Animal Reproduction Science 185 (2017) 109–117

Available online 14 August 2017 0378-4320/ © 2017 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

The association between subclinical mastitis around calving and reproductive

performance in grazing dairy cows
N.A. Villa-Arcilaa, J. Sanchezb, M.H. Rattoc, J.C. Rodriguez-Lecompted, P.C. Duque-Madrida, S.
Sanchez-Ariasa, A. Ceballos-Marqueza,⁎
a Dept of Agric. Sci., Universidad de Caldas, Manizales, Colombia b Dept of Health Mgmt., University of Prince Edward Island,
Charlottetown, PEI, Canada c Dept of Anim. Sci., Universidad Austral de Chile, Valdivia, Chile d Dept of Path. Microbiol.,
University of Prince Edward Island, Charlottetown, PEI, Canada
ARTICLE INFO
Keywords: Intramammary infection Reproductive index Calving-to-conception interval Survival analysis
ABSTRACT
The objective of this study was to evaluate the effect of subclinical mastitis (SCM) on calving-to- first-service interval (CFS),
calving-to-conception interval (CC), and on the number of services per conception (S/C) in grazing Holstein and Normande
cows. Primiparous (n = 43) and mul- tiparous (n = 165) cows were selected from five dairy herds. Two composite milk samples
were aseptically collected from each cow at drying-off, and then every week during the first post- partum month. One sample was
used for somatic cell count (SCC), and the other one for bac- teriological analysis. Cows were followed up to 300 d after calving.
Non-parametric and para- metric survival models, and negative binomial regression were used to assess the association between
SCM, evaluated by SCC and milk culture, and reproductive indices. Staphylococcus aureus, CNS, and Streptococcus uberis were
the most frequent isolated pathogens. Subclinical mastitis in the first month of lactation was not associated with CFS; however,
the CC interval was longer in cows with SCM compared to healthy cows, the former also had a higher number of S/C.
1. Introduction
Bovine mastitis is a costly disease that affects the profitability of the milk industry due to the reduction of milk yield and the
high amount of discarded milk, early culling of cows, and treatment costs. The cost of the udder health programs has been
estimated to be, at least, one third of the total money invested for preventing diseases in dairy herds (Fourichon et al., 2001; Bar
et al., 2008) The reproductive performance of the herd has also an impact on the economic results of the dairy business, as low
reproductive efficiency may cause a decrease in milk production, and in the number of calves born per year (Maizon et al., 2004).
Fertility in dairy herds has declined in the past five decades while milk yield has increased (Walsh et al., 2011). Nevertheless,
how much variation in cow fertility explained by high milk yield remains unclear, as fertility is affected by other factors than milk
yield (e.g. heat detection, nutrition, environment, stress, and cow’s genetics and health status) (Dohoo et al., 2001; LeBlanc,
2010). In recent years, studies on the physiology of reproduction in different species have looked for different explanations on
how physio- logical, immunological and pathological processes have an impact on fertility (Pate et al., 2010; Bromfield and
Sheldon, 2011). For example in cows experiencing intramammary infections (IMI), inflammatory and immune responses are
activated outside the re- productive organs that can lead to embryonic death (Soto et al., 2003).
⁎ Corresponding author at: Calle 65 # 26-10, Manizales, Caldas, Colombia. E-mail address: alejandro.ceballos@ucaldas.edu.co
(A. Ceballos-Marquez).
http://dx.doi.org/10.1016/j.anireprosci.2017.08.010 Received 7 March 2017; Received in revised form 21 July 2017; Accepted 9
August 2017

MARK
N.A. Villa-Arcila et al. Animal Reproduction Science 185 (2017) 109–117
Recent studies found that cows with clinical mastitis had a significant decrease in their first service conception rate, and a
reduced probability of pregnancy compared to healthy cows (Fuenzalida et al., 2015). Likewise, cows with clinical IMI have a
higher number of services per conception (S/C), and longer interval from calving-to-first-service (CFS) or calving-to-conception
(CC) than that of healthy cows. It has been reported that S/C were higher in cows with clinical cases after their first service than
in cows with clinical mastitis before the first service (Gunay and Gunay, 2008). Some other studies have revealed the association
between clinical mastitis and failures to become pregnant after a service (Hertl et al., 2010; Hertl et al., 2014).
Meta-analytic studies suggested that reproductive pathologies occurring around calving, such as retained placenta and ovarian
cysts, might negatively affect the reproductive performance (i.e. increasing of intervals from calving-to-first-service; CFS, and
CC); however, clinical mastitis did not show a significant effect on the reproductive performance (Fourichon et al., 2000).
Nevertheless, an increased production of interleukins and mediators of inflammation (i.e. prostaglandins and cortisol) may
mediate the effect of mastitis, either subclinical or clinical, on the reproductive performance. These compounds may have a
negative effect on the de- velopment and quality of oocytes and corpus luteum, affecting the length of the estrous cycle in cows
(Risco et al., 1999; Schrick et al., 2001).
On the other hand, there are studies that found associations between subclinical mastitis (SCM), as measured by increased
somatic cell counts (SCC) in milk, and impaired reproductive performance (Hudson et al., 2012; Pinedo et al., 2009). Studies
conducted in temperate zones found a reduction of 44% in the risk of pregnancy when cow SCCs increased above 90,000
cells/mL before breeding (Pinedo et al., 2009). Dairy cows in grazing systems in Argentina that had SCM (i.e. SCC > 200,000
cells/mL) were 2.56 times more likely to have more than two services during the breeding season compared to healthy cows
(Gómez-Cifuentes et al., 2014).
Colombia is located in the northwest corner (4° 35′ N, 74° 4′ W) of South America near to the equator. The Colombian
climate is characterized as tropical and isothermal, which causes a significant regional variation in precipitation and temperature
(CENICAFE, 2016) in the pasture-based dairy systems. These systems tend to have gram-positive bacteria dominating effects on
udder health (Ramírez et al., 2014; Reyes et al., 2015; Villa-Arcila et al., 2017), which contrasts to more temperate regions of the
world (Bradley, 2002; Shum et al., 2009). These latter regions have equivocal reports on the effect of specific mastitis pathogens
on the reproductive performance in dairy cows (Fourichon et al., 2000; Fuenzalida et al., 2015).
In Colombian herds Staphylococcus aureus, Streptococcus agalactiae, and Corynebacterium pyogenes are highly prevalent
(Ramírez et al., 2014; Villa-Arcila et al., 2017) but there appear to be no specific studies conducted to evaluate what is the effect
of these infections on the reproductive performance of dairy cows grazing in tropical regions. Therefore, the main objective of
this study was to evaluate the hypothesis that SCM occurring in the first month after calving negatively affects the intervals from
CFS and CC, and the S/C in dairy cows grazing in tropical systems.
2. Materials and methods
A longitudinal study was designed, and conducted between June 2013 and September 2014. Five dairy herds located in
Manizales and Villamaria, Colombia were enrolled. Herds were located at 5° 1′ N and between 75° 22′ and 75° 27′ W. Herds
were selected due to the willingness of the owner to participate in the study, and if the farmers had: permanent veterinary advisory
service, records from each cow of the herd, and a health management plan to prevent infectious reproductive disorders (e.g.
brucellosis, infectious bovine rhinotracheitis, and bovine viral diarrhea), and other infectious diseases, such as leptospirosis, and
foot and mouth disease. These dairy herds were more progressive compared to the average herds in the region. The average herd
size in our study was lower than the mean of 68 lactating cows observed for herds in Manizales and Villamaria, Colombia. The
mean number of lactating cows in our study herds was 41.6 (ranging from 24 to 80).
The sample size for the study was calculated under the assumption of a high incidence risk of IMI around calving in
Colombian dairy herds, which can be as high as 25%, according to unpublished data from DHI records and previous information
from our Laboratory of Milk Quality at Universidad de Caldas. Therefore, the incidence risk of an IMI in cows around calving
was set at 0.20 (exposed) to calculate the required sample size. We expected to have a relative hazard of 0.60 (i.e. hazard in
exposed cows/hazard in unexposed cows), with a confidence of 95% and a study power of 0.80. Consequently, 188 events in total
were required for our study (Schoenfeld, 1983).
Cows (n = 208; primiparous = 43, multiparous = 165) were selected by convenience according to their expected calving date.
Signs of clinical mastitis at drying-off were not observed, and cows had no blind quarters. Cows were assigned to one of two
groups according to their breed: Holstein, n = 151 or Normande, n = 57. A total of 165 cows were multiparous and 43 were
primiparous. The general characteristics of the herds were described elsewhere (Villa-Arcila et al., 2017). Briefly, herds were
managed under rotational grazing systems, and cows received supplementation with concentrates according to milk yield. The
predominant pasture was a mixture of Kikuyu grass (Pennisetum clandestinum), Orchardgrass (Dactylis glomerata L.), and
Yorkshire fog grass (Holcus la- natus). Concentrates used were commercial mixes of cereals, containing 14% to 16% of crude
protein, and approximately 2.9 Mcal of metabolizable energy/kg of dry matter. Concentrates were fed starting three wk before
calving (2 kg/cow/d), and at a rate of 1 kg per 4 kg of milk yield after calving. Mineral supplements and water were available ad
libitum.
2.1. Sampling
Composite milk samples show relatively high sensitivity for the diagnosis of major mastitis pathogens, while the sensitivity
for Strep. uberis and minor pathogens is moderate. Nevertheless, increasing the number of affected quarters improves the
sensitivity of the analysis. The specificity is high no matter what pathogen is involved (Reyher and Dohoo, 2011). Therefore, two
samples of
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composite milk (i.e. milk from all four bovine quarters was collected in a single vial) were aseptically collected one week prior to
drying-off for SCC and microbiological analysis. Then every cow received an intramammary antibiotic for dry cow therapy. Once
again, two milk samples were collected from each cow approximately three d after calving, and then once a week for the first
month. Samples were cooled, and submitted for SCC analysis and, depending on the result, the second sample was used for
microbiological analysis. The SCC cut-off point for deciding the need of bacteriological diagnosis was established at 100,000 and
200,000 cells/mL for primiparous and multiparous cows, respectively (Ceballos et al., 2011). SCC was performed using a
Fossomatic electronic somatic cell counter (Foss Electric Hillerød, Denmark). The SCC was transformed to its natural log
(LnSCC, in thousands/mL) to normalize the distribution (Ali and Shook, 1980).
Bacteriological analyses were performed according to protocols established by the National Mastitis Council (NMC, 1999).
Milk samples were cultured with a sterile cotton swab on blood agar enriched with esculin at 0.1%. Morphology and Gram
staining were used for initial identification. The catalase test was used to differentiate staphylococci from streptococci. Coagulase
test was per- formed on catalase positive cocci to confirm Staphylococcus aureus. Gram-positive and catalase negative cocci were
differentiated by their reaction to the hydrolysis of esculin under ultraviolet light. Christie, Atkins, Munch-Petersen test and
esculin reaction were used to differentiate Streptococcus agalactiae and Strep. dysgalactiae. Esculin reaction and culture in
enterococcosel agar were used for the identification of Strep. uberis. Corynebacterium spp. was identified by their morphology
and Gram stain. Gram-negative bacteria were identified by their morphology and growth in MacConkey agar. Biochemical tests,
such as citrate, indole, motility and oxidase, were used to differentiate gram-negative bacteria.
2.2. Definition of subclinical mastitis
A cow was considered having a SCM at drying-off when SCC was equal or greater than 200,000 cells/mL, and at least one
colony of a mastitis-causing pathogen was isolated. This criterion was not applied to the primiparous cows, as milk samples in
this group were collected only after calving. Cows were sampled at drying-off to have a general idea about the infection status of
the mammary gland; however, this information was not used for statistical analyses.
A case of SCM was established at the beginning of lactation when the mean SCC for the four weeks after calving was equal or
greater than 100,000 or 200,000 cells/mL in primiparous and multiparous cows, respectively, and at least one colony of any
mammary pathogen was isolated in two out of three consecutive bacteriological cultures (i.e. between weeks 1 and 3, or between
weeks 2 and 4 after calving). However, in order to increase the specificity of the bacteriological culture, particularly for NAS
infections, the criterion was to isolate a minimum of two colonies (Dohoo et al., 2011). Milk samples containing more than two
bacterial species were considered contaminated, and not informative of SCM.
2.3. Reproductive parameters
Cows were followed up for 300 d after calving to record their reproductive indices. The interval from CFS was defined as the
number of days between calving and the first insemination date. The first service in each herd did occur after the voluntary
waiting period, which was 40 d after calving in all herds. The interval from CC was defined as the number of days between
calving and the conception date. Artificial insemination was used in all herds. The number of S/C was defined as the number of
services that result in a conception and confirmed by veterinary examination. Conception rate was determined based on the
number of cows that conceived divided by the total number of cows artificially inseminated at the time of pregnancy diagnosis.
Heat detection was conducted in all the farms using a teaser bull with redirection of prepuce and penis. Heat detection rate was
not calculated.
Experienced veterinarians made diagnosis of gestation approximately 45 d after the cows were inseminated. In one of the
herds (n=80 cows), pregnancy diagnosis was made by transrectal ultrasound (Tringa Linear, Vet Pie Medical, Maastricht, The
Netherlands), while in the other four herds the pregnancy was established by rectal palpation and no further checks were made.
2.4. Statistical analysis
All data were initially entered into an Excel spreadsheet (Microsoft Corp., Redmond, WA), and then exported to Stata version
14.1 (StataCorp, College Station, TX, USA) for data cleaning, coding and analysis.
As mentioned above, cows were monitored for 300 d, cows that did not get pregnant during this period were considered
censored. Given that the interest was on the time from calving to either first service or conception, the associations between SCM
and the intervals from CFS and CC were analyzed by survival analysis. Preliminary evaluations were made using Kaplan-Meier
survival graphs according to the absence or presence of SCM. Any eventual differences between the survival functions in cows
with no SCM and those having SCM were analyzed by the Log-rank, Wilcoxon, and Tarone-Ware tests (Dohoo et al., 2009).
Significance level was set at P < 0.05.
The effect of SCM occurring within 30 days in milk on the intervals CFS and CC was then analyzed using parametric survival
models. These models assume a specific form of hazard across time. For example, the exponential model assumes a constant
hazard, while in the Weibull model hazard is monotonic (i.e. may increase or decrease over time) (Dohoo et al., 2009). The data
obtained in this study were assessed using both survival parametric models. The model that fitted the data the best was selected
by its lowest Akaike’s information criterion. The exponentiation of the coefficient for the predictor of interest (i.e. effect of SCM)
produced the hazard ratio. This means that each unit change in the predictor represents a unit of change on the frequency of the
outcome, which in this case was measured as a hazard, assuming a linear relationship between the regressors and the hazard
(Dohoo et al., 2009). Both
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models, exponential and Weibull offer descriptions of the effect statistically equivalent; however, we selected the hazard metrics
for the interpretation, as this might be easier to understand by research-oriented clinicians (Fisher, 1992). Although we used a
para- metric model to analyze our data, the parametric model satisfying the proportional hazard assumption could be written in
the same way as a semi-parametric model following the equation:
h(t) = h
0
(t)*e(β’X) (1)
where h
0
(t) corresponds to the baseline hazard, dependent on time and is assumed to have a specific functional form. The term
e(β’X) corresponds to a non-negative specific function of the subject of the X covariate, not time t dependent and scales the
baseline hazard, which is common to all individuals enrolled in the study. Although the equation of semi-parametric models can
be used to explain the parametric model, the main difference is that parametric models include an intercept term (β
0
) (Dohoo et al., 2009). An evaluation of
the effect of the independent variables: a) SCM around calving (negative, positive), b) herd (categorized 1–5), c) breed (Holstein,
Normande), d) parity (categorized into 1, 2 and 3 or more lactations), e) the average of milk production for the first month of
lactation; and f) days (time; d) after the voluntary waiting period to the occurrence of the event on a continuous scale, was
included in the model. The interaction between SCM and time, modeled as time-varying effects, was evaluated to test whether the
hazard of conception in infected cows changed over time. There was no information about the infection status of the mammary
gland in primiparous before calving, as milk samples were not collected prior to calving in this group. Therefore, statistical
analyses only included data collected after calving in order to consider all cows in the analyses.
The calculation of the Cox-Snell residuals and deviance was used to assess the model fit. Deviance is also useful for
identifying unusual values (outliers) having a high influence on the model. Once the influential points were identified, they were
removed and sensitivity analyses were made to evaluate their effect on the final estimates (Dohoo et al., 2009).
The model of the effect of SCM around calving on the number of S/C was based on the number of cows that conceived.
Therefore, the date of the last service was used to calculate the CC interval, and the number of services to that date was counted.
The dependent variable S/C was analyzed using a zero truncated negative binomial model, which provided values larger than 0.
The effect of the independent variables described above was also included in the model (e.g. herd, SCM at calving, parity, breed,
and average milk yield for the first month of lactation). The general model was as follows:
Log(S/C) = β
0
+β
1
x
1
+β
2
x
2
+ ... + β
n
x
n
(2)
where the number of S/C is expressed on log scale; β
0
corresponds to the intercept, and β
n
corresponds to the coefficient of the
effect when the independent variable x
n
was present. Obtaining Anscombe residuals and Cook’s distance was used to assess the fit of
the model and detect influential points. The significance level was set at P < 0.05.
3. Results
Descriptive statistics of dependent variables and LnSCC are presented in Table 1. The average time between drying-off and
calving was 71 ± 18 d. The average milk yield for the first month of lactation was 30.5 ± 6.3 kg/d in Holsteins and 14.2 ± 4.0
kg/d in Normande cows (P = 0.03).
3.1. Subclinical mastitis
Seventy-four cows out of the 165 multiparous cows were classified as positive for SCM at drying-off. However, there were 13
cows
Table 1 Descriptive statistics (mean ± SD) of the dependent and independent variables and log-transformed somatic cell count
(LnSCC) in cows with no subclinical mastitis (SCM) and cows with SCM in the first month of lactation.
Variable Subclinical mastitis (No. of cowsa)
Negative (n = 139) Positive (n = 66)
Parity 3.3 ± 2.2 5.0 ± 2.6 Milk yield (kg/d)b 26.4 ± 9.3 25.7 ± 9.2 LnSCC 4.41 ± 0.97 6.55 ± 0.96 Intervalsc:
Calving-to-first-service (d) 84 ± 38 86 ± 37 Calving-to-conception (d) 119 ± 67 157 ± 109 Number of cows that conceived: 129
56
Cows pregnant at first service (%) 49.6 42.9 Cows pregnant at second service (%) 25.6 17.9 Cows pregnant at third service or
later (%) 16.3 32.1 Services per conception 1.91 ± 1.27 2.48 ± 1.75
a Milk samples of three cows were contaminated, and not informative of SCM. b Daily average for the first month of lactation. c
These intervals include the 40 d voluntary waiting period.
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Fig. 1. Kaplan-Meier survival function curves for cows without (−) and with (− −) subclinical mastitis (SCM) in the first four
weeks after calving on the interval from calving-to-first service (a; CFS) and on the interval from calving-to-conception (b; CC).
The survival function for CFS did not show any difference according to SCM (a; P > 0.05). Cows with no SCM appeared to be at
a greater risk of conception after calving than positive cows (b; P < 0.05). The follow-up period started once the voluntary
waiting period was over (i.e. day 0 corresponds to the beginning of the breeding period, which is 40 d after calving).
with low SCC (< 200,000 cells/mL) and positive results for bacteriological culture, while 51 cows showed a high SCC (≥200,000
cells/mL) with no isolations for the bacteriological test. These cows did not meet the criteria to classify them as having SCM.
Four milk samples were contaminated and not informative of SCM. Twelve cows (16.2%) remained chronically infected with the
same pathogen after calving.
After calving, a total of 59 milk samples from primiparous cows, and 304 samples from multiparous cows were cultured. In
total, 27 (46%; n = 59) milk samples of primiparous, and 197 (65%; n = 304) of multiparous cows had SCCs higher than the
defined threshold and were culture positive, consequently the SCM was established according to the definition set. In primiparous
cows, there were 10 animals (23.3%; n = 43) with SCM in the first wk after calving. The number of multiparous cows with a new
IMI in the first wk after calving was 18, which corresponded to an incidence risk of 21.0% given that 86 cows were at risk of
infection at drying-off. Based on the number of mastitis pathogens isolations after calving, the most frequentpathogens were
Staph. aureus (29%), followed by CNS (22.6%), Strep. uberis (9.2%), Strep. agalactiae (7.5%), y Cor. bovis (6%). There were
only three isolations of E. coli, one at drying-off and two after calving.
With regard to clinical mastitis, 12 cows (5.8%) presented clinical cases in the first month of lactation. These cases were
caused by Staph. aureus (4/12), CNS (2/12), Strep. uberis (2/12), Strep. agalactiae (1/12), Corynebacterium bovis (1/12),
non-significant growth (1/12). One sample was contaminated.
3.2. Intervals from calving-to-first-service and calving-to-conception
The Kaplan-Meier survival function for cows with and without SCM is shown in Fig. 1. These functions were compared using
different tests that did not yield significant differences for the interval from CFS (Fig. 1a; P > 0.05), while for the interval from
CC the three tests (i.e. Log-rank, Wilcoxon, and Tarone-Ware) indicated that cows with SCM had a lower risk of conception
during the follow-up period (Fig. 2b; P < 0.05).
In both parametric models, exponential and Weibull, the effect of some of the independent variables, such as breed, parity,
average milk yield, and the interaction between SCM around calving and time did not significantly affect the CC interval (P >
0.10). Subsequently, the single effect of SCM and time was evaluated (Table 2). While both models showed a significant effect of
SCM in the
Fig. 2. Predicted survival time for the interval from calving-to-conception (CC) in cows without (−) and with (− −) subclinical
mastitis (SCM) in the first four weeks after calving. The prediction is subsequent to a parametric model with Weibull distribution.
The follow-up period started once the voluntary waiting period was over (i.e. day 0 corresponds to the beginning of the breeding
period, which is 40 d after calving).
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Table 2 Effect of subclinical mastitis (SCM) in the first month of lactation on the interval from calving-to-conception (CC) in
grazing Holstein and Normande cows. The coefficient represents the effect of SCM on the interval (log scale) after controlling for
the effect of herd. The results correspond to parametric survival models with Weibull distribution, time effect was not significant
(P = 0.20) and not showed in the table.
Days after calving Coefficient CI (95%)a P-value
Cows with SCM −0.403b −0.741; −0.065 0.02 Herd: < 0.01
1 Reference 2 −1.248 −1.784; −0.712 3 −0.772 −1.426; −0.118 4 −0.356 −0.844; 0.132 5 0.162 −0.424; 0.749 Constant −6.645
−6.469; −4.821 < 0.01 Shape parameter 1.387 1.236; 1.556
a CI: Confidence interval at 95%. b Hazard ratio: 0.67 [=exp(−0.403)].
first month after calving on the risk of conception in the follow-up, the Weibull model had a lower AIC compared to the
exponential model. At any time after calving, cows with SCM had a lower hazard of conception compared to non infected cows
with a hazard ratio 0.67 [=exp(−0.403)].
The predicted median from CC time was 71 d and 95 d for healthy and infected cows around calving, respectively (Fig. 2).
The shape parameter (p) of the Weibull time distribution was greater than one (Table 2), indicating that the hazard of conception
in both groups of cows was not constant and increased with time after calving (Fig. 3).
Two cows were identified with deviance residuals greater than three. These cows did not have SCM around calving and were
pregnant in the first week of the monitoring period. On the other hand, there was a cow with a deviance residual less than three,
this cow did not show SCM around calving and was not pregnant after 300 d. The latter cow was considered censored. The
Weibull model was fitted again after removing the influential cows, there were no substantial changes either in the magnitude or
direction of the coefficients, and therefore the full dataset was retained.
3.3. Services per conception (S/C)
The overall mean for the number of S/C in pregnant cows was 2.1 ± 1.5. Cows with no SCM had, on average, 1.9 ± 1.3 S/C,
while positive cows had 2.5 ± 1.8 S/C. The initial negative binomial model showed a tendency for higher S/C in cows with SCM
around calving after controlling for herd effect (P = 0.11). Other independent variables such as breed, parity, and average milk
yield in the first month of lactation were not significant. These variables were removed from the final model. Three cows were
highly influential on the model. Those cows did not have SCM and had, on average, five S/C. The subsequent sensitivity analysis
showed a significant effect of SCM around calving on the number of S/C, as infected cows required more services per conception
compared to negative cows (Table 3). Cows with SCM had on average 1.83 more services per conception as compared to healthy
cows.
4. Discussion
The most frequently isolated bacteria were Staph. aureus, CNS, Strep. uberis, Strep. agalactiae y Cor. bovis. The isolations of
Staph. aureus were higher than that observed in other studies that found a quarter-level prevalence up to 18.3%, and an increase in
the
Fig. 3. Representation of the shape parameter (p) obtained in Weibull distribution model for the risk of conception in cows
without (−) and with (− −) subclinical mastitis (SCM) in the first four weeks after calving. The p parameter was greater than 1.0,
indicating that the risk was not constant and was increased with time. The follow-up period started once the voluntary waiting
period was over (i.e. day 0 corresponds to the beginning of the breeding period, which is 40 d after calving).
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Table 3 Effect of subclinical mastitis (SCM) in the first month after calving on the number of services per conception (S/C) in
Holstein and Normande cows. The results of the final negative binomial regression model are presented after removing three
cows highly influential in the model.
Number of S/C Coefficient IC (95%)a P-value
Cows with SCM 0.605 0.172; 1.037 < 0.01 Herd: < 0.01
1 Reference 2 0.705 −0.183; 1.593 3 0.989 0.058; 1.920 4 1.051 0.232; 1.870 5 −0.332 −1.434; 0.770 Constant −1.202 −1.990;
−0.417 < 0.01
a CI: Confidence interval at 95%.
likelihood of being Staph. aureus positive when the cow was infected with CNS (Dufour et al., 2012). One important issue in Co-
lombian dairy herds is their positivity to Strep. agalactiae. A former study conducted in Colombia indicated that the overall
prevalence of bulk tank Strep. agalactiae, weighted by region, was 42% (Keefe et al., 2010). However, the number of
gram-negative isolates was lower compared to studies in temperate regions (Bradley, 2002; Shum et al., 2009). The differences in
the pathogen population between grazing and housed systems might be related to changes in the epidemiology of mastitis,
leading to a higher prevalence of contagious mastitis in tropical dairy herds, characterized mainly by subclinical and chronic
infections. The pressure of infection in these herds was high, as many subclinical cases are not identified and treated.
Subclinical mastitis (i.e. high SCCs and culture positive) in the first month of lactation was not associated with CFS; however,
the CC interval was longer in cows with high SCC compared to healthy cows, and also had a higher number of S/C. Studies
conducted with cows on pasture in Argentina (Gómez-Cifuentes et al., 2014), Chile (Pinedo et al., 2009), and New Zealand
(Compton et al., 2007), which are located in warm temperate zones, have shown an impairment of reproductive performance in
cows with SCM. The main findings in infected cows were an increased interval from CFS and CC, and infected cows needed a
higher number of S/C. A study carried out in a tropical country (Venezuela) indicated that dual-purpose cows with clinical
mastitis had a delay of 62 d and 44 d in the CFS and CC intervals, respectively, finding that clinical mastitis had a stronger effect
on primiparous cows (Nava-Trujillo et al., 2010). Swedish studies found that IMI after calving were associated with longer CFS
and CC intervals, and also with a higher number of S/C (Maizon et al., 2004). Similarly, we detected statistically significant
differences in both CC interval and S/C between healthy and cows with SCM cows around calving; however, we were not able to
observe any significant differences on the CFS interval. Both groups of animals had similar CFS intervals (Table 1) but the small
number of animals in the infected group might have affected our ability to detect such an effect.
Indices of reproductive performance can be related among them; however, under the conditions of this study, SCM did not
appear to affect the resumption of ovarian function after calving but did affect the hazard of conception. Non-detected embryonic
deaths, factors related to the blood progesterone concentration at the time of first service, the presence of chemical mediators that
would affect the viability of the embryo might explain the effect of IMIs on the reproductive performance of the cow, in particular
on the CC interval (Risco et al., 1999; Soto et al., 2003; Huszenicza et al., 2004). Although, it is difficult to rule out a potential
embryo loss in the cows used in this study, we were careful to select and follow an inclusion criteria in all the farms before the
beginning of the study. The farms included in the study had reproductive records that were analyzed for potential reproductive
disorders, such as embryo loss among them. Indeed, a vaccination and health veterinary program was mandatory in every farm to
be included in our study.
The effect of mastitis-causing pathogens on reproductive performance may be explained by the effect of mediators of in-
flammation released during the infections, such as lipopolysaccharides (LPS) and lipoteichoic acid (LTA), and their effects on the
pituitary and gonadal hormones (Hertl et al., 2010; Hertl et al., 2014). These substances might trigger febrile reactions that induce
changes in the pattern of hormone secretion, characterized by inhibiting the gonadotropin release necessary for steroidogenesis
(Huszenicza et al., 2004; Pate et al., 2010). The granulosa cells have receptors for some molecules such as TNFα, interleukin 6
and 8, and chemokines that interfere with the synthesis of estrogens in the dominant follicle (Sakumoto et al., 2003; Herath et al.,
2007; Bromfield and Sheldon, 2011). Cows with chronic SCM showed lower estrogen levels when they are in estrous, which
would favor a delay in ovulation (Lavon et al., 2010; Lavon et al., 2011). Thus changes in the ovarian physiology and
steroidogenesis might explain the delays in the interval from CC associated with SCM (Sakumoto et al., 2003; Lavon et al.,
2011).
 In Colombia, gram-positive bacteria are the most frequent pathogens in milk from cows with SCM. The LTA produced by
Staph. aureus had a higher impact on reproductive physiology compared to the LTA produced by streptococci. This effect was
found in a study where labor in mice was prematurely induced by LTA-producing bacteria, such as Staph. aureus (Kajikawa et al.,
1998). Although the objective of our study was not to evaluate the specific effect of LTA on labor, it might be a cause of low
reproductive performance in cows infected with Staph. aureus, as this bacteria was highly prevalent in our study. Also, endotoxins
produced by other kind of bacteria may induce the release of prostaglandins and cortisol, which would have a negative effect on
the development and quality of the oocyte and the corpus luteum, besides causing a disruption of the estrous cycle (Risco et al.,
1999; Schrick et al., 2001; Hockett et al., 2005). This would confirm the effect of IMIs on reproduction and would indicate that
this effect is mediated by multiple mechanisms, not only at the pituitary level but also at the gonadal level. Therefore, cows with
an IMI would need a larger number of S/C, which in turn increases the interval from CC.
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N.A. Villa-Arcila et al. Animal Reproduction Science 185 (2017) 109–117
The effect of clinical mastitis on reproduction was not evaluated and was beyond of the scope of our study. There were only
12 cases of clinical mastitis. A negative effect of clinical mastitis on conception rates and pregnancy has been reported (Santos et
al., 2004). This effect has been observed when mastitis, no matter the causing pathogen, occurred between the first service date
and pregnancy confirmation. In addition, IMIs affect embryo development (Hansen et al., 2004). Clinical IMIs induce embryonic
death before the pregnancy was confirmed, which may occur due to the action of proinflammatory cytokines (Chebel et al., 2004;
Hansen et al., 2004). Clinical mastitis has been associated also with other reproductive disorders in dairy cows increasing the
interval from CC and in the number of S/C (Huszenicza et al., 2005; Hansen et al., 2004).
The herd effect cannot be ignored; we found a significant effect of this variable on the reproductive indices (Tables 2 and 3).
Herd- level factors that were not evaluated in our study, such as nutrition and methods used for heat detection can affect the
intervals CFS and CC. However, cow-level factors, such as time of calving, milk yield, metabolic diseases occurring around
calving, and the interval between calving regarding to the previous lactation might also have an impact on the intervals from CFS
and from CC (Dohoo et al., 2001). Among the cow-level factors, breed and potential milk yield were the most important factors
that may affect the interval from CFS, which is an indication that genetics influences the reproductive performance (Dohoo et al.,
2001). Same authors concluded that the reproductive performance of individual cows affect the overall performance of the herd.
Nevertheless, we did not find evidence to support that cow-level variables, such as breed, average milk yield around calving or
parity, affected the overall reproductive indices of the herds in this study. Only one cow had dystocia at the time of parturition;
however, neither SCM nor alterations of reproductive parameters were observed in this animal.
5. Conclusion
Under the conditions of this study, the survival models used were suitable for evaluating the effect of SCM on the CFS and CC
intervals. Subclinical mastitis at drying-off was highly prevalent in our study, with some cases remaining chronic after subsequent
calving. However, the number of chronic infections was not enough to stratify the analysis by the duration of SCM.
Gram-positive bacteria caused most of the SCM cases in the first month after calving, which was associated with a longer CC
interval and a higher number of S/C, a pattern observed in both temperate, and now, tropical regions. The pressure of infection
might be higher under the conditions of our study, as contagious pathogens are highly prevalent and chronic infected cows are not
treated; however, other than these conditions, there are no underlying reasons to believe that SCM may affect the reproductive
performance of the cow in a tropical environment in a different way compared to cows in herds from temperate regions. Although
cow-level factors did not affect the indices studied, there was an effect of the overall herd management that should not be
dismissed as a potential explanation for the results. Nevertheless, if the effect was similar under different management and
environmental conditions (i.e. temperate and tropical regions), this means that the weight of the evidence is greater which is very
useful for decision makers. The reproductive performance is the result of the impact of multiple factors, not only explained by
mammary health.
Conflict of interest
None.
Acknowledgements
Authors want to express their gratitude to the Office of the Vice-president of Research and Postgraduate Studies of
Universidad de Caldas (Colombia) for funding this study (Grant # 0182912). We would like to thank also to the owners and
employees of dairy herds enrolled in the study for their willingness to participate, and to all members of the Research Group on
Milk Quality and Veterinary Epidemiology (CLEV) of Universidad de Caldas for their collaboration.
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