Animal Reproduction Science 197 (2018) 22–32

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Animal Reproduction Science

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

Chronic inflammatory and degenerative endometrial lesions in

T
subfertile Criollo Limonero cattle; a B. taurus Latin-American breed
threatened with extinction; A case-control study
⁎
Roberto A. Palomaresa,b, , Juan C. Gutiérrez-Añezc, Sunny Zambranod,
Julio C. Boscan-Ocandoc, Merilio Monteroc, Jesús Camachoc,
Fernando P. Perea-Ganchoue, Maria S. Ferrerb, Jose M. Rodríguez-Márquezc,
German Portillo-Martinezc, Rafaela J. Muñozc, Rosario Godoyc
a
Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
b
Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
c
Unidad de Investigación en Reproducción Animal, Facultad de Ciencias Veterinarias, Universidad del Zulia, Maracaibo, Venezuela
d
Instituto Nacional de Investigaciones Agrarias (INIA), Estación experimental El Laral, Estado Zulia, Venezuela
e
Departamento de Ciencias Agrarias, Núcleo Universitario “Rafael Rangel”, Universidad de Los Andes, Trujillo, Venezuela

A R T IC LE I N F O ABS TRA CT

Keywords: Criollo Limonero is a tropical Bos taurus breed for sustainable dual purpose (milk and beef)
Criollo limonero production in the South-American tropics, which is currently threatened with extinction. The
Repeat breeding objective was to perform a clinical evaluation and histopathological assessment of uterine biopsy
Chronic endometritis samples of repeat breeder (RB) Criollo Limonero cattle to determine the occurrence of patho-
Subfertility
logical conditions as potential causes of subfertility. Twenty-four Criollo Limonero cattle [18
cows (5–13 years old) and 6 heifers (6–7.5 years old)] that had failed to conceive after four or
more services were considered for this study. Additionally, five cows with history of adequate
reproductive performance were used as a control group. Animals were submitted to physical
exam, vaginoscopy, and ultrasonographical evaluation of the reproductive tract. Uterine biopsy
samples were collected for histopathological evaluation. Vaginoscopy revealed that 41.7% of the
RB cattle had abnormal vaginal secretions, while abnormal secretions were not observed in any
control cow. Ultrasonographical examination of the uterus revealed the presence of free uterine
fluid in 20.8% of the RB animals, while none of the control cows had fluid in the uterine lumen.
In addition, ovarian cysts were observed in 25.0% of the RB animals. Histopathological eva-
luation of the endometrial biopsies revealed that mononuclear leukocyte infiltration, dilated
uterine glands, and periglandular fibrosis were the most prevalent lesions in the sub-fertile an-
imals. Chronic endometritis characterized by inflammatory (mononuclear leukocyte infiltration)
and degenerative (dilated glands and periglandular fibrosis) endometrial lesions, and ovarian
cysts were the most frequent reproductive pathologies observed in the studied subfertile Criollo
Limonero cattle, suggesting a strong association with their reduced fertility.

⁎
Corresponding author at: 2200 College Station Rd., Athens, GA, 30602, USA.
E-mail address: palomnr@uga.edu (R.A. Palomares).

https://doi.org/10.1016/j.anireprosci.2018.06.019
Received 12 April 2018; Received in revised form 18 June 2018; Accepted 24 June 2018
Available online 25 June 2018
0378-4320/ © 2018 Elsevier B.V. All rights reserved.
R.A. Palomares et al. Animal Reproduction Science 197 (2018) 22–32

1. Introduction

The Venezuelan Criollo Limonero is a native breed (tropical Bos taurus) derived originally from crossing between Hispanic B.
taurus breeds from the Iberian Peninsula and the Canary Islands brought to South America during the Spanish conquest and colonial
times (Florio-Luis et al., 2011a). This breed is characterized for its great adaptation to extreme warm environments in which other
European Bos taurus breeds have difficulties surviving (Landaeta-Hernández et al., 2011). Additionally, this breed has been de-
monstrated to have an adequate fertility and moderate milk production in grazing systems under tropical conditions, being con-
sidered a promissory breed to develop sustainable cattle production systems in South American tropics (Florio-Luis, 2008). According
to The National Institute for Agricultural Research in Venezuela (INIA), the total Criollo Limonero population is nearly 600 purebred
cattle heads (Florio-Luis et al., 2011a). The absence of accurate genealogic records makes it difficult to quantify the precise number of
purebred cattle (Ramírez, 2005). Additionally, the indiscriminate introduction of breeds that have been selected for high milk
production in template climates has reduced indigenous purebred populations, decreasing the natural genetic variation of the breed.
For these reasons, this Latin-American Criollo breed has been considered threatened with extinction (De Alba, 1987; Mueller-Haye,
1978; Landaeta-Hernández et al., 2011).
Reproductive performance is one of the most important factors determining genetic preservation, and profitability of these herds.
In order to maintain acceptable reproductive performance and farm profitability, it is necessary for cows to have an inter-calving
interval of less than 400 days; calving to conception interval of less than 110 days and less than 4 services per conception (Perea et al.,
2002). Repeat breeding is a major cause of low reproductive performance in tropical dual purpose herds. This condition results in low
pregnancy rates, high number of services per conception, prolonged postpartum intervals, high culling rates, and substantial eco-
nomic losses. A repeat breeder cow is defined as an animal that has failed to conceive after three or more services, returns to estrus
within a normal inter-estrus interval and shows no clinical evidence of reproductive pathologies. The causes of repeat breeding
involve either fertilization failure or early embryonic death (Sreenan and Diskin, 1983; Ayalon, 1984). The complex etiology for this
condition may include a combination of many factors, such as management and nutritional disorders (Peters, 1996), infection and
inflammation of oviductal and uterine environments (Gilbert, 2016), abnormalities in the gametes (Maurer and Echternkamp, 1985),
inadequate luteal function (Mann and Lamming, 2001), delayed ovulation (Båge et al., 2002), and environmental and genetic factors,
among other causes.
Endometritis is the inflammation of the endometrium without systemic clinical signs of disease (Ahmadi et al., 2005; Gilbert,
2016). Because a poor agreement between clinical and cytological findings on evaluation of the vagina and uterus, the condition has
been described as two different entities; cytological endometritis and purulent vaginal discharge (Gilbert, 2016). Disruption of the
anatomical barrier that protect the uterus from infection (i.e. alteration of vulvar conformation, vulvo-vaginal sphincter and cervical
lesions) increases the risk of developing uterine infection and inflammation (Sheldon et al., 2009). This pathology has a substantial
negative effect on reproductive performance of dairy cows reducing conception rates and increasing embryonic loss after in-
semination (Cheong et al., 2011; Gilbert et al., 2005; Dubuc et al., 2010). Subclinical endometritis as a cause of bovine infertility is
not easily diagnosed by a routine clinical and gynecological examination (Ahmadi et al., 2005); therefore, its diagnosis must be based
on an integrated approach that includes other techniques such as ultrasonography, microbial culture, cytology and/or biopsy of the
endometrium (Gilbert, 2016).
The objective of this case-control study was to perform a clinical reproductive examination and histopathological assessment of
uterine biopsy samples in subfertile Criollo Limonero cattle to determine the occurrence of uterine and ovarian pathologies associated
with subfertility.

2. Material and methods

2.1. Location

The study was conducted in the experimental farm “El Laral” of the Instituto Nacional de Investigaciones Agrarias, INIA,
Venezuela (National Institute for Agricultural Research), located in the northwestern region of Maracaibo Lake basin, Venezuela. The
mean daily temperature in the region is 27.8 °C, mean rainfall is around 800 mm/year and relative humidity is 77.3%. The dry season
is from December to April and the rainy season is from May to November. This agro-ecological zone is classified as a dry, tropical
forest.

2.2. Herd management

The farm had a total of 420 purebred animals, from which 120 were females in reproductive age (cows and heifers). The number
of milking cows at any time was between 66 and 76 animals. The cows grazed in a pasture of Guinea grass (Panicum maximum; 10%
crude protein) and German grass (Echinochloa polystachya; 12% crude protein). Additionally, cows were supplemented during milking
with approximately 2 Kg of commercial concentrate feed. Milking was performed manually twice a day. Average milk production of
cows was about 1800 Kg in 244 days. After parturition, cows had a voluntary waiting period of 40 days before breeding. Estrus
detection was done by visual observation for a period of 1 h each morning (6:00 to 7:00 AM) and evening (6:00 to 7:00 PM) with the
help of bulls that were surgically altered with resection of both epididymis tails and penile deviation, so that intromission could not
occur. Animals consistently standing to be mounted were considered in estrus. Cattle were artificially inseminated (AI) 12 h after first
detected in estrus, according to the AM-PM rule. Cattle that had failed to conceive after 3 AI were prophylactically treated with

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intrauterine antibiotics (40 mL of Oxytetracycline, sln 5%) 24 h post AI, plus an injection of 100 μg of gonadorelin acetate (Fertagyl®,
Intervet, Holland) intramuscularly (im) at the time of the fourth AI and then submitted to a controlled natural mating (pen mating) by
a bull with known fertility for subsequent services. The date and time of each AI and natural mating was recorded. Pregnancy
diagnosis was performed by an experienced veterinarian by rectal palpation 60 days after AI. Reproductive performance parameters
were calculated and reported.

2.3. Animals

Twenty-four purebred repeat breeder (RB) Criollo Limonero females (18 cows and 6 heifers) that had failed to conceive after four
or more services (7 ± 0.89 failed services, range: 4–11 services) and were destined for slaughter due to reproductive failure were
submitted to a single time point physical examination, vaginoscopy and ultrasonographical evaluation of the reproductive tract. The
cows were between 5 and 13 years of age (mean age: 9.4 years; median parity: 3.0 calvings and 956.1 ± 110 days postpartum).
Heifers were between 6 and 7.5 years of age (mean age: 6.6 years). Additionally, five cows with history of adequate reproductive
performance (5.5–9 years of age; mean age: 6.8 years; median parity: 2.0 calvings and 114.2 ± 25.5 days postpartum) were also
evaluated and used as a control group. The animals had adequate body condition score (median 3.25; scale 1–5, where 1 is very thing
and 5 is very fat; Edmondson et al., 1989). The animals were cared for in accordance with acceptable practices as for the Care and Use
of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999). The research protocols used in this study were reviewed
and approved by the scientific and animal care committee of the Consejo de Desarrollo Científico y Humanístico (CONDES), of the
University of Zulia, Venezuela.

2.4. Physical exam and vaginoscopy

Animals were submitted to a clinical and reproductive examination following the protocol proposed by Grunert and Gregory
(1984). Rectal temperature, respiratory rate (breaths/min), heart rate (heart beats/min) and signs of depression and dehydration.
The presence of vulvar discharge was recorded. To perform a vaginal examination, the vulvar lips were scrubbed with detergent,
rinsed with abundant water and wiped with a dry paper towel, and a disinfected and lubricated stainless steel vaginoscope was
inserted. With illumination, the vaginal walls and external cervix were assessed for signs of inflammation and the presence of vaginal
secretions.
The character of the vaginal discharge was scored and recorded (0= absent; score 1= clear or translucent mucus; score 2=
mucus containing flecks of white or off-white purulent material; score 3= muco-purulent discharge, containing < 50% white or off-
white mucopurulent materials; score 4= purulent discharge, containing > 50% white or off-white mucopurulent materials (Williams
et al., 2005). The vaginoscope was disinfected with povidone iodine solution and rinsed with water before each examination.

2.5. Clinical pathology

Blood samples were collected from all the animals via coccygeal venipuncture into 10 mL vacuum tubes (Vacutainer, BD
Diagnosis,Franklin Lakes, NJ) with anticoagulant (EDTA) to obtain whole blood. Whole blood was used to determine hematocrit (Hct,
%), hemoglobin (Hgb, gr/dl), red blood cells count (erythrocytes/μL), white blood cells count (WBC/μL), percentage of differential
leukocytes, and presence of hemoparasites using a manual cell count procedure (conventional cyto-morphology) with a Neubauer
chamber and a 40x optical microscope at the Veterinary Clinical Laboratory of the School of Veterinary Medicine at La Universidad
del Zulia.

2.6. Ultrasonography

Transrectal ultrasonography was performed to evaluate the uterine and ovarian status. The examination was accomplished using
a Pie Medical 100 Falco® ultrasound with a linear probe of dual frequency (6.0–8.0 MHz). The diameter of cervix was measured and
the uterus was evaluated to determine the presence of free fluid within the uterine lumen indicative of a possible endometritis
(Kasimanickam et al., 2004). The ovaries were scanned and the presence of ovarian structures such as follicles, corpora lutea (CL) or
cysts was recorded. Additionally, the diameter of the ovarian structures was measured.

2.7. Collection of uterine biopsies and histopathological evaluation

Before taking the endometrial sample, the vulva and perineal areas were scrubbed using iodine scrub and abundant water. The
biopsies were collected using a small uterine biopsy instrument (Hauptner, Solingen, Germany; 70 cm long with an alligator jaw type
basket) for histopathological evaluation. The biopsy instrument in a protective sheath was introduced into the vagina. The instrument
was guided into the cervix by manipulation per rectum. Then, the instrument alone was introduced into the uterus after rupturing the
plastic sheath at the external cervical orifice. Uterine biopsies were obtained from the middle portion of one uterine horn (ap-
proximately 5 cm past the uterine bifurcation) pressing the uterine wall into the biopsy instrument basket by manipulation per
rectum. Care was taken for not damaging the endometrium. Uterine biopsies were fixed in Bouin´s solution and submitted to the
histology laboratory of the College of Veterinary Medicine University of Zulia. Uterine biopsy samples were dehydrated, embedded in
paraffin wax, cut in slices and stained with hematoxilin-eosin for light microscopy examination. Trichromic Masson´s staining was

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carried out for the tissues with evidence of fibrosis to confirm the fibrous inflammatory lesions. Histopathological evaluation of the
uterine biopsies was performed by an experienced pathologist and classified based on the presence and severity of inflammatory and
degenerative lesions according to the criteria reported by Griffin et al. (1974); Hartigan et al. (1974); and Chaffaux et al. (1987):
edema (0: absent; 1:present), aspect of luminal epithelium (1: normal aspect; 2: infiltration mononuclear and polynuclear cells; 3:
partially denuded; 4: totally denuded and may be with constitution of a pyogenic membrane), glandular morphology (1: normal
morphology; 2: dilated glands with modification of the epithelial cells; 3: cystic glands with a flat epithelium), periglandular fibrosis
(1: absent, 2: mild, one or two layers of fibroblasts, 3: moderate, three layers of fibroblasts, and 4: severe, four or more layers of
fibroblasts), mononuclear leukocytes infiltration (1: absent or mild, 2: moderate, 3: severe), polynuclear leukocytes infiltration (1:
absent or mild, 2: moderate, 3: severe), lymphoid aggregates (0: absent, 1: present). Finally, the frequency distribution of the en-
dometrial findings was estimated.

2.8. Statistical analysis

A descriptive statistical analysis was done using the Statistical Analysis System version 9.2 (SAS®). The frequency of each clinical,
ultrasonographical and histopathological finding was calculated through frequency procedure (Proc Freq). Significant differences in
the occurrence of the histopathological lesions were estimated using Chi-square test. Means and standard errors for rectal tem-
perature, respiratory and heart rates, leukocyte counts, as well as diameter of cervix and ovarian structures were determined through
Proc univariate. Comparisons between RB and fertile cows were performed using two sample t-test. For all analyses, values of P <
0.05 were considered significant, and 0.05 < P ≤ 0.10 was considered a tendency.

3. Results

3.1. Physical exam and vaginoscopy

Only one subfertile cow (RB cow # 14) had history of postpartum uterine pathology (puerperal metritis). Most of the cows had
rectal temperature, heart and respiratory rates within the normal range for cattle under tropical conditions (Da Costa et al., 2015).
One RB cow had high temperature (41.9 °C), respiratory rate (> 40 breaths/min), and heart rate (> 80 beats/min), which indicated
an alteration of their health status. This cow also had low Hct, Hgb concentration and showed abundant number of the protozoa
Trypanosoma vixax in the blood smear. Two control cows had high heart and respiratory rates and one of these had anemia related to
the presence of Trypanosoma vixax.
Evident vulvar discharge was observed in 12.5% (3/24) of the RB group (Table 1). The vaginoscopic exam revealed that 50.0%
(12/24) of the RB cattle had clear translucent mucus, 16.6% (4/24) mucus containing flecks, 20.8% (5/24) muco-purulent and 4.2%
(1/24) purulent vaginal secretions. Two animals (8.3%) did not show vaginal secretions. Thus, 41.7% (10/24) of the RB group had
mucous, muco-purulent or purulent vaginal secretions. On the other hand, abnormal secretions were not observed in any cow of the
control group (P = 0.07; Table 1).

3.2. Clinical pathology

Hematocrit, hemoglobin and red blood cell count were not different between groups (Table 2). Four of the RB animals showed
blood profiles consistent with anemia (Hct < 24%, red blood cell count < 5 × 106 cells/μL, or Hgb concentration < 8 g/dL) (Kramer,
2000; Jones and Allison, 2007). No differences were found for total leukocyte counts between groups (RB: 7220 cells /μL and control:
6900 cells /μL). A high proportion of animals were infected with Trypanosoma vivax [47.8 (11/23) and 40.0% (2/5), for RB and

Table 1
Clinical and ultrasonographical evaluation of the genital tract of repeat breeder (n = 24) and fertile (n = 5) Criollo Limonero cattle.
Repeat breeder cattle Fertile cows

Clinical evaluation n % n %
Vulvar discharge 3 12.5 a 0 0a
No vulvar discharge 21 87.5 a 5 100 a
Presence of abnormal vaginal mucus 10 41.7 a 0 0b
Presence of clear vaginal mucus 12 50.0 a 1 20.0 a
Absence of mucus 2 8.3 c 4 80.0 d
Ultrasound evaluation
a a
Cervix diameter in cm (mean ± SE) 24 2.98 ± 0.16 5 2.89 ± 0.14
Uterine fluid 5 20.8 a 0 0a
Normal uterine pattern 19 79.2 a 5 100 a
Ovarian Cysts 6 25.0 a 0 0a
Normal ovarian structures 18 75.0 a 5 100 a
Corpus luteum 9 37.5 a 1 20.0 a
Follicles ≥ 10 mm 9 37.5 a 4 80.0 a

a,b c,d
(P = 0.07) (P < 0.001).

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Table 2
Blood profile of repeat breeder and fertile Criollo Limonero cattle.
Group n Hct Hgb Erythrocytes WBC Incidence of T. vivax
Gr/dl Cells x 106/μL Cells x 103/μL
(%) n %

Repeat breeder cattle 23 27.1 9.1 5.6 7.2 11/23 47.8

Fertile cows 5 24.0 8.0 5.2 6.9 02/5 40.0

No significant differences between groups.

control groups, respectively].

3.3. Ultrasonography

The mean cervix diameter was not different between groups (2.98 ± 0.16 and 2.89 ± 0.14 cm for RB and control groups,
respectively). Free fluid (anechoic pattern) was observed in the uterine lumen of 20.8% RB cows (5/24). None of the fertile cows
showed free fluid in the uterine lumen (Table 1). Further, 25.0% (6/24) of RB animals had ovarian cysts (mean diameter:
25.41 ± 0.9 mm). Mature corpora lutea were found in 37.5% (9/24) of RB cattle (mean diameter: 20.2 ± 1.2 mm); while 37.5% (9/
24) had follicles > 10 mm as a main ovarian structure (mean diameter: 16.6 ± 1.2 mm; Table 1). All control cows had medium to
large follicles (14.1 ± 0.9 mm diameter) and one cow had a CL (24.5 mm diameter). No control cows had evidence of ovarian cysts.

3.4. Histopathology

Endometrial biopsy was not accomplished in 3 RB animals (2 cows and 1 heifer) due to difficulties to pass the biopsy instrument
through the cervix. It was presumed that cervical abnormalities could have been the cause of their infertility. Endometrial edema was
found in 57.1% (12/21) of the uterine biopsy samples of the RB animals (Table 3; Fig. 1B). The luminal epithelium was partially and
totally denuded in 66.7% and 9.5% of the animals with impaired fertility (Fig. 1C–F). In some of these animals, the necrosis of the
luminal epithelium was associated with a significant leukocyte infiltration in the subcellular stratum.
A significantly high proportion of RB cattle had moderate (57.1%; 12/21) or severe (23.8%; 5/21) diffuse mononuclear leukocyte
infiltration in the uterine stroma (Table 3; Fig. 3A–C). The majority of the endometrial biopsies (80.9%) revealed absent or mild

Table 3
Endometrial lesions of repeat breeder and fertile cattle from a Criollo Limonero herd.
Endometrial Lesions Repeat breeder cattle Fertile control cows

n % n %

Edema
Present 12 57.1 4 80.0
Absent 9 42.9 1 20.0
Epithelium
Normal 1 4.7 1 20.0
Leukocyte infiltration 4 19 1 20.0
Partially denuded 14 66.7 2 40.0
Totally denuded 2 9.5 1 20.0
Uterine glands
Normal 4 19 2 40.0
Dilated 13 61.9 3 60.0
Cystic 4 19 0 0
Periglandular fibrosis
Absent 4 19 2 40.0
Mild 3 14.3 1 20.0
Moderate 10 47.6 2 40.0
Severe 4 19 0 0
Mononuclear infiltration
Absent or mild 4 19.0 4 80.0
Moderate 12 57.1 1 20.0
Severe 5 23.8 0 0
Polynuclear infiltration
Absent or mild 17 80.9 4 80.0
Moderate 2 9.5 1 20.0
Severe 2 9.5 0 0
Lymphoid Aggregates
Present 9 42.9 3 60.0
Absent 12 57.1 2 40.0

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R.A. Palomares et al. Animal Reproduction Science 197 (2018) 22–32

Fig. 1. Photomicrographs showing uterine epithelium and stroma of repeat breeder cows from a Criollo Limonero herd (H&E). A normal epithelium
(arrows heads) and stroma (arrow). B endometrial edema (arrows) and subepithelial inflammatory infiltration (arrow heads). C-D partially and totally
(arrow heads) denuded epithelium. E–F epithelium totally denuded (arrows) with severe inflammatory infiltration (arrow heads).

polymorphonuclear infiltration under the epithelium and around the endometrial glands. Only 19% of the animals had a significant
degree of polymorphonuclear leukocytes infiltration (Fig. 3D). Lymphoid aggregates or nodules were observed in 42.9% of the
animals (Table 3). Nodules consisted of masses of mononuclear leukocytes accumulated to form spherical or oval structures which
were distributed within the endometrial stroma (Fig. 3E and F). Interestingly, lymphoid aggregates were found in the endometria of
three control animals.
Only 19.04% of the RB animals had normal endometrial glands (Table 3). Normal glands had cuboidal-tall epithelial cells and
were embedded in fibroblasts and lax connective tissue trabeculae in the endometrial stroma. Some leukocytes were also dispersed in
the connective tissue (Fig. 2A). The endometrial glands were dilated showing signs of degeneration in 61.9% of the RB animals (13/
21; Fig. 2B and C). Thus, the lumen of the glands was increased in size and the periglandular connective tissue was hypertrophied in
most of these animals (8/13). Moreover, 19.04% (4/21) of the endometrial biopsies from RB animals had cystic endometrial glands (3
cows and 1 heifer; Table 3), which had wide branched lumen and low cuboidal flattened epithelium (Fig. 2D and E). Some of the
dilated and cystic glands were occluded showing secretions and numerous leukocytes in the lumen. Some endometrial glands were
dilated in 3 control cows, but cystic glands were not evidenced in the control group. Moderate or severe periglandular fibrosis (3 or
more layers of fibroblasts) was found in 66.7% (14/21) of the RB cows (Fig. 2F). Periglandular fibrosis was observed as spindle-
shaped cells closely packed and parallel to each other and which had slender nuclei. In some instances, glands were nested in small
groups surrounded by the fibrous tissue. Two out of five control cows had moderate periglandular fibrosis. A summary of the main

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Fig. 2. Photomicrographs from histological sections showing endometrial glands of different repeat breeder cows from a Criollo Limonero herd (H&
E). A normal glands (arrows). B–C dilated glands (arrows) with inflammatory cells (solid arrows) and secretions in the lumen (arrow heads). D–E
cystic branched glands (arrows). F severe periglandular fibrosis stained with Manson trichromic (solid arrows).

clinical, ultrasonographical and histopathological findings for each animal is presented in Table 4.

4. Discussion

This is the first report focused on a clinical examination and histopathological evaluation of uterine biopsy samples in subfertile
Criollo Limonero cattle. This cattle breed represents an important genetic resource for sustainable milk and beef production under
tropical conditions, and is currently threatened with extinction (De Alba, 1987; Mueller-Haye, 1978; Landaeta-Hernández et al.,
2011). Investigation and development of strategies for enhancing reproductive performance to promote genetic preservation of South
American Criollo breeds with specific adaptations is warranted (FAO/UNEP, 1987). Overall, the studied herd had a low reproductive
performance (first service conception rate: 45.8%; percentage of repeat breeder cows: 25%; services per conception: 3.4 and average
calving to conception interval: 155 days) when compared to acceptable values reported for dual purpose herds under tropical
conditions (Gonzalez-Stagnaro et al., 2002).
In this study all the repeat breeder animals showed some degree of uterine inflammatory and/or degenerative lesions. Therefore,
poor fertility in this cattle group could be associated with the observed endometrial alterations. Main causes of low fertility in cattle
involve a combination of several factors related to unsuccessful ovulation, fertilization failure and embryonic death. Endometritis is
the superficial inflammation of the endometrium extending no deeper than the stratum spongiosum, without systemic signs

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Fig. 3. Photomicrographs showing infiltration of inflammatory cells in repeat breeder cows from a Criollo Limonero herd. A–C severe infiltration of
mononuclear inflammatory cells in stroma (arrows). D periglandular polynuclear leukocyte infiltration (arrows). E–F lymphoid aggregates (arrows).

(BonDurant, 1999). It has been recognized as the most likely cause of increased services per conception, prolonged calving to
conception interval and low conception rates in cows (Borsberry and Dobson, 1989; Heuwieser et al., 2000; LeBlanc et al., 2002;
Ahmadi et al., 2005). In previous studies, cows with endometritis have shown about 20% lower conception rates, longer calving to
conception intervals and higher reproductive culling rates than control cows (Borsberry and Dobson, 1989; LeBlanc et al., 2002;
Sheldon and Dobson, 2004). Furthermore, a more recent study showed that repeat breeder cows have an increased culling risks, from
8.1% to 35.2% (Bonneville-Hébert et al., 2011). In contrast, other studies showed that culling risk was not affected by the occurrence
of cytological endometritis and/or purulent vaginal discharge (Dubuc et al., 2011).
Ten repeat breeder cattle had abnormal cervico-vaginal secretions, while only three showed purulent vulvar discharges.
Examination of the vagina for the presence of secretions (manually or by using a vaginoscope) is a practical technique for the
diagnosis of endometritis (Bretzlaff, 1987; Sheldon and Dobson, 2004; LeBlanc et al., 2002). The character of the vaginal mucus can
be scored and correlated with the bacterial growth density in the uterus, being useful for prognosis about the likely success of
treatment (Sheldon and Dobson, 2004). However, the absence of uterine discharge by vaginoscopic examination is not truly in-
dicative of absence of endometritis (Kasimanickam et al., 2004). Moreover, although visible abnormal vaginal exudate may ac-
company more severe forms of endometritis, it has become clear that cows may have visible reproductive tract secretions (i.e. from
cervicitis or vaginitis) without endometritis (Gilbert, 2016). Uterine fluids were evidenced in five out of 24 repeat breeder animals.
Ultrasound evaluation of the endometrium and uterine content is a diagnostic technique that could be used to identify some, but not
all cases of subclinical endometritis (Kasimanickam et al., 2004; LeBlanc, 2008). The volume of the intrauterine fluid identified using

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Table 4
Summary of main clinical and histopathological findings in repeat breeder (RB) cows and heifers compared to normal fertile control cows.
Animal Group Ovarian Cysts Vulvar Discharge Abnormal Vaginal Uterine Fluid Mod-sev MNL Mod-sev Peri-glandular
Mucus Infiltration Fibrosis

1 RB cow – – + – + +
2 RB cow + – – – + +
3 RB cow – – + – + +
4 RB cow + + + + + +
5 RB cow – – – – + +
6 BR cow – – – + + +
7 RB cow + – + + + –
8 RB cow – – – – – +
9 RB cow – – – + + +
10 RB cow – + – + – +
11 BR cow – – + – + –
12 RB cow – + – – + –
13 RB cow + – – – + –
14 RB cow – – – – + +
15 RB cow – – – – – –
16 RB cow – – – – + –
17 RB cow – – + – NA NA
18 RB cow – – – – NA NA
19 RB heifer – – – – – +
20 RB heifer + – + – + +
21 RB heifer + – + – + +
22 RB heifer – – – – + +
23 RB heifer – – + – + –
24 RB heifer – – + – NA NA
25 Control – – + – – –
26 Control – – – – – +
27 Control – – – – – –
28 Control – – – – + –
29 Control – – – – – +

NA: biopsy not available. MNL: Mononuclear Leukocytes.

ultrasonography in combination with endometrial cytology effectively assisted in the diagnosis of subclinical endometritis in prior
studies and were significantly correlated with uterine bacterial growth, reduction in the relative pregnancy rate and increased time to
achievement of pregnancy (Mateus et al., 2002; Kasimanickam et al., 2004).
In the present study, a high percentage of the sub-fertile animals had disrupted uterine epithelium and dilated or cystic en-
dometrial glands. The morphological alterations of the glands and endometrium in the abnormal uteri can be caused by occlusion of
the lumen of the affected glands, which prevents glandular secretions from reaching the uterine lumen, remaining within the glands
(Cupps, 1973). Cystic endometrial gland degeneration was not observed in the fertile cows. Thus, this chronic degenerative lesion
was associated with poor reproductive performance, as previously reported (Ahmadi et al., 2005; Chaffaux et al., 1987; Cupps, 1973).
In the current case-control study, a significant proportion of animals had moderate or severe diffuse mononuclear infiltration dis-
tributed throughout the endometrium and/or around the glands, which could be associated with the chronicity of the inflammatory
condition. Chronic endometritis has been previously characterized by infiltration of mononuclear inflammatory cells and the pre-
sence of cystic glands and periglandular fibrosis (Ahmadi et al., 2005). In contrast to the diffuse mononuclear leukocyte infiltration,
lymphoid aggregates in the submucosa were evidenced in both, subfertile and fertile animals, suggesting that their presence may not
be associated with endometrial damage or interfere with normal fertility (Chaffaux et al., 1987; Moss et al., 2002).
In this study, the observed chronic endometrial lesions might have been associated with the age of the studied population
(subfertile cows: 5 to 13 years and subfertile heifers: 6 to 7.5 years). Interestingly, subfertile cows were remarkably older (27.4
months) than the control cows. Age has been associated with changes in genitalia conformation (St. Jean et al., 1988; Pouret, 1982).
As animals get older the conformation of the reproductive tract changes affecting the barriers that protect the uterus from infection
(vulva, vulvo-vaginal sphincter and cervix). Abnormal vulvar conformation such as horizontal vulva (age/parity related), increases
the risk of urovagina (St. Jean et al., 1988; Gautam and Nakao, 2009) pneumovagina (Goncagul et al., 2012) and vaginitis due to
fecal contamination, which ultimately lead to severe endometritis and infertility (Gautam and Nakao, 2009). Age of the subfertile
animals in this study was significantly greater than the average age of cows and heifers of other B. taurus breeds within a regular
reproductive program in commercial dairy operations. Therefore, it is possible that changes in conformation of the external genitalia
may have impaired the host defense mechanisms, particularly by disruption of the physical barriers and local immune function
(Sheldon et al., 2009) predisposing the animals to suffer endometritis. Despite this study was focused on clinical and histopatho-
logical evaluation of the reproductive tract, conformation of the vulvar lips and vulvo-vaginal sphincter was not evaluated.
A high number of cows with dilated/cystic glands (17/21) also had moderate to severe mononuclear infiltration (13/17, 76.4%).
Similarly, Cupps (1973) found that 20 out of 58 (34.5%) infertile animals had abnormal uterine glands combined with leukocyte

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R.A. Palomares et al. Animal Reproduction Science 197 (2018) 22–32

infiltration, indicating that the presence of eosinophils and lymphocytes, individually or in combination, was strongly associated with
modified uterine glands. The results of this study agree with previous studies by Moss et al. (2002) which showed that periglandular
fibrosis was present in high proportion (17/30, 56%) of poor breeder cows compared with normal breeders (3/25, 12%). Severe
periglandular fibrosis was not found in any fertile animal of our study, which is in agreement with Chaffaux et al. (1987) who
reported that 85% of the fertile animals did not have severe endometritis and periglandular fibrosis, but 92% of the animals showing
severe leukocyte infiltration and fibrosis were infertile (Chaffaux et al., 1987).
In the current study, 25.0% of the subfertile cattle had ovarian cysts (OC). The percentage of cows with intrauterine fluid (20.8%),
or muco-purulent or purulent discharge (25.0%) was similar. Moreover, vaginal abnormal secretions (mucous, muco-purulent or
purulent) were observed in 4 out of 6 animals with OC. All cattle with ovarian cysts showed chronic endometritis characterized by
moderate to severe mononuclear leukocyte infiltration, altered glandular structure and periglandular fibrosis. These findings suggest
a possible connection between endometritis and OC in these cows. A higher risk for developing OCs was previously reported in cows
with untreated endometritis with mucopurulent or purulent fetid discharge (Tsousis et al., 2009; Mateus et al., 2002). Endotoxins or
endometrial damage associated with endometritis are thought to be involved in the pathogenesis of OC by disrupting the hy-
pothalamic-pituitary-ovarian axis (Tsousis et al., 2009). Endotoxins and bacteria stimulate the release of endometrial PGF2α as part of
the inflammatory response. Cows with endometritis that later developed OC had higher blood PGFM and cortisol concentrations than
control cows. PGFM can induce the release of ACTH and stimulate adrenal activity (Bosu and Peter, 1987). The increase in cortisol
was associated with decreased LH secretion, altering ovarian function and ovulation, and leading to OCs (Bosu and Peter, 1987). The
more severe the infection, the more significant the effect on ovarian function (Bosu and Peter, 1987).
Systemic signs of disease were not observed in most of the animals examined in this study. Moreover, animals with histological
lesions of endometritis did not show significant variation in the total blood leukocyte counts compared with the range reported for
bovines (Kramer, 2000). Despite Criollo Limonero cattle have been reported to be tolerant to Trypanosoma (Florio-Luis et al., 2011b),
the high incidence of Trypanosoma vivax infection observed in this herd might have contributed with the poor reproductive per-
formance. Trypanosoma vivax is a pathogenic hemoparasite found in Africa, South and Central America and the Caribbean, especially
in regions occupied by the biological vector, the tsetse fly. Serious reproductive disorders might appear during the acute or chronic
periods of Trypanosoma infection, including abortions, irregular estrous, heat repetitions, placental retention, delay in uterine in-
volution, infertility, perinatal mortality, stillbirths, and weakness of newly born calves (Bezerra and Batista, 2008; Cadioli et al.,
2012; Dagnachew et al., 2015). Additionally, immunosuppression is a well-documented general feature of infection with Trypano-
soma in cattle, humans and mice (Mansfield, 1989; De Baetselier, 1996; Taylor, 1998) and thus may reduce the host's ability to
develop a protective immune response after infections.

5. Conclusion

In this study chronic endometritis, characterized by vaginal and uterine secretions, and the presence of moderate to severe
mononuclear leukocyte infiltration, dilated endometrial glands, and moderate to severe periglandular fibrosis was the most common
reproductive pathology observed in the subfertile Criollo Limonero cattle, which indicates a strong association with conception
difficulties in these animals. In addition, ovarian cysts were evidenced in high proportion of cows with endometritis, suggesting a
possible connection between these two reproductive pathologies.

Conflict of interest

The authors declare that they have no conflict of interest. All applicable international, national, and/or institutional guidelines for
the care and use of animals were followed.

Acknowledgments

This study was conducted under cooperative agreement between The University of Georgia, Unidad de Investigaciones en
Reproducción Animal (FCV-LUZ) and Estación Carrasquero-INIA. The authors would like to express their most sincere thanks to the
Consejo de Desarrollo Científico y Humanístico de la Universidad del Zulia (CONDES-LUZ) and INIA for providing financial support
for this research.

References

Ahmadi, M.R., Khodakaram-Tafti, A., Nazifi, S., Ghaisari, H.R., 2005. The comparative evaluation of uterine and cervical mucosa cytology with endometrial histo-
pathology in cows. Comp. Clin. Pathol. 14, 90–94.
Ayalon, N., 1984. The repeat breeding problem. Proceedings of the Xth International Congress of Animal Reproduction and Artificial Insemination 10-14 June 41–50
(Abstract).
Båge, R., Gustafsson, H., Larsson, B., Forsberg, M., Rodríguez-Martínez, H., 2002. Repeat breeding in dairy heifers: follicular dynamics and estrous cycle characteristics
in relation to sexual hormone patterns. Theriogenology 57, 2257–2269.
Bezerra, F.S., Batista, J.S., 2008. Efeitos da infecção por Trypanosoma vivax sobre a reprodução: uma revisão. Acta Vet. Br. 61–66.
Bondurant, R.H., 1999. Inflammation in the bovine female reproductive tract. J. Anim. Sci. 77, 101–110.
Bonneville-Hébert, A., Bouchard, E., Du Tremblay, D., Lefebvre, R., 2011. Effect of reproductive disorders and parity on repeat breeder status and culling of dairy cows
in Quebec. Can. J. Vet. Res. 75, 147–151.
Borsberry, S., Dobson, H., 1989. Periparturient diseases and their effect on reproductive performance in five dairy herds. Vet. Rec. 124, 217–219.
Bosu, W.T.K., Peter, A.T., 1987. Evidence for a role of intrauterine infections in the pathogenesis of cystic ovaries in postpartum dairy cows. Theriogenology 28,

31
R.A. Palomares et al. Animal Reproduction Science 197 (2018) 22–32

725–736.
Bretzlaff, K., 1987. Rationale for treatment of endometritis in the dairy cow. Vet. Clin. North. Am. Food Anim. Pract. 3, 593–607.
Cadioli, F.A., Barnabé Pde, A., Machado, R.Z., Teixeira, M.C., André, M.R., Sampaio, P.H., Fidélis Junior, O.L., Teixeira, M.M., Marques, L.C., 2012. First report of
Trypanosoma vivax outbreak in dairy cattle in São Paulo state, Brazil. Rev. Br. Parasitol. Vet. 21, 118–124.
Chaffaux, S., Recorbet, Y., Bhat, P., Crespeau, F., Thibier, M., 1987. Biopsies de l’endometre au cours su post-partum pathologique chez la vache. Rec. Med. Vet. 63,
199–209.
Cheong, S.H., Nydam, D.V., Galva˜o, K.N., Crosier, B.M., Gilbert, R.O., 2011. Cow-level and herd-level risk factors for subclinical endometritis in lactating Holstein
cows. J. Dairy Sci. 94, 762–770.
Cupps, P.T., 1973. Uterine changes associated with impaired fertility in the dairy cow. J. Dairy Sci. 56, 878–884.
Da Costa, A.N., Feitosa, J.V., Montezuma Jr, P.A., de Souza, P.T., de Araújo, A.A., 2015. Rectal temperatures, respiratory rates, production, and reproduction
performances of crossbred Girolando cows under heat stress in northeastern Brazil. Int. J. Biometeorol. 59, 1647–1653.
Dagnachew, S., Bezie, M., Terefe, G., Abebe, G., Barry, J.D., Goddeeris, B.M., 2015. Comparative clinico-haematological analysis in young Zebu cattle experimentally
infected with Trypanosoma vivax isolates from tsetse infested and non-tsetse infested areas of Northwest Ethiopia. Acta Vet. Scand. 57, 24–34.
De Alba, J., 1987. Criollo cattle of Latin American. Hogdes, J. (Ed.), Proceeding of the Second Meeting of the Food and Agriculture Organization of the United Nations/
United Nations Environment Programme (FAO/UNEP) 17–39.
De Baetselier, P., 1996. Mechanisms underlying trypanosome-induced T-cell immunosuppression. In: Mustafa, A.S., Al-Attiyah, R.J., Nath, I., Chugh, T.D. (Eds.), T-Cell
Subsets and Cytokines Interplay in Infectious Diseases, S Karger, pp. 124–139 Basel, Switzerland.
Dubuc, J., Duffield, T.F., Leslie, K.E., Walton, J.S., LeBlanc, S.J., 2010. Definitions and diagnosis of postpartum endometritis in dairy cows. J. Dairy Sci. 93, 5225–5233.
Dubuc, J., Duffield, T.F., Leslie, K.E., Walton, J.S., Leblanc, S.J., 2011. Effects of postpartum uterine diseases on milk production and culling in dairy cows. J. Dairy Sci.
94, 1339–1346.
Edmondson, A.J., Lean, I.J., Weaver, L.D., Farver, T., Webster, G., 1989. A body condition scoring chart for Holstein dairy cows. J. Dairy Sci. 72, 68–78.
FASS, 1999. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. FASS Inc., Champaign, IL.
Florio-Luis, J., 2008. Uso de los bovinos criollos en cruzamientos con otras razas bovinas en américa Latina, con énfasis en ganadería doble propósito. In: González-
Stagnaro, C., Madrid-Bury, N., Soto-Belloso, E. (Eds.), desarrollo Sostenible de la Ganadería Doble Propósito. Fundación GIRARZ. Ediciones Astro Data S.A.
Maracaibo, Venezuela, pp. 58–69.
Florio-Luis, J., Contreras, G., Zambrano, S., Fajardo, J., Fuenmayor, A., 2011a. Criollo limonero’s national preservation and animal breeding program at Venezuela
Bolivarian Republic. Actas Iberoamericanas de Conservación Animal 1, 117–122.
Florio-Luis, J., Tamasaukas, R., Agudo, L., 2011b. Trypanotolerance in bovine livestock at Bolivarian Republic of Venezuela: emphasis Creole cattle. Actas
Iberoamericanas de Conservación Animal 1, 304–308.
Food and Agriculture Organization of the United Nations/ United Nations Environment Programme (FAO/UNEP), 1987. Principles for genetic improvement of
indigenous animals in the tropics. Recommendations of the expert panel. Section a: animal production and health. Paper 66. Animal genetic resources. Strategies
for improved use and conservation. Proceedings of the 2nd Meeting of the FAO/UNEP Expert Panel Pp. 3.
Gautam, G., Nakao, T., 2009. Prevalence of urovagina and its effects on reproductive performance in Holstein cows. Theriogenology 71, 1451–1461.
Gilbert, R.O., 2016. Management of reproductive disease in dairy cows. Vet. Clin. North. Am. Food Anim. 32, 387–410.
Gilbert, R.O., Shin, S.T., Guard, C.L., Erb, H.N., Frajblat, M., 2005. Prevalence of endometritis and its effects on reproductive performance of dairy cows.
Theriogenology 64, 1879–1888.
Goncagul, G., Seyrek Intas, K., Kumru, I.H., Seyrek Intas, D., 2012. Prevalence and accompanying signs of pneumovagina and urovagina in dairy cows in the Southern
Marmara region. Tierarztl Prax Ausg G Grosstiere Nutztiere 40, 359–366.
Gonzalez-Stagnaro, C., Madrid-Bury, N., Goicochea-Llaque, J., Rodríguez-Urbina, M., 2002. Methodology and implementation of bovine reproductive control in
production medicine and total quality programs. Rev. Cient., FCV-LUZ 12, 730–741.
Griffin, J.F., Hartigan, P.J., Nunn, W.R., 1974. Non-specific uterine infection and bovine fertility. I. Infection patterns and endometritis during the first seven weeks
post-partum. Theriogenology 1, 91–106.
Grunert, E., Gregory, R.M., 1984. Diagnóstico e terapêutica da infertilidade na vaca. Porto Alegre: Sulina. 163 Pp.
Hartigan, P.J., Murphy, J.A., Nunn, W.R., Griffin, J.F., 1974. The technical importance of procedures in the preparation of histological specimens from the bovine
endometrium. Br. Vet. J. 130, 164–168.
Heuwieser, W., Tenhagen, B.A., Tischer, M., Lühr, J., Blum, H., 2000. Effect of three programmes for the treatment of endometritis on the reproductive performance of
a dairy herd. Vet. Rec. 146, 338–341.
Jones, M.L., Allison, R.W., 2007. Evaluation of the ruminant complete blood cell count. Vet. Clin. North. Am. Food Anim. 23, 377–402.
Kasimanickam, R., Duffield, T.F., Foster, R.A., Gartley, C.J., Leslie, K.E., Walton, J.S., Johnson, W.H., 2004. Endometrial cytology and ultrasonography for the
detection of subclinical endometritis in postpartum dairy cows. Theriogenology 62, 9–23.
Kramer, J.W., 2000. Normal hematology of cattle, sheep, and goats. In: Feldman, B.F., Zinkl, J.G., Jain, N.C. (Eds.), Schalm’s Veterinary Hematology, 5th edition.
Lippincott Williams & Wilkins, Philadelphia, pp. 1075–1084.
Landaeta-Hernández, A., Zambrano-Nava, S., Hernández-Fonseca, J.P., Godoy, R., Calles, M., Iragorri, J.L., Añez, L., Polanco, M., Montero-Urdaneta, M., Olson, T.,
2011. Variability of hair coat and skin traits as related to adaptation in Criollo Limonero cattle. Trop. Anim. Health Prod. 43, 657–663.
LeBlanc, S.J., 2008. Postpartum uterine disease and dairy herd reproductive performance: a review. Vet. J. 176, 102–114.
LeBlanc, S.J., Duffield, T.F., Leslie, K.E., Bateman, K.G., Keefe, G.P., Walton, J.S., Johnson, W.H., 2002. Defining and diagnosing postpartum clinical endometritis and
its impact on reproductive performance in dairy cows. J. Dairy Sci. 85, 2223–2236.
Mann, G.E., Lamming, G.E., 2001. Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in
cows. Reproduction 121, 175–180.
Mansfield, J.M., 1989. Immunology of African trypanosomiasis. In: Wyler, D.G. (Ed.), Modern Parasite Biology. WH Freeman and Company, New York, pp. 222–246.
Mateus, L., da Costa, L.L., Bernardo, F., Silva, J.R., 2002. Influence of puerperal uterine infection on uterine involution and postpartum ovarian activity in dairy cows.
Reprod. Domest. Anim. 37, 31–35.
Maurer, R.R., Echternkamp, S.E., 1985. Repeat-breeder females in beef cattle: influences and causes. J. Anim. Sci. 61, 624–636.
Moss, N., Lean, I.J., Reid, S.W., Hodgson, D.R., 2002. Risk factors for repeat-breeder syndrome in New South Wales dairy cows. Prev. Vet. Med. 54, 91–103.
Mueller-Haye, B., 1978. Possibilities of International and National Action Programmes for the Conservation and Evaluation of Animal Genetic Resources in Latin
America With Special Reference to the Criollo Cattle. FAO, Rome, Italy pp. 14.
Perea, F., Soto, E., González, C., 2002. Días vacíos y producción de leche en vacas mestizas. In: González-Stagnaro, C., Soto-Belloso, E., Ramírez-Iglesia, L. (Eds.),
Avances en la Ganadería de Doble Propósito, (Fundación GIRARZ, Ediciones Astro Data S.A., Maracaibo. Venezuela), pp. 401–416.
Peters, A.R., 1996. Herd management for reproductive efficiency. Anim. Reprod. Sci. 42, 455–464.
Pouret, E.J.M., 1982. Surgical technique for the correction of pneumo and urovagina. Equine Vet. J. 14, 249–250.
Ramírez, L.N., 2005. El Ganado Criollo Limonero: Una raza en peligro de extinción? Mundo Pecuario 1, 30–31.
Sheldon, I.M., Dobson, H., 2004. Postpartum uterine health in cattle. Anim. Reprod. Sci. 82, 295–306.
Sheldon, I.M., Cronin, J., Goetze, L., Donofrio, G., Schuberth, H.J., 2009. Defining postpartum uterine disease and the mechanisms of infection and immunity in the
female reproductive tract in cattle. Biol. Reprod. 81, 1025–1032.
Sreenan, J.M., Diskin, M.G., 1983. Early embryonic mortality in the cow; Its relationship with progesterone concentration. Vet. Rec. 112, 517–521.
St. Jean, F.G., Hull, B.L., Robertson, J.T., Hoffsis, G.F., Haibel, G.K., 1988. Urethral extension for correction of urovagina in cattle: a review of 14 cases. Vet. Surg. 17,
258–262.
Taylor, K.A., 1998. Immune responses of cattle to African trypanosomes: protective or pathogenic? Int. J. Parasitol. 28, 219–240.
Tsousis, G., Sharafi, R., Hoedemaker, M., 2009. Associations between the clinical signs of chronic endometritis with ovarian cysts and body condition loss in German
Holstein Friesian cows. J. Vet. Sci. 10, 337–341.
Williams, E.J., Fischer, D.P., Pfeiffer, D.U., England, G.C., Noakes, D.E., Dobson, H., Sheldon, I.M., 2005. Clinical evaluation of postpartum vaginal mucus reflects
uterine bacterial infection and the immune response in cattle. Theriogenology 63, 102–117.

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