Trop Anim Health Prod (2014) 46:1435–1439

DOI 10.1007/s11250-014-0661-y

REGULAR ARTICLES

Incidence of subclinical endometritis and its effects

on reproductive performance of crossbred dairy cows
Luisa Cunha Carneiro & Adolfo Firmo Ferreira &
Mariana Padua & João Paulo Saut &
Antonio Sergio Ferraudo & Ricarda Maria dos Santos

Received: 7 November 2013 / Accepted: 19 August 2014 / Published online: 4 September 2014
# Springer Science+Business Media Dordrecht 2014

Abstract In dairy cattle, uterine infections are not life threat- were not influenced by the presence of subclinical endometri-
ening and often unavoidable; however, they reduce fertility tis in crossbred dairy cows.
and increase the production costs of properties. The aim of this
study was to evaluate the incidence of subclinical endometritis Keywords Cytobrush . Neutrophils . Uterine infection .
from 32 to 70 days in milk (DIM) and its effects on the Reproduction
reproductive performance of crossbred dairy cows. Lactating
cows (Holstein/Gir; n=172), with no history of retained pla-
centa, without clinical signs of uterine infection were used. Introduction
The body condition score (BCS) was evaluated on a scale
from 1 to 5. Ultrasound examination was performed to eval- In dairy cows, after the voluntary waiting period, the repro-
uate uterine lining and ovarian activity, while vaginal mucus ductive performance depends on many factors, such as body
was analyzed by gloved hand. The diagnosis of subclinical condition score (BCS), heat detection, insemination tech-
endometritis was performed by endometrial cytobrush tech- nique, semen quality, and uterine health (Noakes et al.
nique. The samples were collected, stained, and examined 2002). The resumption of ovarian cyclicity is one of the most
microscopically; positive cases for subclinical endometritis important events for dairy cows to recover to their reproduc-
were considered with the presence of ≥5 % of neutrophils. tive potential (Kaneko and Kawakami 2009).
Later, the cows were submitted to conventional artificial in- Within the postpartum period, the uterus tolerates the
semination or timed artificial insemination. The incidence of growth of lots of bacteria, such as aerobic and anaerobic.
subclinical endometritis in the herd was 26 %, and this was not The proportion of infected animals or uterine bacterial amount
affected by the season of calving, presence of corpus luteum, usually increases between the seventh and 14th day postpar-
DIM, and parity. Cows with a BCS ≤2.50 had a higher tum. This observation leads to the assumption that there is not
incidence of subclinical endometritis. The conception rate to only a bacterial contamination during postpartum that is re-
first insemination and pregnancy rate at 150 days postpartum sponsible for uterine problems (Sheldon 2004).
During 4 weeks after parturition, the cows immune system
is extremely challenged, most of the cows in the early post-
L. C. Carneiro (*)
partum period develop a non-pathological endometritis, and
Faculty of Agricultural Sciences and Veterinary, UNESP, 1270, the lochia and placenta are usually eliminated by the second
Machado de Assis St, Uberlândia, Minas Gerais 38400112, Brazil week postpartum (Thatcher et al. 2006).
e-mail: luisacunhacarneiro@hotmail.com About 35 % of dairy cows may be clinically affect in the
A. F. Ferreira : M. Padua : J. P. Saut : R. M. dos Santos
first 21 days postpartum (metritis), and approximately 10–
Faculty of Veterinary Medicine, Federal University of Uberlandia, 20 % will remain infected or develop endometritis (Borsberry
3637, República do Piratini St, Uberlândia, Minas Gerais 38402028, and Dobson 1989; McDougall et al. 2007; Sheldon et al.
Brazil 2009).
Inflammatory and endometrial cells can be collected by
A. S. Ferraudo
Faculty of Agricultural Sciences and Veterinary, UNESP, Jaboticabal, swab, in order to diagnose the presence of a subclinical
SP 14884-900, Brazil endometritis, uterine biopsy (most common in mares), uterine
1436 Trop Anim Health Prod (2014) 46:1435–1439

lavage, or by cytobrush technique (Kasimanickam et al. ovarian activity, follicles and/or corpus luteum (CL), an ultra-
2005). Barlund et al. (2008) stated that cytobrush technique sound examination was performed.
is a useful tool and it is considered the most reliable test. The uterine mucus was evaluated by gloved hand accord-
Subclinical endometritis is most commonly diagnosed by ing to Pleticha et al. (2009) and classified as follows: grade 0
uterine cytology and affects about 35–50 % of cows between (no discharge), grade 1 (clear mucus), grade 2 (mucus with
35 and 60 days in milk (DIM) and is associated with a pus flakes), grade 3 (mucopurulent discharge), grade 4 (puru-
reduction in pregnancy rate, increase in time to conception lent discharge), and grade 5 (fetid purulent discharge)
(30–88 days), and increase in 20 % of cows that are not (adapted from McDougall et al. 2007).
pregnant by 300 DIM (Kasimanickam et al. 2004; Gilbert The method selected to diagnose subclinical endometritis
et al. 2005). was the cytobrush technique, where a gynecologic modified
Some important economic losses are caused by uterine sterile brush was introduced into the uterine body through the
infections in dairy farms, since they increase calving interval, vagina then turned approximately one quarter turn to obtain
number of artificial insemination (AI) per conception, culling cellular material from the endometrium. This method was
rate and decrease milk production (Fourichon et al. 2000; used only in animals with clear mucus (without clinical uter-
Drillich et al. 2005). ine infection).
Based on this information, the aim of this study was to After sample collection, the slides were identified (animal
evaluate the incidence of subclinical endometritis between 32 number, date of collection, and property) and prepared by
and 70 days postpartum and their effects on the reproductive rolling the cytobrush with the collected material on a clean
performance of crossbred dairy cows. microscope slide. The slides were air-dried and transported to
the laboratory to be stained by May-Grunwald-Giemsa meth-
od (Vallada 1999) and subsequently subjected to a microscop-
ic evaluation.
Materials and methods The microscopic evaluation was based on the number of
neutrophils present in each slide. Two independent observers
The study was submitted to the Ethics Committee on Animal counted 200 cells under a microscope (Olympus®, CHT-2
Use at the Federal University of Uberlandia and subsequently Optical Co. Ltd, Japan) by ×400 magnification in each blade.
approved (082/11) for this experiment. The cells were classified into epithelial cells, polymorphonu-
All data were collected on a commercial dairy farm, located clear cells (neutrophils), large mononuclear cells (presumed to
in Centralina, Minas Gerais, Brazil, from April 2010 to June be macrophages), and small mononuclear cells (presumed to
2011. For this experiment, 172 crossbred dairy cows (Hol- be lymphocytes). Subclinical endometritis was defined as
stein/Gir) were used. All the cows were lactating milked twice being positive in all those animals that had more than 5 % of
daily (average 17 kg milk/day). During winter, the cows were neutrophils in each slide (Gilbert et al. 2005).
fed with corn or sorghum silage, and during summer, they After evaluation and material collection, the cows were
were managed in rotational grazing system, properly sup- submitted to a reproductive management program. This
plemented with concentrate, mineral salt, and access to consisted in a conventional artificial insemination (AI) or
water ad libitum. The cows were not subjected to any type timed artificial insemination (TAI). The TAI protocol
of environmental control (ventilation, heating, cooling, or consisted of the following: day (D) 0: intramuscular injection
others). The animal zoonotic calendar was regularly of 2.0 mg estradiol benzoate (2.0 mL Estrogin®, Farmavet)
followed to all the state laws for dairy herd (recommended and insertion of a progesterone (P4) device with 1.9 g of P4
vaccination and vermifugation in accordance to the state (CIDR®, Zoetis Animal Health); D7: intramuscular injection
law was performed). of 12.5 mg diniprost tromethamine, PGF2a analog (2.5 mL
Only animals that did not have a history of retained pla- Lutalyse®, Zoetis Animal Health); D9: P4 device removal and
centa, puerperal metritis, or other diseases that required anti- muscular injection of 0.5 mg of estradiol cypionate (0.5 mL of
biotic treatment before collecting uterine material were select- ECP®, Zoetis Animal Health); D11: TAI (adapted from
ed. All animals with clinical uterine infection were excluded Meneghetti et al. 2006). All animals were inseminated with
from the study, based on the presence of a muco-purulent previously analyzed semen.
vaginal discharge or the presence of echogenic contents in Pregnancy diagnosis was performed by ultrasound (Emperor
the uterus during ultrasound examination (Emperor-Vet ® 820-Vet®) 28 days after artificial insemination technique was
820) (adapted from LeBlanc et al. 2002a). fulfilled. All cows that showed embryo with heartbeat was
Reproductive data and endometrial cytology samples were considered to be pregnant. Forty five days after insemination,
collected between 32 and 70 days postpartum. The BCS was pregnancy diagnosis was confirmed by rectal palpation.
evaluated (scale of 1—very thin to 5—obese; Ferguson et al. The effects of season of calving (spring/summer versus
1994). To evaluate the characteristics of the uterine wall, fall/winter), BCS, presence of CL, DIM at the time of
Trop Anim Health Prod (2014) 46:1435–1439 1437

collection, and parity (first versus second versus third or more The presence of subclinical endometritis did not affect
lactations) on the incidence of subclinical endometritis were conception rate at first AI (P > 0.05). Pregnancy rate at
analyzed by logistic regression (Proc Logistic of SAS). The 150 days postpartum was analyzed, and there was no signif-
effect of subclinical endometritis on conception rate to first AI icant difference (P>0.05) between cows with subclinical en-
and pregnancy rate at 150 days postpartum was analyzed by dometritis and those without subclinical endometritis (Fig. 1).
log-rank test by Statistic program, where survival analyses
curves were made. Differences detected at P≤0.05 were con-
sidered significant. Discussion

The prevalence of subclinical endometritis is quite variable

among herds. The diagnosis of endometritis is herd-specific
Results and depends on the sensitivity and specificity of the diagnostic
method. The average of subclinical endometritis in this study
The prevalence of subclinical endometritis in crossbred dairy was 26 %, while Madoz et al. (2013) studied the prevalence of
cows at 32 and 70 DIM in this experiment was 26 % (44/172), subclinical endometritis in grazing dairy cows at 21 to
and the results are summarized in Table 1. Season (spring/ 62 DIM, with 5 % of PMN, and found an average around
autumn versus summer/winter) had no effect on the incidence 17 %. Although a shorter prevalence of subclinical endome-
of subclinical endometritis (P>0.05). Cows with BCS ≤2.50 tritis was reported by these researchers, endometritis diagnosis
showed a higher incidence of subclinical endometritis was performed in the same method (cytobrush technique).
(32.43 %) compared with those with BCS ≥2.75 (16.39 %) Barlund et al. (2008) also found a lower incidence than
(P<0.05). In this work, the presence or absence of CL had no observed in this study. They evaluated 189 dairy cows at 28–
effect on the incidence of subclinical endometritis (P>0.05). 41 days postpartum using the cytobrush technique and ob-
In this experiment, the interval between calving and material served that only 11.1 % had more than 8 % of polymorpho-
collection had no effect on the incidence of subclinical endo- nuclear cells as classified positive for subclinical endometritis.
metritis, even if these evaluations ranged from 32 to 70 days Still, a lower incidence was reported by Dubuc et al. (2010)
postpartum (fifth and tenth week). Parity (first versus second that evaluated 1,295 dairy cows at 35 days postpartum and
versus third or more) did not affect the incidence of subclinical found 18.7 % of cows positive for subclinical endometritis
endometritis (P>0.05). (considering positive those with ≥6 % neutrophils on endo-
metrial samples).
Kasimanickam et al. (2004) also used the cytobrush meth-
Table 1 Effects of season, body condition score, presence of corpus
luteum, days postpartum, and parity on the incidence of subclinical od as a diagnostic in cows around 20–33 days postpartum.
endometritis in crossbred dairy cows, Centralina-MG They found 37 % of cows positive for cytology endometritis;
however, they considered those cows with more than 18 % of
Variable Incidence of subclinical P value
endometritis %
neutrophils in each sample positive. They also related that the
incidence of subclinical endometritis at 20–33 and 34–47 days
Season postpartum in 228 dairy cows was not influenced by season,
Spring/summer 32.35 (22/68) 0.176 as in this study.
Fall/winter 23.08 (24/104) It was expected that those cows that calved in summer had
Body condition score a higher rate of subclinical endometritis than those that calved
≤2.50 32.43 (36/111) 0.025 in winter, due to increased stress and greater challenge of
≥2.75 16.39 (10/61)
environmental contamination; however, no effect of season
Presence of corpus luteum on the incidence of subclinical endometritis was reported in
Without corpus luteum 33.33 (21/63) 0.146 this experiment (P>0.05). On a study conducted by Senosy
With corpus luteum 22.94 (25/109)
and Osawa (2013), no influence of season on the incidence of
Days postpartum
subclinical endometritis was reported, while there was an
30–39 days 27.59 (08/29) 0.560
40–49 days 28.00 (21/75) effect on the PMN% on positive animals diagnosed between
50–59 days 27.66 (13/47)
the hot season and the temperate season.
More than 60 days 19.05 (04/21)
In this study, it was observed that cows with a BCS ≤2.50
Parity
showed higher prevalence of subclinical endometritis
(P<0.05) than those cows with BCS ≥2.75. In a study con-
First 33.33 (13/39) 0.097
Second 33.33 (14/42) ducted by Priest et al. (2013), they also found a lower BCS in
Third or more 20.88 (19/91) cows with subclinical endometritis, when compared to those
without it. In some studies conducted by Bacha and Regassa
1438 Trop Anim Health Prod (2014) 46:1435–1439

Fig. 1 Survival curves for non- Cumulative Proportion Surviving (Kaplan-Meier)

pregnant cows (%) related to days 100%
after parturition, in a dairy herd of 90%
172 Holstein cows, according to

NON-PREGNANT COWS
80%
the presence (POSITIVE) or ab- 70%
sence (NEGATIVE) of subclinical
60%
endometritis
50%
40%
30%
20%
10% NEGATIVE
POSITIVE
0%
20 40 60 80 100 120 140
DAYS AFTER PARTURITION

(2009), the BCS also had an effect on the occurrence of sub- uterine infection during the eighth week after parturition when
clinical endometritis when evaluated during 4 weeks after par- compared to those cows with a healthy uterus.
turition. They found that 64.5 % of the cows with a BCS ≤2.50 In a study with 201 milk cows, Kaufmann et al. (2009)
had subclinical endometritis, and in those cows with a BCS considered positive for subclinical endometritis cows with
≥2.50, only 28.6 % was positive for subclinical endometritis. more than 15 % of PMN cells in samples collected after AI
According to Dubuc et al. (2010), more cows had subclin- around 65 days postpartum and reported a conception rate at
ical endometritis when their BCS was lower than 2.75. Also, first AI of 43.3 % for all cows examined by cytobrush tech-
Dubuc et al. (2011) in another study observed that cows with a nique; also, those cows with 0–15 % of neutrophils in endo-
BCS less than 2.75 were more likely to develop any kind metrial samples or those negative for subclinical endometritis
of uterine disease. had higher conception rate at first service when compared to
Roche et al. (2013) in their studies about BCS during those with more than 15 % of neutrophils (positive for endo-
calving, observed that cows with low and medium BCS had metrial cytological).
proportionately more polymorphonucleated cells in their uter- Madoz et al. (2013) found that subclinical endometritis
ine secretions at 3 and 5 weeks postpartum than those with diagnosed at 21 to 62 DIM decreased the hazard for pregnan-
high BCS, indicating that both low and medium BCS at cy and increased the calving-to-conception interval by 30 days
calving will increase the risk of uterine disease. compared with normal cows; also, Galvao et al. (2009) re-
Williams et al. (2005) concluded that uterine infection also ported that cows with subclinical endometritis had a reduction
affects ovarian function; however, in this work, the presence of 26 % in pregnancy rate at 300 days of lactation, when
or absence of CL had no effect on the incidence of subclinical compared with those without subclinical endometritis, al-
endometritis. In a study conducted by Burke et al. (2010), though, in this study, the presence of subclinical endometritis
using the endometrial cytobrush technique, noticed that cyclic did not influence the pregnancy rate.
cows diagnosed with low amounts of PMN cells ovulated Furthermore, Gilbert et al. (2005) certified that the positive
early (between 63 and 70 days postpartum) when compared diagnosis for subclinical endometritis decreased the propor-
with those with high levels of these cells, which took longer to tion of pregnant cows at 300 days postpartum when compared
ovulate. It is important to notice that the presence of CL affects with those cows negative for subclinical endometritis (63.0
the recovery of the uterus after parturition, directly interfering versus 89.0 %, respectively). Also, Dubuc et al. (2011) dem-
in the future reproductive performance. onstrated that the presence of subclinical endometritis was a
According to Gilbert et al. (2005), cows parity did not key factor in pregnancy rate, with an increase up to 16 days in
influence the prevalence of endometritis, as showed in this days open.
study. However, LeBlanc et al. (2002b) reported that endo- The incidence of subclinical endometritis in crossbred
metritis was more prevalent in mature cows (in their third or dairy cows in this study was 26 %. Calving season, presence
more lactations) than in those in the first and second lactation. of corpus luteum, DIM, and parity did not affect the incidence
Bacha and Regassa (2009) observed that the number of of subclinical endometritis, while cows with BCS ≤2.50 had a
cows with subclinical endometritis decreased in the eighth higher incidence. The presence of subclinical endometritis did
week postpartum when compared with the fourth week post- not affect conception rate at first AI and pregnancy rate at
partum. They found a total of 18 positive cows in the fourth 150 days postpartum in crossbred dairy cows.
week postpartum and 13 of them remained being positive in
the eighth week. This indicated that cows that had uterine Acknowledgments The authors acknowledge the Research Support
infection during the fourth week postpartum had twice risk of Foundation of Minas Gerais state, Brazil, for the financial support.
Trop Anim Health Prod (2014) 46:1435–1439 1439

Conflict of interest The authors declare that they have no conflict of Kaufmann, TB, Drillich, M, Tenhagen, BA, Forderung, D, Heuwieser, W,
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