula estimated by 𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪 
was [12] 

𝑵𝑵Ο  =    z
2×p(1-p)

 e2   

z =  critical value of the desired level of confidence (i.e. 95%) its z-value is 1.96 

e =  margin of error i.e., is 5% 

p =  estimated proportion of an attribute that is present in the population (that can be a maximum of 50%) 

n0 =  sample size/proportion of unknown population 

now, 

𝑵𝑵Ο  =    z
2×p(1-p)

 e2   

= 
(1.96)2*0.5(1-0.5)

 (0.05)2  A  = ~385 cattle’s 

First, calculate the proportion and then use the formulation for the population correction factor to calculate 
the exact sample size (formula) 

𝑵𝑵 =  
n0×N

 n0+(N-1)  

n =  sample size of known population 

n0 =  proportion of the unknown population 

N =  known population  

Total Population of cattle in Kathmandu District is about 52,470  

Now, 

𝑪𝑪 =  
n0×N

 n0+(N-1)  

=  
385×52470

 385+(52470-1)      = ~383 cattle’s 

Therefore, the total sample size is 383 but due to the pandemic of Lumpy Skin Disease (LSD), only 307 sam-
ples were collected.  

2.3 Sampling Technique 

Simple random sampling was adapted as it favors the unbiased selection of animals [12] and a total of 307 
samples were collected from cattle at the heat from different farms in the Kathmandu district.  



Cluj Vet J 2024, 29, 3 23 of 30 
 

 

Out of total 307 samples, 12 (Budhanilkantha Municipality), 18 (Chandragiri Municipality), 10 (Dakshinkali 
Urban Municipality), 35 (Kageshwori Manahara Municipality), 6 (Kathmandu Metropolitan City), 123 (Kir-
tipur Municipality), 10 (Nagarjun Municipality), 9 (Shankharapur Urban Municipality), 11(Tarkeshwor Mu-
nicipality), 56 (Tokha Municipality), were collected.  (Table 1) 

Table 1: No. of sample collection from different municipalities of Kathmandu District 

Municipality of Kathmandu District Total number of samples collected 

Budhanilkantha Municipality 12 

Chandragiri Municipality 18 

Dakshinkali Municipality 10 

Gokarneshwor Municipality 35 

Kageshwori Manahara Municipality 17 

Kathmandu Metropolitan City 6 

Kirtipur Municipality 123 

Nagarjun Municipality 10 

Shankharapur Municipality 9 

Tarkeshwor Municipality 11 

Tokha Municipality 56 

Total 307 

2.4 Experimental Design 

The present study was conducted at different farms of Kathmandu District during the period from May 1 to 
September 1 2023. Several 307 crossbred Holstein Friesian cattle of 246 repeat breeding cows presented for 
treatment and 61 clinically normal cows presented for artificial insemination at their first service, were se-
lected for sample collection (shown in supplementary figure S1).  

All of the animal's estrus cervical mucus was collected 8 to 12 hours after the first signs of behavioral estrus. 
Animals were properly restrained for this purpose. For rectal feces evacuation, a full-sleeve gloved left hand 
was inserted per rectum and lubricated (with sterile liquid paraffin/oil). The vulva and the perineum were 
both properly cleaned with soap and water, dried with soft absorbent cotton, and then disinfected with 70% 
alcohol. The vagina had been penetrated with a 10 mL sterilized pipette and a 20 mL disposable syringe was 
linked to the pipette's exterior pointed end. 

The pipette was guided through the vaginal canal to the cervical os or mid cervix and was grabbed by the 
left hand already introduced per rectum then cervical mucus was aspirated from the cervical os or mid cervix 
and then transferred to a sterilized test tube. 



Cluj Vet J 2024, 29, 3 24 of 30 
 

 

For WST, 1 mL of estrus cervical mucus was heated with an equal volume of 5% sodium hydroxide up to 
boiling point (shown in supplementary figure S2) and after cooling the intensity of color changes was stud-
ied and graded as normal (no color), mild infection (light yellow color), moderate infection (yellow color) 
and severe infection (dark yellow color) [13]. (shown in supplementary figure S3) 

In animals that were positive for the WST, the cervical mucus was sent for bacteriological culture, and iso-
lation and identification of the organisms were carried out based on cultural, morphological, colony charac-
teristics, motility, and biochemical reactions.  

3. Results 

From the WST out of 307 samples there are 246 repeat breeding cattle, 56(22.76%) showed no color, 
147(59.75%) showed light yellow, 29(11.78%) showed Yellow, and 14(5.69%) showed Dark Yellow color with 
a normal, mild, moderate, and severe grade of infection. The control group (normal cyclic cattle) shows 
52(85.24%) with no color change and 9(14.75%) shows light yellow with normal and mild grades of infection. 
(Table 2)  

Table 2: Results of WST showing grades of infection based on color intensity between repeat breeders 
and normal cyclic (Control group) animals 

Color Intensity Grade of Infection      Repeat Breeding cattle Control group 

N                   % N                      % 

No color (0)     Normal  56 22.76% 52 85.24% 

Light Yellow  +     Mild  147 59.75% 9 14.75% 

Yellow   ++    Moderate  29 11.78% - - 

Dark Yellow   +++    Severe 14 5.69% - - 

Overall  246 100 61 100 

 

Out of 246 repeat breeding cattle, 147 show light yellow color, 29 show yellow color, 14 show dark yellow color, and 56 
show no color change whereas, in the control group 52 show no color change, and 9 show light yellow color. (Figure 2) 

 

 

 

 

 

 



Cluj Vet J 2024, 29, 3 25 of 30 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2: The above bar diagram represents the repeat breeding and control group of cattle about color intensity 
according to WST 

A total number of positive cases in Budhanilkantha Urban Municipality 9(3.65%), Chandragiri Urban Municipality 
14(5.69%), Dakshinkali Urban Municipality 7(2.84%), Dakshinkali Urban Municipality 7(2.84%), Gokarneshwor Urban 
Municipality 28(11.38%), Kirtipur Urban Municipality 105(42.68%), Kageshwori Manahara Urban Municipality 
12(4.47%), Kathmandu Metropolitan City 5(2.03%), Nagarjun Urban Municipality 5(2.03%), Shankharapur Urban Mu-
nicipality 9(3.65%), Tarkeshwor Urban Municipality 7(2.84%), and Tokha Urban Municipality 45(18.29%). (Table 3) 

Table 3: Total number of positive cases from 11 different municipalities 

Municipality of Kathmandu District  Total number of positive cases  Percentage of positive cases  

Budhanilkantha Urban Municipality 9 3.65% 

Chandragiri Urban Municipality 14 5.69% 

Dakshinkali Urban Municipality 7 2.84% 

Gokarneshwor Urban Municipality 28 11.38% 

Kageshwori Manahara Urban Municipality 12 4.47% 

Kathmandu Metropolitan City 5 2.03% 

Kirtipur Urban Municipality 105 42.68% 

56 52

147

9

29

14

0

20

40

60

80

100

120

140

160

Repeat breeding cattle     Control Group

to
ta

l n
um

be
r o

f c
at

tle
s 

No colour Light yellow Yellow Dark Yellow



Cluj Vet J 2024, 29, 3 26 of 30 
 

 

Nagarjun Urban Municipality 5 2.03% 

Shankharapur Urban Municipality 9 3.65% 

Tarkeshwor Urban Municipality 7 2.84% 

Tokha Urban Municipality  45 18.29% 

Total 246 100 

Out of 307 samples collected, Kirtipur Municipalities show the highest percentage of positive cases 105 (42.68%), fol-
lowed by Tokha Municipalities 45 (18.29%), and Gokarneshwor Municipalities 28 (11.38%), while Kathmandu Metro-
politan City shows the lowest percentage of positive cases 5(2.03%), followed by Nagarjun Municipalities 5(2.03%). 
(Figure 3) 

 

 

 

The sample which shows mild (+), moderate (++), and severe (+++) grades of infection from the WST were cultured. i.e., 
190 samples were cultured. In bacterial culture, different bacteria were isolated i.e., E.coli 55(28.94%), Pseudomonas spp. 
7(3.68%), Staphylococcus spp. 39(20.52%), Streptococcus spp. 26(13.68%), Corynebacterium spp. 10(5.26%), Klebsiella spp. 
9(4.73%), and mix bacterial growth were 44(23.15%).Pseudomonas spp. 7(3.68%), Klebsiella spp. 9(4.73%), Corynebacterium 
spp. 10(5.26%), E. coli 55 (28.94%), Staphylococcus spp. 39(20.52%), Streptococcus spp. 26(13.68%), and mix bacterial growth 
were 44(23.15%) seen in (Figure 4).  

42.68%

18.29%

11.28%

3.65%

5.69%

2.84%

4.47%

2.03%

2.03%

3.65%

2.84%

0.00% 5.00% 10.00% 15.00% 20.00% 25.00% 30.00% 35.00% 40.00% 45.00%

Kirtipur  Municipality

Tokha  Municipality

Gokarneshwor  Municipality

Budhanilkantha  Municipality

Chandragiri  Municipality

Dakshinkali  Municipality

Kageshwori Manahara  Municipality

Kathmandu Metropolitan City

Nagarjun  Municipality

Shankharapur  Municipality

Tarkeshwor  Municipality

11 municipalities of Kathamndu District

Figure 3: Total percentage of positive cases in Repeat breeding cattle of different Municipalities 
of Kathmandu 

 

            
    

 

            
    

 

            
    

No. of positive cases in percentage 



Cluj Vet J 2024, 29, 3 27 of 30 
 

 

 

 

 

 

 

 

 

 

 

 

 

              Figure 4: Total percentage of bacteria present in the culture of repeat-breeding cattle 

 

4. Discussion 

From the results of WST, it can be inferred that only 9(14.75%) animals in the control group showed infection 
and the remaining 52(85.24%) animals were free of infection; however, the majority of repeat breeding ani-
mals showed infection 190(77.22%) and only 56(22.76%) of animals were free of infection. Furthermore, on 
bacterial culture, we found E.coli 55 (28.947%), Staphylococcus spp. 39(20.526%), Streptococcus spp. 26(13.684%), 
Corynebacterium spp. 10(5.263%), Pseudomonas spp. 7(3.684%), Klebsiella spp. 9(4.736%), and mix bacterial 
growth were 44(23.157%).  

This result is consistent with the results of the White Side Test in repeat breeder crossbred Jersey cows, which 
were recorded as 16.67, 66.67, 09.26, and 7.41 percent color pattern of no color, light yellow, yellow, and dark 
yellow type, respectively. These results were interpreted as negative, mild, moderate, and severe.  The var-
ious bacteria that were isolated and cultured from repeat breeding cattle included Staphylococcus spp. 16 
(21.05%), E. coli 14 (18.42%), Bacillus spp. 10 (13.16%), Corynebacterium spp. 10 (13.16%), Pseudo-monas spp. 8 
(10.53%), Proteus spp. 8 (10.53%), Klebsiella spp. 6 (7.89%), and Streptococcus spp. 4 (5.26%). Of the 46 (76.67%) 
repeat-breeding cows, a single organism was identified, whereas mixed infections involving many types of 
organisms were found in 14 (23.33%) [9] 

The results of the present study were more or less in agreement where the majority of aerobic bacteria 
(84.72%) are present in the cervical mucus of repeat-breeding cows with subclinical endometritis. Staphylo-
coccus aureus isolates have been shown to make up the biggest proportion of all facultative anaerobic bacte-
rial species isolates, followed by E. coli, Streptococcus spp., Enterobacter spp., Proteus spp., and Pseudomonas spp. 
[14]. 

The reported color reaction on WST in 92.85% of cervical mucus samples of normal animals indicating sub-
clinical uterine infections which differ from our result where 77.22% were positive for WST in repeat breed-
ing cattle [8]. The recorded color changes in 100% samples of cervical mucus in repeat breeding cows suffer-
ing from endometritis which differ from our result [15]. The variance in the infection rate between research 



Cluj Vet J 2024, 29, 3 28 of 30 
 

 

may be attributable to the severity of the infections being studied, sample size and demographic variation, 
and the agro-climatic conditions in the regions where the studies were conducted. 

The proportion of positive cases was highest in Kirtipur Municipalities, where it was 105 (42.68%), and low-
est in Kathmandu Metropolitan City and Nagarjun Municipalities, where it was 5(2.03%) and 5(2.03%), re-
spectively. Less sample collection and a smaller number of cattle being raised may be to reasons for the 
disparity in the results. 

5. Conclusions 
The findings from this study underscore the significant prevalence of nonspecific bacterial genital in-

fections in repeat breeding cattle, as evidenced by the WST. With 77.22% of repeat breeders testing positive 
for infection, compared to a mere 14.75% in the control group, it is clear that these infections pose a major 
concern for cattle reproductive health. The predominance of specific bacterial pathogens, particularly E. coli 
and Staphylococcus spp., highlights the urgent need for targeted management strategies to mitigate their 
impact on fertility. Geographical analysis reveals notable disparities in infection rates across different mu-
nicipalities, suggesting that local management practices, environmental conditions, and herd health proto-
cols may significantly influence the occurrence of these infections. This variability emphasizes the im-
portance of region-specific interventions and monitoring systems to enhance reproductive performance in 
cattle. The WST proves to be a valuable diagnostic tool for identifying subclinical endometritis in cattle, 
facilitating early intervention and potentially improving reproductive outcomes. By employing such a rapid 
and cost-effective test, farmers can make informed decisions regarding treatment and management, ulti-
mately leading to better herd health and productivity. 

Implications for South Asia 
These findings are particularly relevant for South Asian nations, such as Nepal and India, where agriculture 
and dairy production are vital components of the economy. The high prevalence of reproductive infections 
in cattle can lead to significant economic losses due to reduced milk yield and fertility. By utilizing the WST, 
farmers can quickly identify infected animals and implement appropriate management strategies, thus en-
hancing milk production and overall herd health. Improving reproductive health in dairy cattle not only 
increases milk availability, which is crucial for food security, but also supports the livelihoods of millions of 
smallholder farmers dependent on dairy for their income. Enhanced reproductive efficiency can contribute 
to the sustainability of dairy farming in these regions, ensuring that farmers can meet the growing demand 
for dairy products while maintaining the health of their livestock. Future research should focus on elucidat-
ing the relationship between specific bacterial pathogens and reproductive failure, as well as exploring ef-
fective management practices to reduce the incidence of genital infections in cattle populations. These efforts 
will be crucial in advancing cattle health and productivity, ultimately contributing to sustainable livestock 
management practices that benefit both farmers and the broader agricultural economy in South Asia. By 
prioritizing the health of livestock, these nations can secure a more resilient agricultural framework capable 
of withstanding economic and environmental challenges. 
 
 

 
 
 
 
 
 
 
 
  



Cluj Vet J 2024, 29, 3 29 of 30 
 

 

Supplementary Materials (Figures) 
 

  
 
 
 

 
 

  
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 

 
Reference 

 

Figure S1: Collection of cervical mucus 
for sample 

 

     
   

 

     
   

 

     
   

Figure S2: Laboratory Examination of 
samples 

 

    
  

 

    
  

 

    
  

Figure S3: Changes in colour after WST 
 



Cluj Vet J 2024, 29, 3 30 of 30 
 

 

 
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