Short communication: Efficacy of a second intrauterine cephapirin infusion for the treatment of purulent vaginal discharge and endometritis in postpartum dairy cows

Abstract

The objective of this study was to quantify the efficacy of a second intrauterine cephapirin treatment administered 14 d after the initial one on subsequent reproductive performance of postpartum dairy cows affected by purulent vaginal discharge (PVD) or endometritis (ENDO). In total, 4,140 Holstein cows from 30 commercial herds were enrolled in a randomized clinical trial. At 36 (±7) d in milk, cows were examined using the Metricheck device to diagnose PVD. An endometrial cytology sample was also collected from each cow to perform a leukocyte esterase test for diagnosing ENDO. Diagnosis of PVD and ENDO was done cow-side. Cows diagnosed with PVD or ENDO were assigned to receive 1 of 2 treatments: (1) a single intrauterine cephapirin infusion (500 mg of cephapirin benzathin; Metricure, Merck Animal Health, Kirkland, QC, Canada) at the time of initial examination or (2) a single intrauterine infusion at the time of initial examination and a second one 14 d later. Subsequent reproductive and culling events were collected until 200 d in milk. Statistical analyses were performed using univariable and multivariable mixed logistic regression models. In cows affected by PVD, a second intrauterine cephapirin infusion increased the pregnancy risk at first insemination in comparison with cows that only received one treatment (28.0 vs. 38.8%). In cows affected by ENDO, a second treatment also increased the pregnancy risk at first insemination compared with cows that only received one treatment (30.3 vs. 39.2%). Overall, these results demonstrate that administering a second intrauterine cephapirin infusion 14 d after the initial treatment in postpartum cows affected by PVD or ENDO did improve their subsequent reproductive performance.

Full text

ABSTRACT
The objective of this study was to quantify the ef-
ficacy of a second intrauterine cephapirin treatment
administered 14 d after the initial one on subsequent
reproductive performance of postpartum dairy cows
affected by purulent vaginal discharge (PVD) or en-
dometritis (ENDO). In total, 4,140 Holstein cows from
30 commercial herds were enrolled in a randomized
clinical trial. At 36 (±7) d in milk, cows were exam-
ined using the Metricheck device to diagnose PVD. An
endometrial cytology sample was also collected from
each cow to perform a leukocyte esterase test for di-
agnosing ENDO. Diagnosis of PVD and ENDO was
done cow-side. Cows diagnosed with PVD or ENDO
were assigned to receive 1 of 2 treatments: (1) a single
intrauterine cephapirin infusion (500 mg of cephapirin
benzathin; Metricure, Merck Animal Health, Kirkland,
QC, Canada) at the time of initial examination or
(2) a single intrauterine infusion at the time of initial
examination and a second one 14 d later. Subsequent
reproductive and culling events were collected until 200
d in milk. Statistical analyses were performed using
univariable and multivariable mixed logistic regression
models. In cows affected by PVD, a second intrauterine
cephapirin infusion increased the pregnancy risk at
first insemination in comparison with cows that only
received one treatment (28.0 vs. 38.8%). In cows af-
fected by ENDO, a second treatment also increased
the pregnancy risk at first insemination compared with
cows that only received one treatment (30.3 vs. 39.2%).
Overall, these results demonstrate that administering a
second intrauterine cephapirin infusion 14 d after the
initial treatment in postpartum cows affected by PVD
or ENDO did improve their subsequent reproductive
performance.
Key words: dairy cow, cephapirin, purulent vaginal
discharge, endometritis
Short Communication
Purulent vaginal discharge (PVD) and endometritis
(ENDO) are common postpartum reproductive tract
diseases in dairy cows (Cheong et al., 2011; Dubuc and
Denis-Robichaud, 2017) and have a negative effect
on subsequent reproductive performance (LeBlanc et
al., 2002a; Dubuc et al., 2010; de Boer et al., 2015).
Multiple studies have shown the efficacy of intrauter-
ine cephapirin (CEPHA) administration for treating
PVD and ENDO (LeBlanc et al., 2002b; Kasimanickam
et al., 2005; Denis-Robichaud and Dubuc, 2015b). In
these studies, a single infusion of CEPHA improved the
subsequent reproductive performance of treated cows
compared with untreated ones. Over the last 20 yr,
the availability of cow-side tests for diagnosing PVD
and ENDO has increased the awareness about these
diseases on dairy farms. Interestingly, veterinarians
and dairy producers frequently using CEPHA infusion
are questioning if there is a benefit on reproductive
performance when administering a second CEPHA
treatment during the weeks following the initial one.
Unfortunately, no data are currently available to an-
swer this question.
Therefore, the objective of this study was to quantify
the efficacy of a second CEPHA treatment, adminis-
tered 14 d after the initial one, on subsequent repro-
ductive performance of postpartum dairy cows affected
by PVD or ENDO. Our hypothesis was that cows
receiving 2 CEPHA treatments would have improved
reproductive performance compared with cows that
only received one treatment.
Cows from 30 commercial Holstein dairy herds were
enrolled in a randomized clinical trial approved by the
Animal Care Committee of the Université de Montréal
(20-Rech-2061). Herd selection was based on conve-
nience for being located within 1 h of St-Hyacinthe
(QC, Canada), for being a client of the bovine ambula-
tory clinic of the Faculté de médecine vétérinaire of the
Université de Montréal (St-Hyacinthe, QC, Canada),
for having a history of elevated PVD or ENDO preva-
lence, for being visited by a veterinarian every 2 wk
Short communication: Efficacy of a second intrauterine
cephapirin infusion for the treatment of purulent vaginal
discharge and endometritis in postpartum dairy cows
J. Dubuc,* V. Fauteux, M. Villettaz-Robichaud, J.-P. Roy, M. Rousseau, and S. Buczinski
Faculté de médecine vétérinaire, Université de Montréal, 3200 rue Sicotte, Saint-Hyacinthe, Québec, J2S 2M2, Canada
J. Dairy Sci. 104
https://doi.org/10.3168/jds.2020-19537
© 2021, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Received August 27, 2020.
Accepted October 15, 2020.
*Corresponding author: Jocelyn.dubuc@ umontreal .ca

Journal of Dairy Science Vol. 104 No. 3, 2021
(herd health medicine program), and for using a sys-
tematic ovulation synchronization protocol for the first
insemination of cows during their lactation (around 70
DIM).
Once participation was agreed upon, herds were vis-
ited every 14 d by an animal health technician. During
farm visits, cows were enrolled at 36 (±7) DIM and
were examined for reproductive tract diseases. Cows
having a “do not breed” status were not enrolled. After
identifying the cows, the vulva was cleaned with paper
towels and water, and a Metricheck device (Simcro,
Hamilton, New Zealand) was used to assess vaginal
discharge. Scoring was defined as 0 = no discharge,
1 = clear mucus, 2 = mucus with flecks of pus, 3 =
mucopurulent discharge, 4 = purulent discharge, and
5 = foul-smelling discharge (McDougall et al., 2007).
Diagnosis of PVD was based on having a vaginal score
≥3. Once Metricheck testing was completed, a cytologi-
cal sample from the endometrium located in the body
of the uterus was collected using the cytobrush tech-
nique (Kasimanickam et al., 2004). Once the cytobrush
was retrieved from the uterus, a leukocyte esterase
test was performed. The cytobrush was immerged into
a vial containing 1 mL of physiological saline (NaCl
0.9% Irrigation, Baxter Corporation, Mississauga, ON,
Canada), and a leukocyte esterase strip (Multistix 10
SG, Bayer Corporation, Elkart, IN) was dipped in the
solution (Denis-Robichaud and Dubuc, 2015a). Scor-
ing was done after a waiting period of 2 min and was
defined as 0 = negative, 0.5 = trace of leukocytes, 1 =
small amount of leukocytes, 2 = moderate amount of
leukocytes, and 3 = large amount of leukocytes (Couto
et al., 2013). Diagnosis of ENDO was based on having
a leukocyte esterase test score ≥1 (Denis-Robichaud
and Dubuc, 2015a). Diagnosis of PVD and ENDO were
both done cow-side.
All cows that were not affected by PVD or ENDO
at 36 (±7) DIM were enrolled in the unaffected group.
Within each participating herd, all cows diagnosed with
PVD or ENDO were randomly allocated (coin toss)
to 1 of 2 treatment groups. Cows in the first group
(CEPH1X) received a single CEPHA intrauterine
infusion (500 mg of cephapirin benzathin; Metricure,
Merck Animal Health, Kirkland, QC, Canada) at the
time of initial examination. Cows in the second group
(CEPH2X) received a single CEPHA intrauterine
infusion (500 mg of cephapirin benzathin; Metricure,
Merck Animal Health, Kirkland, QC, Canada) at the
time of initial examination and a second one (500 mg of
cephapirin benzathin; Metricure, Merck Animal Health,
Kirkland, QC, Canada) 14 d later (50 ± 7 DIM). Farm-
ers were blinded to disease diagnosis and treatment al-
location, whereas the research staff were not.
Treatment group allocation was stratified based on
reproductive tract disease status (PVD-CEPH1X
vs. PVD-CEPH2X; ENDO-CEPH1X vs. ENDO-
CEPH2X). Cows simultaneously having PVD and
ENDO were enrolled in both strata and allocated to
the same treatment group (CEPH1X or CEPH2X)
by performing only one coin toss. In the end, there
were 5 treatment groups of cows in this experiment
(unaffected, PVD-CEPH1X, PVD-CEPH2X, ENDO-
CEPH1X, and ENDO-CEPH2X). A sample size of 350
cows per treatment group was estimated for the study
based on finding a difference of 10% in pregnancy risk
at first insemination (PRFI) between treatment groups
(CEPH1X: 30%; CEPH2X: 40%), with 95% confidence
and 80% power (Dohoo et al., 2003). Therefore, 700
cows with PVD and 700 cows with ENDO needed to
be recruited. Based on an estimated PVD prevalence of
17%, a total of 4,200 cows would have been necessary.
Based on an estimated ENDO prevalence of 20%, a
total of 3,500 cows would have been necessary. In the
end, a total of 4,200 cows was targeted for this study as
the most limiting sample size.
In all herds, timed AI was exclusively used for breed-
ing. The voluntary waiting period in these herds was
50 DIM. Pregnancy diagnosis was performed by tran-
srectal palpation (using ultrasonography when needed)
between 32 and 46 d after insemination. Reproductive
and culling events were monitored until 200 DIM.
All statistical analyses were performed using SAS
version 9.4 (SAS Institute Inc., Cary, NC). The unit
of interest in this study was the cow. Individual cow
data such as parity (first, second, third, or greater),
season of calving (winter: January to March; spring:
April to June; summer: July to September; fall: Octo-
ber to December), as well as reproductive and culling
events were collected. Descriptive statistics were com-
puted using the FREQ procedure. Prevalence of parity
group, season of calving, PVD, ENDO, culling, and
PRFI were computed overall and within each stratified
subgroup (unaffected, PVD-CEPH1X, PVD-CEPH2X,
ENDO-CEPH1X, and ENDO-CEPH2X). Univariable
associations between PRFI and each aforementioned
variable were computed using mixed logistic regres-
sion models accounting for herd clustering (random
intercept; PROC GLIMMIX). A multivariable mixed
logistic regression model accounting for herd clustering
as a random effect (PROC GLIMMIX) was built. This
model considered PRFI as the dependent variable and
treatment and disease strata as the main independent
variables. Parity and season of calving were offered to
the model as confounders and were kept if their ef-
fect was greater than 10% (Maldonado and Greenland,
1993). Least squares means were computed from the fi-
Dubuc et al.: SHORT COMMUNICATION: EFFICACY OF A SECOND CEPHAPIRIN TREATMENT

Journal of Dairy Science Vol. 104 No. 3, 2021
nal model, and differences between least squares means
were calculated using a Tukey-Kramer test.
A total of 4,248 Holstein cows from 30 herds were en-
rolled in this study. Most participating herds (n = 21)
were housed in tiestall facilities (9 herds were housed
in freestall facilities). Of all the cows initially enrolled,
108 cows were excluded because they were not bred
by 100 DIM (do not breed status) or they were culled
before pregnancy diagnosis after the first insemination.
Therefore, 4,140 cows were used for data analysis. The
median herd size was 86 cows (minimum: 58, maximum:
421). Overall, the prevalence of PVD and ENDO at
first examination (36 ± 7 DIM) was 25.1% (n = 1,038)
and 37.6% (n = 1,557), respectively. The median herd
prevalence of PVD was 24.3% (minimum: 13.4%, maxi-
mum: 52.9%) and 38.5% for ENDO (minimum: 14.5%,
maximum: 68.7%). The prevalence of the unaffected
group was 47.0% (n = 1,946), and 401 cows simultane-
ously had PVD and ENDO. Of all the cows with PVD
at first examination (n = 1,038), 522 (50.3%) and 516
(49.7%) cows were allocated to CEPH1X and CEPH2X
treatment groups, respectively. Of all the cows with
ENDO at first examination (n = 1,557), 772 (49.6%)
and 785 (50.4%) cows were allocated to the CEPH1X
and CEPH2X treatment groups, respectively. Descrip-
tive statistics of the studied population are presented
in Table 1.
In univariable models, parity group, season of calv-
ing, treatment group, and disease status were associ-
ated with PRFI (P < 0.05). In the multivariable model,
parity group, season of calving, treatment group,
and disease status were associated with PRFI (P <
0.05). Least squares means from the final model are
presented in Figure 1. This figure shows that CEPHA
treatment increased PRFI in cows affected by PVD at
first examination (CEPH1X: 28.0%; CEPH2X: 38.8%;
P = 0.01). A similar effect of CEPHA treatment was
noted in cows affected by ENDO at first examination
(CEPH1X: 30.3%; CEPH2X: 39.2%; P = 0.03). Cows
in the unaffected group had a PRFI of 43.2%.
This study is the first one to report a benefit of
administering a second CEPHA infusion treatment in
postpartum cows affected by PVD and ENDO. Based
on our results, the magnitude of this benefit for PVD
was an increase of 10 percentage points in PRFI in com-
parison with cows only given one CEPHA infusion. For
ENDO, it was an increase of 9 percentage points. When
interpreting these data, it is important to keep in mind
that all cows had their first insemination performed fol-
lowing a systematic ovulation synchronization protocol.
This approach was chosen as a way to control for the
stage of lactation at which first inseminations occurred.
It is unclear if similar results would have been found
when using estrus detection for first insemination.
Because of the study design, the benefit of a second
treatment can only be compared with cows receiving
one CEPHA treatment (positive control group) or to
cows unaffected by any reproductive tract diseases. It
was not the objective of the present study to quan-
tify the effect of administering one CEPHA treatment
(i.e., compared with no treatment at all); such stud-
ies have been performed in the past (LeBlanc et al.,
2002b; Runciman et al., 2009; Denis-Robichaud and
Dubuc, 2015b). The sample size needed for each ad-
ditional treatment group in our study was 350 cows
affected by PVD; the same number applied for ENDO.
Unfortunately, it was not logistically feasible to include
additional treatment groups in the present study as
the study size was already large enough. Future studies
on this topic should therefore consider a study design
that allows such investigation, if feasible. Future studies
should also consider including bacteriological culture
in their investigation to properly diagnose the pres-
ence of uterine pathogens and possibly allow specific
Dubuc et al.: SHORT COMMUNICATION: EFFICACY OF A SECOND CEPHAPIRIN TREATMENT
Table 1. Descriptive statistics from a randomized clinical trial investigating the efficacy of a second intrauterine cephapirin treatment (in
comparison with only one treatment) in 4,140 postpartum dairy cows
Item Unaffected
Overall
Category
Purulent vaginal discharge
Endometritis
CEPH1X CEPH2X CEPH1X CEPH2X CEPH1X CEPH2X
N 1,946 1,294 1,301 522 516 772 785
Parity group (%)
132.1 32.4 32.7 32.8 32.2 32.5 32.7
2 30.8 31.1 30.6 32.0 30.1 31.0 30.5
3+ 37.1 36.6 36.7 35.2 37.7 36.5 36.8
Season of calving (%)
Winter 21.8 22.3 22.0 22.0 22.4 22.9 21.7
Spring 23.6 23.5 23.7 23.0 24.1 23.2 23.5
Summer 28.0 27.6 27.9 27.8 27.7 27.4 28.3
Fall 26.6 26.6 26.4 27.2 25.8 26.4 26.6

Journal of Dairy Science Vol. 104 No. 3, 2021
identification. Unfortunately, the current study did not
include bacteriological testing, which led to the use of
cephalosporin in cows without identification of patho-
gens. Considering the current worldwide pressure about
prudent antibiotic drug use, this strategy might need to
be revisited and refined in the future.
Although diagnosing ENDO with the leukocyte es-
terase test is convenient and can be done cow-side, one
should keep in mind that the results of the present
study might not have been exactly the same if using the
endometrial cytology test (i.e., microscope slide read-
ing). Leukocyte esterase and endometrial cytology are
both well accepted diagnostic tests for ENDO and their
results are associated with detrimental effects on the
subsequent reproductive performance of cows (Couto
et al., 2013; Denis-Robichaud and Dubuc, 2015a). It
remains unclear if conducting the present study with
endometrial cytology instead of the leukocyte esterase
test would have provided different results. Further re-
search should clarify this point.
It was not logistically and financially possible to
diagnose prolonged anovulation in cows during the
present study. It has been shown that prolonged an-
ovulation and postpartum reproductive tract diseases
are associated (Krause et al., 2014; Vieira-Neto et al.,
2014). Another study also suggested that the effect of
intrauterine CEPHA treatment on PRFI is modulated
by the anovulation status of cows, the magnitude of
the effect being greater in ovular cows than in anovular
cows (Denis-Robichaud and Dubuc, 2015b). It is highly
possible that the benefit of a second CEPHA treatment
is also modulated by prolonged anovulation. Unfortu-
nately, this relationship cannot be explored in our data.
This aspect should be explored in future studies.
In conclusion, the administration of a second intra-
uterine CEPHA infusion 14 d after the initial treatment
improved the subsequent reproductive performance of
postpartum cows affected by PVD or ENDO. In the
context of our study, the benefit of a second treatment
was an increase of 10 and 9 percentage points in PRFI
of cows affected by PVD and ENDO, respectively.
ACKNOWLEDGMENTS
This project was financially supported by Merck
Animal Health (Kirkland, QC, Canada), “Fonds de
Recherche du Québec–Nature et Technologies” (Qué-
bec, QC, Canada), and “Réseau Québécois en Repro-
duction” (Université de Montréal, St-Hyacinthe, QC,
Canada). The authors acknowledge the technical work
provided by Jean-Philippe Pelletier (St-Hyacinthe, QC,
Canada) and the participating dairy producers for their
contribution to this study. The authors also express
gratitude to Guy Boisclair (Victoriaville, QC, Canada)
Dubuc et al.: SHORT COMMUNICATION: EFFICACY OF A SECOND CEPHAPIRIN TREATMENT
Figure 1. Least squares means (±SEM) of intrauterine cephapirin treatment groups at 36 (±7) DIM on pregnancy risk at first insemina-
tion stratified by reproductive tract disease status (unaffected; purulent vaginal discharge: PVD; endometritis: ENDO) in 4,140 Holstein cows
enrolled in a randomized clinical trial. Different lowercase letters (a, b) indicate a significant difference (P < 0.05). CEPH1X refers to a single
treatment of intrauterine cephapirin at 36 (±7) DIM. CEPH2X refers to a treatment of intrauterine cephapirin at 36 (±7) DIM and a second
one at 50 (±7) DIM.

Journal of Dairy Science Vol. 104 No. 3, 2021
and Jantijn Swinkels (Boxmeer, the Netherlands) for
their great interest in this study. The authors have not
stated any conflicts of interest.
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ORCIDS
J. Dubuc https: / / orcid .org/ 0000 -0003 -1013 -8372
V. Fauteux https: / / orcid .org/ 0000 -0002 -8786 -2038
M. Villettaz-Robichaud https: / / orcid .org/ 0000 -0001 -9685 -1827
J.-P. Roy https: / / orcid .org/ 0000 -0002 -0444 -2303
M. Rousseau https: / / orcid .org/ 0000 -0002 -2702 -434X
S. Buczinski https: / / orcid .org/ 0000 -0002 -8460 -4885
Dubuc et al.: SHORT COMMUNICATION: EFFICACY OF A SECOND CEPHAPIRIN TREATMENT