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Semen quality of stallions challenged with the Kentucky 84 strain of equine arteritis virus
Theriogenology 82 (2014) 1068–1079
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Semen quality of stallions challenged with the Kentucky 84 strain of equine arteritis virus Juliana R. Campos a, Patrick Breheny b,1, Reno R. Araujo c, Mats H.T. Troedsson a, Edward L. Squires a, Peter J. Timoney a, Udeni B.R. Balasuriya a, * a
Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, Lexington, Kentucky, USA Department of Biostatistics, University of Kentucky, Lexington, Kentucky, USA c University of Sao Paulo, Laboratory of Molecular Morphophysiology and Development, FZEA-USP, Pirassununga, Sao Paulo, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 January 2014 Received in revised form 11 June 2014 Accepted 5 July 2014
Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. Some strains of EAV can cause fever, leukopenia, and dependent edema of the limbs, scrotum, and preputium in the acutely infected stallion. We hypothesized that fever and scrotal edema observed during the acute phase of the infection, but not the presence of EAV, have an adverse effect on semen quality. A group of seven stallions were intranasally inoculated with the Kentucky 84 (KY84) strain of EAV. Stallions were monitored for clinical signs of EVA until 42 days postinoculation (dpi). Semen was collected every other day for the first 15 days and 2 times a week up to 79 dpi. Additional samples were collected at 147, 149, and 151 dpi. Semen from each stallion was evaluated on the basis of motion characteristics, total number of spermatozoa, membrane integrity, and morphology. Virus infectivity titers were determined in RK-13 cells. Significant decreases in sperm quality were observed between 9 and 76 dpi. LOWESS (locally weighted scatterplot smoothing) curves for each horse were fit and integrated to quantify spermatozoa exposure to fever, virus, and edema over a period of 67 days before each ejaculation. Linear mixed models were then fit to isolate the effects of each factor on semen quality. Scrotal edema and fever were found to exert independent effects on all the semen quality parameters (P 0.002), whereas virus seems to exert little to no direct effect, as virus titers remained high long after semen quality returned to baseline. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Equine arteritis virus EAV Equine viral arteritis EVA Semen quality Semen evaluation
1. Introduction Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. First isolated from a lung of an aborted
* Corresponding author. Tel.: þ1 859 218 1124; fax: þ1 859 257 8542. E-mail address: [email protected] (U.B.R. Balasuriya). 1 Present address: Department of Biostatistics, College of Public Health, 145 North Riverside Drive, University of Iowa, Iowa City, IA 52242, USA. 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.07.004
fetus in 1953 [1,2], EAV is transmitted mainly by respiratory [2,3] and venereal routes [4]. Although most EAV strains cause subclinical infection after infection, some can cause moderate to severe clinical signs [5–7]. The clinical signs of EVA are characterized by fever, nasal and ocular discharge, conjunctivitis, depended edema, leukopenia, and abortion in pregnant mares [2,3]. The 1984 EVA outbreak in Kentucky’s Thoroughbred breeding population generated widespread interest and major concern to the equine industry [3]. Epidemiologic data collected during that outbreak strongly confirmed establishment of long-term persistence of EAV in stallions and the
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importance of the carrier stallion in the dissemination and perpetuation of the virus. After EAV infection, a variable proportion of stallions (30%–70%) can become carriers and continuously shed virus in their semen for varying periods [4,8]. The virus persists in the male reproductive tract, specifically in the accessory glands with highest infectivity titers found in the ampulla of the vas deferens [9], despite the presence of high titers of neutralizing antibodies to EAV in serum [4,8,9]. Isolation of virus from the preejaculatory fluid of semen from persistently infected stallions was not successful, whereas it could be recovered from the sperm-rich fraction of the ejaculate [8]. The precise mechanism of EAV persistence in the reproductive tract of stallions is unclear; however, it has been shown that establishment and maintenance of infection are testosterone dependent [10,11]. Persistently infected stallions play a major role in the transmission of EAV to mares either by natural breeding or artificial insemination with fresh-cooled or frozen semen [4,8,12]. Recently, it has been reported under experimental conditions that EAV can be transmitted to naive recipient mares via embryo transfer from a donor mare inseminated with EAVinfective semen [13]. Several viruses have been detected in the semen of a number of animal species, some of which can have direct or indirect detrimental consequences on the male reproductive tract and on semen [14]. Semen quality of stallions experimentally infected with the Kentucky 84 (KY84) strain of EAV was decreased for a period of time after inoculation with the virus, but it was not confirmed that the negative effect on semen parameters was because of the direct effect of the presence of virus in semen [15]. Thus, the objectives of this study were to determine (1) the effect on various semen parameters of stallions experimentally infected with the KY84 strain of EAV using computer-assisted sperm analysis (CASA) and differential interference contrast (DIC) microscopy and (2) if possible changes observed in semen quality result from direct effects of the presence of virus in the semen or from the indirect effects of fever and scrotal edema during the acute phase of infection in the stallion. 2. Materials and methods 2.1. Cells and viruses The high passage rabbit kidney cell line (RK-13 KY; passage level 399–409) was maintained in Eagle’s minimum essential medium (EMEM; Mediatech, Inc., Manassas, VA, USA) supplemented with 10% ferritin-supplemented bovine calf serum (Hyclone Laboratories, Logan, UT, USA), 100 U/mL penicillin and streptomycin (Mediatech, Inc.), and 1 mg/mL amphotericin B (Sigma–Aldrich, St. Louis, MO, USA). The virulent KY84 strain of EAV was used as the challenge virus [6,16]. The KY84 strain of EAV has been shown to establish persistent infection in the reproductive tract of stallions and to cause moderate to severe clinical signs of EVA infection in horses [9]. The modified live virus vaccine strain of EAV (ARVAC; Zoetis Animal Health Inc., Kalamazoo, MI, USA) was used as challenge virus in the microneutralization assay.
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2.2. Stallions Seven sexually mature (4–16-year old) stallions of mixed breed were included in the study (stallions IDs: L136–L142). Horses were obtained from a local commercial vendor and acclimated to their new environment for approximately 2 months before the study commenced. During this period, animals were accommodated in individual paddocks and trained to mount a mare or a phantom for collection of semen into an artificial vagina. All stallions exhibited good libido and normal sexual behavior. All were confirmed seronegative (titer <1:4) for EAV neutralizing antibodies several times before intranasal inoculation with the KY84 strain of EAV using a previously described protocol [17]. The animals were housed in individual stalls in an isolation facility for the duration of the study at the University of Kentucky Maine Chance Farm, Lexington, KY, USA. The study was carried out in accordance with an Institutional Animal Care and Use Committee–approved protocol at the University of Kentucky, Lexington, KY, USA (protocol number 2011-0888). 2.3. Experimental inoculation of stallions and clinical examination Stallions were inoculated intranasally with 3.75 105 plaque-forming units (PFU) of the KY84 strain of EAV in 5.0 mL of EMEM using a fenestrated catheter passed via the posterior nares into the nasopharynx as previously described [5]. All stallions were examined and clinical parameters were recorded by the same veterinarian. Scrotal edema was classified on a scale from 0 to 5 and recorded as absent (0), mild (1–2), moderate (3), and severe (4–5). Preinoculation (7, 5, and 2 days before experimental challenge) clinical examinations were performed once daily to determine baseline values for body temperature and also to certify that the parameters were within normal limits before experimental challenge of the stallions. Specifically, fever and scrotal edema were monitored twice daily (every 12 hours) for the first 15 days after infection, and the highest value of the day was recorded. Clinical signs continued to be monitored once per week during the following 4 weeks of the experiment (at 21, 28, 35, and 42 days postinoculation [dpi]). Blood samples were collected at 0, 2, 4, 6, 8, 10, 12, 14, 21, 28, 35, and 42 dpi to determine individual serum neutralizing antibody responses to EAV. The neutralizing antibody titers were determined as described by Senne et al. [17]. 2.4. Semen collection Two ovariectomized mares previously vaccinated with the commercial modified live virus vaccine against EVA (ARVAC; Zoetis Animal Health, Inc.) (respective antibody titers 1:256 and 1:512) were used to “tease” the stallions. Each stallion was allowed to mount either one of the mares or a breeding phantom to enable semen collection in an artificial vagina (Botucatu model; Botupharma, Botucatu, SP, Brazil). The artificial vagina was disinfected with a disinfectant (Roccal-D Plus; Pfizer Inc., New York, NY, USA) between collections. A disposable liner lubricated with
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sterile lubricant (Priority Care; First Priority Inc., Elgin, IL, USA) was used to collect semen from each stallion to avoid cross-contamination between ejaculate samples. Semen was collected from each stallion three times per week for a period of 3 weeks, and discarded, before evaluation of samples commenced. Semen was collected and evaluated 7, 5, and 2 days before challenge and every other day during the first 15 dpi and twice weekly until 79 dpi. Additional samples were collected and evaluated at 147, 149, and 152 dpi. Preinoculation semen samples were obtained to determine baseline values for percentage of total and progressively motile sperm cells (TMOT and PMOT, respectively), total number of spermatozoa (TNS), curvilinear velocity (VCL), percentage of live spermatozoa (LS), and percentage of morphologically normal spermatozoa (MNS). For the purpose of monitoring virus persistence in the reproductive tract of the stallions, semen was collected for virus isolation (VI) approximately once a month, specifically on 86, 107, 128, 149, 170, and 198 dpi (6.5 months), after challenge. 2.5. Semen processing and CASA The total number of spermatozoa in gel-free semen was estimated by measuring the volume with a graduated cylinder and the initial sperm concentration with a spectrophotometer-based instrument (SpermaCue; Minitube of America Inc., Verona, WI, USA). An aliquot of each ejaculate was fixed in 10% buffered formalin for analysis of sperm morphology. The gel-free semen was extended to contain approximately 25 106 spermatozoa/mL with a warm (37 C) commercial skim milk–based extender containing gentamicin (EQUIPRO; Minitube of America Inc.) and evaluated using a CASA instrument (Sperm Vision 3.5 Software; Minitube of America Inc.) to determine the percent of TMOT and PMOT, and VCL. A total of six microscopic fields per sample were selected for computerassisted analysis by the same examiner. 2.6. Membrane integrity The integrity of the spermatozoa plasmatic membrane was determined using eosin-nigrosin staining (Hancock Stain, Animal Reproduction Systems; Dupree Inc., Chino, CA, USA). Duplicate smears stained with Hancock stain were prepared for spermatozoa membrane integrity evaluation, as previously described [18]. The percentage of LS was determined by counting 200 cells per slide for each of two slides, and the average number of live (unstained) vs. dead (stained) cells was recorded. When the number of spermatozoa was low because of low concentration in a sample, less than 200 spermatozoa were counted and the percentage number was calculated. All slides were prepared and counted by the same individual who was blinded to the nature of the samples being analyzed.
(ZeissAxio Imager Upright Microscope). For analysis, 1.5 mL aliquots of fixed semen were applied to a microscope slide and overlaid with a cover glass. Individual spermatozoa were assigned to only one morphologic category even if they exhibited several abnormalities. When more than one defect was seen in the same cell, the most proximal defect was recorded. A total of 100 spermatozoa per sample were examined. All slides were counted by the same trained individual who was blinded to the samples being analyzed. 2.8. Virus isolation Virus isolation from raw gel-free semen samples was attempted in the RK-13 KY cell line according to a standard protocol used by the World Organisation for Animal Health (OIE) Reference Laboratory at Maxwell H. Gluck Equine Research Center, University of Kentucky [17]. Briefly, after sonication, serial decimal dilutions (101–104) of each sample supernatant were prepared in EMEM (Cellgro; Mediatech, Inc.). One milliliter of each dilution was inoculated into a 25-cm2 flask containing a confluent monolayer of RK-13 KY cells grown in supplemented EMEM and overlaid with the supplemented EMEM containing 0.75% carboxymethylcellulose (Sigma–Aldrich). Flasks not showing cytopathic effect after 4 days were subinoculated onto new RK-13 monolayers using a 1-mL culture supernatant as the inoculum. Tissue culture fluid (TCF) was harvested and stored at 80 C for viral RNA extraction. First and second passage flasks were stained with a 0.2% crystal violet solution in buffered formalin on Day 4 after inoculation for counting plaques and calculating the virus titer. The same individual performed all attempted VIs. Virus isolates were, later, confirmed by an EAV specific TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR) assay as previously described [19]. 2.9. Microneutralization assay The neutralizing antibody titers of the test sera were determined by microneutralization assay as described by Senne et al. [17]. 2.10. Real-time RT-PCR Briefly, viral RNA was directly isolated from 50 mL of TCF using a commercial RNA isolation kit (MagMAX-96 Viral RNA Isolation Kit; Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Viral RNA was eluted in 50 mL of nuclease-free water and stored at 80 C. The RNA extracted from TCF derived from RK-13 cells inoculated with semen samples that were negative for EAV, and from nuclease-free water were included as negative controls. Viral RNA extracted from TCF containing the KY84 strain of EAV was used as a positive control. A one-tube TaqMan real-time RT-PCR assay was performed as described by Miszczak et al. [19].
2.7. Sperm morphology 2.11. Statistical analysis Sperm morphology (normal morphology, head, acrosome, mid-piece and tail abnormalities, detached heads, and proximal and distal droplets) was assessed by DIC
Locally weighted polynomials (LOWESS [locally weighted scatterplot smoothing]) were fitted to the raw
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Fig. 1. Average smoothed curve of (A) body temperature (in degree Celsius) of stallions, and (B) virus titer in semen after experimental inoculation with the KY84 strain of EAV. The shaded area represents the 95% confidence interval.
data for each horse to produce smooth curves [20]. Pointwise averages of these curves were taken, and pointwise standard deviations (SDs) used to construct confidence bands. One-way ANOVA analyses were used to quantify onset and recovery, with “onset” defined as the earliest dpi for which the average response differed significantly (P<0.05) from baseline, and “recovery” defined as the latest dpi for which the average response failed to differ significantly from the baseline. To quantify spermatozoa exposure to each potentially causative factor (fever, edema, and virus concentration), these curves were then integrated over a period of 67 days before each sperm quality measurement. Regression models were then fitted for each semen quality outcome to determine the effects of each explanatory factor when adjusting for the other factors. To account for within-stallion correlation among the measurements, linear mixed-effect models were used. All analyses were carried out using R software for statistical computing and graphics (http://www.r-project.org). 3. Results 3.1. Clinical signs All seven stallions developed moderate to severe clinical signs of EVA after experimental inoculation with the KY84 strain of EAV which included any combination of fever (>38.6 C [101.5 F]), dependent edema of the limbs, scrotum and prepuce, periorbital edema, nasal and ocular discharge, photophobia, dyspnea, anorexia, decreased libido and congestion, petechiae, or ecchymosis of the oral mucous membranes. All stallions had fever ranging from 38.7 C to 40.8 C (101.7 F to 105.6 F) starting from 1 to 4 dpi and lasting until 8 to 9 dpi (5–8 days). On average, fever was observed from 3 to 8 dpi (Fig. 1). Highest body temperatures were observed among the stallions between 5 and 7 dpi. The severity and duration of edema varied between stallions. Five of seven stallions (L136–L140; 71.4%) developed moderate to severe scrotal edema starting from 4 to 7 dpi and persisting until 9 to 15 dpi (3–10 days) (Table 1).
Two stallions (L141 and L142) did not develop any edema of the scrotum. All stallions showed decreased libido during the acute phase of infection. The time to drop the penis and the number of times each stallion had to jump the mare or the phantom before ejaculation increased. All the clinical signs together with the moderate to severe edema in the hind limbs of the stallions observed during the acute phase of infection made it difficult for them to mount, which contributed to the increased time needed to obtain an ejaculate. However, none of the seven stallions failed to mount the mare or the phantom. One stallion (L142) failed to ejaculate and instead provided only the pre-ejaculatory Table 1 Scrotal edema of stallions (n ¼ 7) before and after inoculation with the KY84 strain of EAV. Days postinoculation (dpi)
Stallions IDs L136
L137
L138
L139
L140
L141
L142
7 5 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 21 28 35 42
0 0 0 0 0 0 0 0 0 0 þþ þþ þþ 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 þþ þþ þþþ þþþ þþþ þþþ þþþ þþ þþ þþ 0 0 0 0
0 0 0 0 0 0 0 0 0 þ þþþ þþþ þþþ þþþ þþ þþ þþ þþ 0 0 0 0 0
0 0 0 0 0 0 0 þ þ þ þþ þþ þþ þþ þþ þ þ 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 þþ þþþ þþþ þþþ þþþ þþþ þþþ þþ þ 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0, edema absent; þ, mild edema; þþ, moderate edema; þþþ, severe edema.
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Fig. 2. Smoothed average response in semen quality parameters after experimental inoculation of stallions with the KY84 strain of EAV. (A) Total motile spermatozoa (%TMOT); (B) progressive motile spermatozoa (%PMOT); (C) total number of spermatozoa (TNS); (D) curvilinear velocity (VCL); (E) live spermatozoa (%LS); and (F) morphologically normal spermatozoa (%MNS). The shaded area represents the 95% confidence interval.
fluid at 1 and 30 dpi. All stallions seroconverted to EAV starting from 8 dpi and maintained high serum neutralizing antibody titers until at least 42 dpi (1:64 to >1:512). 3.2. Evaluation of semen for virus shedding After experimental inoculation of the stallions with the KY84 strain of EAV, semen samples from each stallion were
tested for EAV shedding by VI in cell culture until 198 dpi. All stallions shed EAV in their semen starting at 5 dpi and continuing to shed at least until 107 dpi (3.5 months) (Fig. 1). Infectivity titers in their semen varied from 1 101 to 1.88 107 PFU/mL during the acute phase of infection. After the acute phase of infection, all but two of the seven stallions continued to shed very high titers of virus in their semen until 107 dpi. The exceptions were stallions L138
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Table 2 (A) Average onset and recovery times (days postinoculation [dpi]) of semen quality parameters after experimental inoculation of stallions (n ¼ 7) with the KY84 strain of EAV; (B) average onset and recovery times of abnormalities in spermatozoa. (A) Semen parameters Semen quality parameters
Total motility
Progressive motility
Total number of spermatozoa
Percentage of morphologic normal spermatozoa
Percentage of live spermatozoa
Onset (dpi) Recovery (dpi)
15 65
15 65
44 62
9 76
13 23
(B) Morphologic abnormalities in spermatozoa Defects in spermatozoa
Head
Detached head
Acrosome
Mid-piece
Tail
Proximal droplet
Distal droplet
Onset (dpi) Recovery (dpi)
23 69
20 65
58 72
13 23
41 48
13 27
9 13
Note that this table is based on an ANOVA test comparing the average response with baseline and does not indicate that all stallions had biologically relevant declines in these parameters during these time periods.
and L137, which shed low titers of virus equivalent to 6 101 and 8 101 PFU/mL, respectively, at 107 dpi. Those stallions were no longer semen shedders of virus at 128 and 149 dpi, respectively. Stallion L142 stopped shedding virus sometime between 170 and 198 dpi. Four of seven stallions (57%) continued to shed high titers of virus at least until 198 dpi (L136, L139, L140, and L141). 3.3. Evaluation of sperm motion characteristics and total number of spermatozoa A total of 201 sequential semen samples (29 per stallion from stallions L136–L141, and 27 from stallion L142) were collected over a period of 12 weeks, including 21 samples (three per stallion) before the experimental inoculation of stallions with the KY84 strain of EAV. Only one stallion (L142) failed to ejaculate at 1 and 30 dpi. Three semen samples from each stallion collected before experimental inoculation (7, 5, and 2 days) were used to establish the normal semen characteristics (baseline values) for TNS per ejaculate, percentage of TMOT and PMOT, VCL, percentage of LS, and percentage of MNS. Before inoculation, the mean baseline values for TNS, TMOT, PMOT, VCL, LS, and MNS were 5.87 109 spermatozoa; 75.6%; 66.1%; 141.6 mm/s, 90%; and 53.5%, respectively. Decrease in at least one of the parameters analyzed for semen quality was observed in all stallions after inoculation. On average, semen quality of stallions inoculated with the KY84 strain of EAV was reduced between 9 and 76 dpi and then returned to baseline (Fig. 2). With the exception of one stallion (L141), TMOT, PMOT, VCL, TNS, and the percentage of LS followed a similar trend in the other six stallions. Stallion L141 showed a drop only in MNS after challenge with virus, but never showed a decrease in the other semen parameters. The alteration in the number of MNS followed a similar trend in all seven stallions, including L141. The percentage of TMOT and PMOT, VCL, and TNS across all stallions was averaged for each collection and compared with the baseline average before virus inoculation (Fig. 2). There was more than 50% reduction in TMOT and PMOT of sperm between 30 and 51 dpi. On average, semen motility decreased (TMOT and PMOT) between 15 and 65 dpi and returned to preinoculation values around 65 dpi. Equine
arteritis virus infection of the stallions also had an effect on the VCL of spermatozoa, with decreased mean value observed between 23 and 34 dpi. Total number of spermatozoa reduced on average between 44 and 62 dpi. Two stallions, L139 and L142, had azoospermia at 34, 37, 41, 48, 51, and 55 dpi, and 44, 48, and 51 dpi, respectively. The TNS count returned to baseline values at around 62 dpi. Average onset and recovery times (dpi) of semen quality parameters after experimental inoculation of stallions are shown on Table 2. 3.4. Membrane integrity The percentages of spermatozoa with intact plasmatic membrane (LS) across all stallions were averaged for each collection and compared with the baseline average before inoculation with the KY84 strain of EAV. Before inoculation, all stallions had at least 81% LS and the mean baseline value for LS was 90% (Fig. 2). Changes in membrane integrity observed after experimental inoculation of the stallions followed a similar trend in all stallions, with the exception of stallion L141, as mentioned previously. The percentage of LS decreased on average between 13 and 23 dpi. 3.5. Evaluation of sperm morphology The percentages of MNS across all stallions were averaged for each collection and compared with the baseline average before inoculation with the KY84 strain of EAV (Fig. 2). All stallions had at least 50% MNS before experimental inoculation with the exception L136 (48%) and L141 (41%). Morphologic abnormalities in spermatozoa followed a similar trend in all seven stallions, including L141, with an overall reduction in the percentage of MNS observed from 9 to 76 dpi. The abnormalities in spermatozoa across all stallions were averaged for each collection and compared with the baseline average before inoculation (Fig. 3). Baseline values for all sperm abnormalities were seen on a frequency lower than 10%, with the exception of mid-piece defects, which were observed at a high frequency (25.10%). An increase in a wide variety of morphologic alterations in spermatozoa was observed after inoculation of the stallions and individual abnormalities were often higher than 10%. The
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Fig. 3. Smoothed average abnormalities (%) in spermatozoa after experimental inoculation of stallions with the KY84 strain of EAV. (A) Detached spermatozoa; (B) head abnormalities; (C) proximal droplets; (D) distal droplets; (E) mid-piece abnormalities; (F) acrosome abnormalities; and (G) tail abnormalities.
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Fig. 4. Representative picture of morphologic abnormalities observed in spermatozoa after experimental inoculation of stallions with KY84 strain of EAV. (A) Normal spermatozoa; (B) detached spermatozoa; (C) mid-piece abnormality (bent); (D) tail abnormalities (bent and coiled); (E) proximal droplet; (F) distal droplet; (G) head abnormality; (H) acrosome abnormality; and (I–L) multiple abnormalities in the same spermatozoon. Ten percent formalin-fixed nonstained semen samples viewed by differential interference contrast microscopy.
abnormalities that mostly increased after the inoculation of the stallions were detached heads, followed by head and proximal droplet defects, tail, mid-piece, and finally acrosome. Although there was an increase in acrosome and tail defects, those abnormalities were never detected at a frequency higher than 10%. A decrease in the percentage of distal droplets was seen between 20 and 58 dpi. During the period of the most dramatic decrease (23–58 dpi) in the percentage of MNS individual sperm frequently had three or more abnormalities, although only the most proximal was counted (Fig. 4). In summary, after inoculation of the stallions with the KY84 strain of EAV, all stallions had a significant decrease in the number of MNS accompanied by a consequent increase in the number of abnormalities in spermatozoa. On average, alteration in semen morphology occurred between 9 and 76 dpi. Average onset and recovery times (dpi) of morphologic abnormalities in spermatozoa after experimental inoculation of stallions are shown in Table 2. 3.6. Effects of fever, edema, and virus on semen quality The timing of the changes in semen quality indicates that fever and edema play a direct role in loss of semen quality, whereas the mere presence of virus does not
(Fig. 5). The onset of high temperatures and scrotal edema in the acute phase of the infection are consistently followed by a drop in semen quality over the next 2 months (roughly the duration of spermatogenesis). Virus concentrations, on the other hand, remain high, well after the semen quality parameters have returned to baseline. These observational conclusions were borne out in formal analyses using linear mixed models (Fig. 6). These models estimated the effect of a 1 SD change in fever, scrotal edema and virus exposure on various semen quality parameters, given that the other two exposures remained constant. Thus, a 1 SD change in temperature over the course of the sperm cycle is estimated to result in a 16% drop in normal morphology, an 11% decline in TMOT, a 14% decline in PMOT, a 4% decrease in VCL, and a 3% decrease in the number of LS. The equivalent change in edema is estimated to result in a 14% drop in normal morphology, a 10% decline in TMOT, a 12% decline in PMOT, a 6% decrease in VCL, and a 2% decrease in the number of LS. The estimated effects of changes in viral concentration, on the other hand, were insignificant for all the aforementioned semen quality parameters. The only significant association involving viral concentration involves TNS; a 1 SD increase in exposure to viral concentration over the course of spermatogenesis is estimated to result in a 7% decrease in TNS. The explanation
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Fig. 5. Overlay of smoothed average responses for body temperature, scrotal edema, and semen virus titers along with semen quality parameters. All variables have been put on a common axis representing the number of standard deviations away from baseline.
for this can be seen in Figure 5, in which TNS returns to baseline much later than the other parameters, and thus more closely matches the timing of viral exposure. 4. Discussion Using an objective assessment of semen quality (CASA), this study revealed that semen parameters are negatively affected after experimental inoculation of stallions with the KY84 strain of EAV. These results agree with the findings of Neu et al. [15] that deterioration in semen quality after experimental inoculation of stallions with the same strain of EAV appears to be temporary, and that the duration of reduced semen quality corresponded approximately with the duration of spermatogenesis in the horse [21]. In the study of Neu et al. [15], it was not clear if the negative effect on semen parameters was because of the direct effect of the presence of the virus in semen. The loss of stallions from that study at intervals made statistically significant results difficult to obtain. In addition, the use of a subjective method of semen analysis could have allowed the results to be susceptible to bias. Our findings indicate that edema and fever exert independent deleterious effects on semen quality, whereas viral concentrations, which remained high long after semen quality returned to baseline, appear to exert little to no direct effect. After challenge, all stallions shed virus in the semen starting from 5 until at least 107 dpi. Thus, the presence of virus in semen was constant throughout the experimental observation period. High body temperature and scrotal edema caused by infection were not
constant, however, and were observed sometime between 1 and 15 dpi during the acute phase of the disease. After 6 to 13 days of exposure to the clinical consequences of fever and/or scrotal edema, deleterious effects on spermatogenesis were noticed. Yet, it is not possible to entirely rule out the direct effect of the virus infection on semen quality given that, in a previous study, the KY84 strain of EAV was isolated from the testis of stallions euthanized up to 20 dpi [9]. Nonetheless, it is unlikely that virus, rather than the combined effect of fever and scrotal edema, was responsible for the detrimental effect seen in semen quality, because microscopic lesions observed in the reproductive tract of stallions during the acute phase of EAV infection were a result of viral replication and lysis of endothelial cells, and changes in germ cells were never reported [9]. In contrast, histopathologic changes in germ cells of testis exposed to heat were detected in different species [22]. It is well known that testicular temperature, 3 C to 5 C cooler than the body, is required in most mammals for normal spermatogenesis to occur [21,22]. Previous studies assessing the effects of increased testicular temperature in different species such as mice [23], rams [24], boars [25], bulls [26], and stallions [27,28] have shown that transient and chronic exposure of the testis to elevated temperatures can disrupt spermatogenesis. When testicular temperature is elevated because of conditions such as fever, testicular trauma, inflammation, and edema, the thermoregulatory mechanism necessary to cool the testis and allow for normal spermatogenesis is disrupted [29]. Under these conditions, metabolism and oxygen demand increase at a
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Fig. 6. Linear mixed models for the effect of body temperature (Temp), virus titer (Log Virus), and scrotal edema (Edema) on sperm quality measurements over a period of 67 days before semen collection. (A) Total motile spermatozoa (%TMOT); (B) progressive motile spermatozoa (%PMOT); (C) total number of spermatozoa (TNS); (D) curvilinear velocity (VCL); (E) live spermatozoa (%LS); and (F) morphologically normal spermatozoa (% MNS).
greater rate than blood flow from the testicular artery to the testes, and testicular hypoxia occurs with consequent detrimental effects on sperm production and quality [21,22]. In this study, with the exception of one stallion (L141) that showed a drop only in the number of MNS, semen of the other six stallions experimentally infected with EAV showed a significant decrease in all parameters evaluated for semen quality (TMOT and PMOT; TNS; LS; MNS; VCL) between 9 and 76 dpi. Stallion L141 had fever but not scrotal edema during the acute phase of the disease. Perhaps, the fever in this animal was not sufficient to elevate the intrascrotal temperature to a degree needed to interfere with motility, TNS, LS, and VCL, but it was sufficient to decrease the percentage of MNS. Neu et al. [15] observed a similar outcome in a group of stallions experimentally infected with the KY84 strain of EAV, which experienced a decrease in the percentage of MNS but not in motility, and suggested that mechanisms important in the formation or maintenance of normal sperm structure may be the most sensitive to an increase in temperature. Spermatogenesis in stallions takes 57 days [21], and this is followed by the epididymal transit of spermatozoa, a process that lasts 8 to 11 days [30]. Therefore, spermatozoa evaluated
anytime between 9 and 76 dpi were at different stages of development at the time of exposure to adverse conditions (1–15 dpi). It is possible that various stages of developing spermatozoa (spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and mature sperm cells) were sensitive to the effects of high body temperature and scrotal edema observed during the acute phase of EAV infection. However, most of the morphologic defects were observed at around 41 dpi. Taking into consideration the length of spermatogenesis, the lifespans of primary (19 days) and secondary spermatocytes (0.7 days), spermatids with round nuclei (8.7 days), and elongated spermatids (10.1 days), as well as knowing that epididymal transit is approximately 9 days [31], the appearance of morphologically abnormal spermatozoa mostly at 41 dpi suggests that the cells that were more dramatically affected at the time of testicular injuries were primary and secondary spermatocytes. These results are similar to the results found by Freidman et al. [27], in which primary and secondary spermatocytes were the germ cells most adversely affected after increased testicular temperature resulting from 24 and 48 hours insulation of stallions’ testis. In the assessment of morphologic abnormalities, spermatozoa were assigned only to one morphologic category
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and the most proximal defect was prioritized over the distal defects. The defects seen in spermatozoa were varied and all were increased after experimental inoculation of the stallions with virus, with the exception of the percentage of distal droplets, which was decreased. The spermatozoa with distal droplets decreased significantly between 23 and 58 dpi; however, this decrease did not necessarily mean that their frequency had dropped, but rather, that another abnormality had increased. The period in which distal droplet abnormalities were significantly decreased coincided with the period of the most dramatic decrease (23–58 dpi) in the percentage of MNS, when an increase in the multiple defects in the spermatozoa was seen. The most dramatic increase was seen in spermatozoa with detached heads with an 11-fold increase occurring at 41 dpi, compared with baseline, followed by head abnormalities with a sixfold increase at 51 dpi. Detached heads and head defects are among those abnormalities that appear to have a deleterious effect on fertility, and although proximal droplets were also observed at a high frequency after experimental inoculation of the stallions, the droplets seem to have a minor effect on fertility [32]. It is worth noting that our use of onset and recovery is based on hypothesis tests regarding changes from baseline and are only intended to convey a rough sense of the overall timing of changes in semen quality. They are neither guided by scientific judgment concerning levels of change thought to be biologically meaningful nor do they convey the variability between apparent onset and recovery times for four stallions. For example, although edema was observed on average from 6 to 15 dpi, two stallions never experienced edema. 4.1. Conclusions Fever and scrotal edema are the most likely cause of deterioration of semen quality after experimental inoculation of stallions with the KY84 strain of EVA. This reduction in quality was great enough to cause temporary subfertility in the stallions. In addition to the risk of acutely EAVinfected stallions becoming long-term carriers and shedding virus in semen for years, the deterioration in semen quality of acutely EAV-infected stallions can cause considerable economic loss associated with the temporary removal of these animals from breeding activity for as long as 3 to 4 months, until semen quality returns to normal. Acknowledgments This work was supported by the Hildegard Rosa Shapiro Endowed Equine Research Fund endowment fund at the Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky and the Agriculture and Food Research Initiative competitive grant number 2013-68004-20360 from the USDA-National Institute of Food and Agriculture. The authors would like to thank the staff at the University of Kentucky’s Maine Chance Farm for their excellent care of the horses and Dr. Elizabeth Woodward at the Maxwell H. Gluck Equine Research Center for helping with the collection of clinical samples.
References [1] Doll ER, Bryans JT, McCollum WH, Crowe ME. Isolation of a filterable agent causing arteritis of horses and abortion by mares; its differentiation from the equine abortion (influenza) virus. Cornell Vet 1957;47:3–41. [2] Doll ER, Bryans JT, Knappenberger RE. An outbreak of abortion caused by the equine arteritis virus. Cornell Vet 1957;47: 69–75. [3] Timoney PJ, McCollum WH. Equine viral arteritis. Vet Clin North Am Equine Pract 1993;9:295–309. [4] Timoney PJ, McCollum WH, Roberts AW, Murphy TW. Demonstration of the carrier state in naturally acquired equine arteritis virus infection in the stallion. Res Vet Sci 1986;41:279–80. [5] Balasuriya UBR, Snijder EJ, Heidner HW, Zhang J, ZevenhovenDobbe JC, Boone JD, et al. Development and characterization of an infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus. J Gen Virol 2007;88:918–24. [6] McCollum WH, Timoney PJ. Experimental observation on the virulence of isolates of equine arteritis virus. Dubai, United Arab Emirates. In: Proceedings of the 8th international conference on equine infectious diseases 1999. p. 558–9. [7] Balasuriya UBR, Go YY, MacLachlan NJ. Equine arteritis virus. Vet Microbiol 2013;167:93–122. [8] Timoney PJ, McCollum WH, Murphy TW, Roberts AW, Willard JG, Carswell GD. The carrier state in equine arteritis virus infection in the stallion with specific emphasis on the venereal mode of virus transmission. J Reprod Fertil Suppl 1987;35:95–102. [9] Neu SM, Timoney PJ. Persistent infection of the reproductive tract in stallions experimentally infected with equine arteritis virus. In: Proceedings of the 5th conference on equine infectious diseases. Lexington: Kentucky; 1988. p. 149–54. [10] McCollum WH, Little TV, Timoney PJ, Swerczek TW. Resistance of castrated male horses to attempted establishment of the carrier state with equine arteritis virus. J Comp Pathol 1994;111: 383–8. [11] Little TV, Holyoak GR, McCollum WH, Timoney PJ. Output of equine arteritis virus from persistently infected stallions is testosterone dependent. Cambridge, United Kingdom. In: Proceedings of the 6th international conference on equine infectious diseases 1992. p. 225–9. [12] Balasuriya UB, Hedges JF, Nadler SA, McCollum WH, Timoney PJ, MacLachlan NJ. Genetic stability of equine arteritis virus during horizontal and vertical transmission in an outbreak of equine viral arteritis. J Gen Virol 1999;80:1949–58. [13] Broaddus CC, Balasuriya UBR, Timoney PJ, White JLR, Makloski C, Torrisi K, et al. Infection of embryos following insemination of donor mares with equine arteritis virus infective semen. Theriogenology 2011;76:47–60. [14] Dejucq N, Jégou B. Viruses in the mammalian male genital tract and their effects on the reproductive system. Microbiol Mol Biol Rev 2001;65:208–31. [15] Neu SM, Timoney PJ, Lowry SR. Changes in semen quality in the stallion following experimental infection with equine arteritis virus. Theriogenology 1992;37:407–31. [16] McCollum WH, Timoney PJ. The pathogenic qualities of the 1984 strain of equine arteritis virus. In: Proceedings Grayson Foundation International Conference of Thoroughbred Breeders Organizations Equine Viral Arteritis. Dromoland Castle, County Clare, Ireland, October 31 & November 1, 1984. p. 34–84. [17] Senne DA, Pearson JE, Cabrey EA. Equine viral arteritis: a standard procedure for the virus neutralization test and comparison of results of a proficiency test performed at five laboratories. In: Proceedings of the 89th annual meeting of the United States Animal Health Association 1985. p. 29–34. [18] Hancock JL. A staining technique for the study of temperature-shock in semen. Nature 1951;167:323–4. [19] Miszczak F, Shuck KM, Lu Z, Go YY, Zhang J, Sells S, et al. Evaluation of two magnetic-bead-based viral nucleic acid purification kits and three real-time reverse transcription-PCR reagent systems in two TaqMan assays for equine arteritis virus detection in semen. J Clin Microbiol 2011;49:3694–6. [20] Cleveland W. Robust locally weighted regression and smoothing scatterplots. J Am Stat Assoc 1979;74:829–36. [21] Brito LF. Evaluation of stallion sperm morphology. Clin Tech Equine Pract 2007;6:249–64. [22] Setchell BP. The Parkes lecture. Heat and the testis. J Reprod Fertil 1998;114:179–94.
J.R. Campos et al. / Theriogenology 82 (2014) 1068–1079 [23] Pérez-Crespo M, Pintado B, Gutiérrez-Adán A. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice. Mol Reprod Dev 2008;75:40–7. [24] Rasooli A, Taha Jalali M, Nouri M, Mohammadian B, Barati F. Effects of chronic heat stress on testicular structures, serum testosterone and cortisol concentrations in developing lambs. Anim Reprod Sci 2010;117:55–9. [25] Wettemann R, Wells ME, Johnson RK. Reproductive characteristics of boars during and after exposure to increased ambient temperature. J Anim Sci 1979;49:1501–5. [26] Karabinus DS, Vogler CJ, Saacke RG, Evenson DP. Chromatin structural changes in sperm after scrotal insulation of Holstein bulls. J Androl 1997;18:549–55. [27] Freidman R, Scott M, Heath SE, Hughes JP, Daels PF, Tran TQ. The effects of increase testicular temperature on spermatogenesis in the stallion. J Reprod Fertil Suppl 1991;44:127–34.
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[28] Love CC, Kenney RM. Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. Biol Reprod 1999; 60:615–20. [29] Johnson L, Blanchard TL, Varner DD, Scrutchtield WL. Factors affecting spermatogenesis in the stallion. Theriogenology 1997;48: 1199–216. [30] Swierstra EE, Pickett BW, Gebauer MR. Spermatogenesis and duration of transit of spermatozoa through the excurrent ducts of stallions. J Reprod Fertil Suppl 1975;23:53–7. [31] Swierstra MR, Gebauer MR, Pickett BW. Reproductive physiology of the stallion I. Spermatogenesis and testis composition. J Reprod Fertil 1974;40:113–23. [32] Love CC, Varner DD, Thompson JA. Intra- and inter-stallion variation in sperm morphology and their relationship with fertility. J Reprod Fertil Suppl 2000;56:93–100.
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Semen quality of stallions challenged with the Kentucky 84 strain of equine arteritis virus Juliana R. Campos a, Patrick Breheny b,1, Reno R. Araujo c, Mats H.T. Troedsson a, Edward L. Squires a, Peter J. Timoney a, Udeni B.R. Balasuriya a, * a
Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, Lexington, Kentucky, USA Department of Biostatistics, University of Kentucky, Lexington, Kentucky, USA c University of Sao Paulo, Laboratory of Molecular Morphophysiology and Development, FZEA-USP, Pirassununga, Sao Paulo, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 January 2014 Received in revised form 11 June 2014 Accepted 5 July 2014
Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. Some strains of EAV can cause fever, leukopenia, and dependent edema of the limbs, scrotum, and preputium in the acutely infected stallion. We hypothesized that fever and scrotal edema observed during the acute phase of the infection, but not the presence of EAV, have an adverse effect on semen quality. A group of seven stallions were intranasally inoculated with the Kentucky 84 (KY84) strain of EAV. Stallions were monitored for clinical signs of EVA until 42 days postinoculation (dpi). Semen was collected every other day for the first 15 days and 2 times a week up to 79 dpi. Additional samples were collected at 147, 149, and 151 dpi. Semen from each stallion was evaluated on the basis of motion characteristics, total number of spermatozoa, membrane integrity, and morphology. Virus infectivity titers were determined in RK-13 cells. Significant decreases in sperm quality were observed between 9 and 76 dpi. LOWESS (locally weighted scatterplot smoothing) curves for each horse were fit and integrated to quantify spermatozoa exposure to fever, virus, and edema over a period of 67 days before each ejaculation. Linear mixed models were then fit to isolate the effects of each factor on semen quality. Scrotal edema and fever were found to exert independent effects on all the semen quality parameters (P 0.002), whereas virus seems to exert little to no direct effect, as virus titers remained high long after semen quality returned to baseline. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Equine arteritis virus EAV Equine viral arteritis EVA Semen quality Semen evaluation
1. Introduction Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. First isolated from a lung of an aborted
* Corresponding author. Tel.: þ1 859 218 1124; fax: þ1 859 257 8542. E-mail address: [email protected] (U.B.R. Balasuriya). 1 Present address: Department of Biostatistics, College of Public Health, 145 North Riverside Drive, University of Iowa, Iowa City, IA 52242, USA. 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.07.004
fetus in 1953 [1,2], EAV is transmitted mainly by respiratory [2,3] and venereal routes [4]. Although most EAV strains cause subclinical infection after infection, some can cause moderate to severe clinical signs [5–7]. The clinical signs of EVA are characterized by fever, nasal and ocular discharge, conjunctivitis, depended edema, leukopenia, and abortion in pregnant mares [2,3]. The 1984 EVA outbreak in Kentucky’s Thoroughbred breeding population generated widespread interest and major concern to the equine industry [3]. Epidemiologic data collected during that outbreak strongly confirmed establishment of long-term persistence of EAV in stallions and the
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importance of the carrier stallion in the dissemination and perpetuation of the virus. After EAV infection, a variable proportion of stallions (30%–70%) can become carriers and continuously shed virus in their semen for varying periods [4,8]. The virus persists in the male reproductive tract, specifically in the accessory glands with highest infectivity titers found in the ampulla of the vas deferens [9], despite the presence of high titers of neutralizing antibodies to EAV in serum [4,8,9]. Isolation of virus from the preejaculatory fluid of semen from persistently infected stallions was not successful, whereas it could be recovered from the sperm-rich fraction of the ejaculate [8]. The precise mechanism of EAV persistence in the reproductive tract of stallions is unclear; however, it has been shown that establishment and maintenance of infection are testosterone dependent [10,11]. Persistently infected stallions play a major role in the transmission of EAV to mares either by natural breeding or artificial insemination with fresh-cooled or frozen semen [4,8,12]. Recently, it has been reported under experimental conditions that EAV can be transmitted to naive recipient mares via embryo transfer from a donor mare inseminated with EAVinfective semen [13]. Several viruses have been detected in the semen of a number of animal species, some of which can have direct or indirect detrimental consequences on the male reproductive tract and on semen [14]. Semen quality of stallions experimentally infected with the Kentucky 84 (KY84) strain of EAV was decreased for a period of time after inoculation with the virus, but it was not confirmed that the negative effect on semen parameters was because of the direct effect of the presence of virus in semen [15]. Thus, the objectives of this study were to determine (1) the effect on various semen parameters of stallions experimentally infected with the KY84 strain of EAV using computer-assisted sperm analysis (CASA) and differential interference contrast (DIC) microscopy and (2) if possible changes observed in semen quality result from direct effects of the presence of virus in the semen or from the indirect effects of fever and scrotal edema during the acute phase of infection in the stallion. 2. Materials and methods 2.1. Cells and viruses The high passage rabbit kidney cell line (RK-13 KY; passage level 399–409) was maintained in Eagle’s minimum essential medium (EMEM; Mediatech, Inc., Manassas, VA, USA) supplemented with 10% ferritin-supplemented bovine calf serum (Hyclone Laboratories, Logan, UT, USA), 100 U/mL penicillin and streptomycin (Mediatech, Inc.), and 1 mg/mL amphotericin B (Sigma–Aldrich, St. Louis, MO, USA). The virulent KY84 strain of EAV was used as the challenge virus [6,16]. The KY84 strain of EAV has been shown to establish persistent infection in the reproductive tract of stallions and to cause moderate to severe clinical signs of EVA infection in horses [9]. The modified live virus vaccine strain of EAV (ARVAC; Zoetis Animal Health Inc., Kalamazoo, MI, USA) was used as challenge virus in the microneutralization assay.
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2.2. Stallions Seven sexually mature (4–16-year old) stallions of mixed breed were included in the study (stallions IDs: L136–L142). Horses were obtained from a local commercial vendor and acclimated to their new environment for approximately 2 months before the study commenced. During this period, animals were accommodated in individual paddocks and trained to mount a mare or a phantom for collection of semen into an artificial vagina. All stallions exhibited good libido and normal sexual behavior. All were confirmed seronegative (titer <1:4) for EAV neutralizing antibodies several times before intranasal inoculation with the KY84 strain of EAV using a previously described protocol [17]. The animals were housed in individual stalls in an isolation facility for the duration of the study at the University of Kentucky Maine Chance Farm, Lexington, KY, USA. The study was carried out in accordance with an Institutional Animal Care and Use Committee–approved protocol at the University of Kentucky, Lexington, KY, USA (protocol number 2011-0888). 2.3. Experimental inoculation of stallions and clinical examination Stallions were inoculated intranasally with 3.75 105 plaque-forming units (PFU) of the KY84 strain of EAV in 5.0 mL of EMEM using a fenestrated catheter passed via the posterior nares into the nasopharynx as previously described [5]. All stallions were examined and clinical parameters were recorded by the same veterinarian. Scrotal edema was classified on a scale from 0 to 5 and recorded as absent (0), mild (1–2), moderate (3), and severe (4–5). Preinoculation (7, 5, and 2 days before experimental challenge) clinical examinations were performed once daily to determine baseline values for body temperature and also to certify that the parameters were within normal limits before experimental challenge of the stallions. Specifically, fever and scrotal edema were monitored twice daily (every 12 hours) for the first 15 days after infection, and the highest value of the day was recorded. Clinical signs continued to be monitored once per week during the following 4 weeks of the experiment (at 21, 28, 35, and 42 days postinoculation [dpi]). Blood samples were collected at 0, 2, 4, 6, 8, 10, 12, 14, 21, 28, 35, and 42 dpi to determine individual serum neutralizing antibody responses to EAV. The neutralizing antibody titers were determined as described by Senne et al. [17]. 2.4. Semen collection Two ovariectomized mares previously vaccinated with the commercial modified live virus vaccine against EVA (ARVAC; Zoetis Animal Health, Inc.) (respective antibody titers 1:256 and 1:512) were used to “tease” the stallions. Each stallion was allowed to mount either one of the mares or a breeding phantom to enable semen collection in an artificial vagina (Botucatu model; Botupharma, Botucatu, SP, Brazil). The artificial vagina was disinfected with a disinfectant (Roccal-D Plus; Pfizer Inc., New York, NY, USA) between collections. A disposable liner lubricated with
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sterile lubricant (Priority Care; First Priority Inc., Elgin, IL, USA) was used to collect semen from each stallion to avoid cross-contamination between ejaculate samples. Semen was collected from each stallion three times per week for a period of 3 weeks, and discarded, before evaluation of samples commenced. Semen was collected and evaluated 7, 5, and 2 days before challenge and every other day during the first 15 dpi and twice weekly until 79 dpi. Additional samples were collected and evaluated at 147, 149, and 152 dpi. Preinoculation semen samples were obtained to determine baseline values for percentage of total and progressively motile sperm cells (TMOT and PMOT, respectively), total number of spermatozoa (TNS), curvilinear velocity (VCL), percentage of live spermatozoa (LS), and percentage of morphologically normal spermatozoa (MNS). For the purpose of monitoring virus persistence in the reproductive tract of the stallions, semen was collected for virus isolation (VI) approximately once a month, specifically on 86, 107, 128, 149, 170, and 198 dpi (6.5 months), after challenge. 2.5. Semen processing and CASA The total number of spermatozoa in gel-free semen was estimated by measuring the volume with a graduated cylinder and the initial sperm concentration with a spectrophotometer-based instrument (SpermaCue; Minitube of America Inc., Verona, WI, USA). An aliquot of each ejaculate was fixed in 10% buffered formalin for analysis of sperm morphology. The gel-free semen was extended to contain approximately 25 106 spermatozoa/mL with a warm (37 C) commercial skim milk–based extender containing gentamicin (EQUIPRO; Minitube of America Inc.) and evaluated using a CASA instrument (Sperm Vision 3.5 Software; Minitube of America Inc.) to determine the percent of TMOT and PMOT, and VCL. A total of six microscopic fields per sample were selected for computerassisted analysis by the same examiner. 2.6. Membrane integrity The integrity of the spermatozoa plasmatic membrane was determined using eosin-nigrosin staining (Hancock Stain, Animal Reproduction Systems; Dupree Inc., Chino, CA, USA). Duplicate smears stained with Hancock stain were prepared for spermatozoa membrane integrity evaluation, as previously described [18]. The percentage of LS was determined by counting 200 cells per slide for each of two slides, and the average number of live (unstained) vs. dead (stained) cells was recorded. When the number of spermatozoa was low because of low concentration in a sample, less than 200 spermatozoa were counted and the percentage number was calculated. All slides were prepared and counted by the same individual who was blinded to the nature of the samples being analyzed.
(ZeissAxio Imager Upright Microscope). For analysis, 1.5 mL aliquots of fixed semen were applied to a microscope slide and overlaid with a cover glass. Individual spermatozoa were assigned to only one morphologic category even if they exhibited several abnormalities. When more than one defect was seen in the same cell, the most proximal defect was recorded. A total of 100 spermatozoa per sample were examined. All slides were counted by the same trained individual who was blinded to the samples being analyzed. 2.8. Virus isolation Virus isolation from raw gel-free semen samples was attempted in the RK-13 KY cell line according to a standard protocol used by the World Organisation for Animal Health (OIE) Reference Laboratory at Maxwell H. Gluck Equine Research Center, University of Kentucky [17]. Briefly, after sonication, serial decimal dilutions (101–104) of each sample supernatant were prepared in EMEM (Cellgro; Mediatech, Inc.). One milliliter of each dilution was inoculated into a 25-cm2 flask containing a confluent monolayer of RK-13 KY cells grown in supplemented EMEM and overlaid with the supplemented EMEM containing 0.75% carboxymethylcellulose (Sigma–Aldrich). Flasks not showing cytopathic effect after 4 days were subinoculated onto new RK-13 monolayers using a 1-mL culture supernatant as the inoculum. Tissue culture fluid (TCF) was harvested and stored at 80 C for viral RNA extraction. First and second passage flasks were stained with a 0.2% crystal violet solution in buffered formalin on Day 4 after inoculation for counting plaques and calculating the virus titer. The same individual performed all attempted VIs. Virus isolates were, later, confirmed by an EAV specific TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR) assay as previously described [19]. 2.9. Microneutralization assay The neutralizing antibody titers of the test sera were determined by microneutralization assay as described by Senne et al. [17]. 2.10. Real-time RT-PCR Briefly, viral RNA was directly isolated from 50 mL of TCF using a commercial RNA isolation kit (MagMAX-96 Viral RNA Isolation Kit; Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Viral RNA was eluted in 50 mL of nuclease-free water and stored at 80 C. The RNA extracted from TCF derived from RK-13 cells inoculated with semen samples that were negative for EAV, and from nuclease-free water were included as negative controls. Viral RNA extracted from TCF containing the KY84 strain of EAV was used as a positive control. A one-tube TaqMan real-time RT-PCR assay was performed as described by Miszczak et al. [19].
2.7. Sperm morphology 2.11. Statistical analysis Sperm morphology (normal morphology, head, acrosome, mid-piece and tail abnormalities, detached heads, and proximal and distal droplets) was assessed by DIC
Locally weighted polynomials (LOWESS [locally weighted scatterplot smoothing]) were fitted to the raw
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Fig. 1. Average smoothed curve of (A) body temperature (in degree Celsius) of stallions, and (B) virus titer in semen after experimental inoculation with the KY84 strain of EAV. The shaded area represents the 95% confidence interval.
data for each horse to produce smooth curves [20]. Pointwise averages of these curves were taken, and pointwise standard deviations (SDs) used to construct confidence bands. One-way ANOVA analyses were used to quantify onset and recovery, with “onset” defined as the earliest dpi for which the average response differed significantly (P<0.05) from baseline, and “recovery” defined as the latest dpi for which the average response failed to differ significantly from the baseline. To quantify spermatozoa exposure to each potentially causative factor (fever, edema, and virus concentration), these curves were then integrated over a period of 67 days before each sperm quality measurement. Regression models were then fitted for each semen quality outcome to determine the effects of each explanatory factor when adjusting for the other factors. To account for within-stallion correlation among the measurements, linear mixed-effect models were used. All analyses were carried out using R software for statistical computing and graphics (http://www.r-project.org). 3. Results 3.1. Clinical signs All seven stallions developed moderate to severe clinical signs of EVA after experimental inoculation with the KY84 strain of EAV which included any combination of fever (>38.6 C [101.5 F]), dependent edema of the limbs, scrotum and prepuce, periorbital edema, nasal and ocular discharge, photophobia, dyspnea, anorexia, decreased libido and congestion, petechiae, or ecchymosis of the oral mucous membranes. All stallions had fever ranging from 38.7 C to 40.8 C (101.7 F to 105.6 F) starting from 1 to 4 dpi and lasting until 8 to 9 dpi (5–8 days). On average, fever was observed from 3 to 8 dpi (Fig. 1). Highest body temperatures were observed among the stallions between 5 and 7 dpi. The severity and duration of edema varied between stallions. Five of seven stallions (L136–L140; 71.4%) developed moderate to severe scrotal edema starting from 4 to 7 dpi and persisting until 9 to 15 dpi (3–10 days) (Table 1).
Two stallions (L141 and L142) did not develop any edema of the scrotum. All stallions showed decreased libido during the acute phase of infection. The time to drop the penis and the number of times each stallion had to jump the mare or the phantom before ejaculation increased. All the clinical signs together with the moderate to severe edema in the hind limbs of the stallions observed during the acute phase of infection made it difficult for them to mount, which contributed to the increased time needed to obtain an ejaculate. However, none of the seven stallions failed to mount the mare or the phantom. One stallion (L142) failed to ejaculate and instead provided only the pre-ejaculatory Table 1 Scrotal edema of stallions (n ¼ 7) before and after inoculation with the KY84 strain of EAV. Days postinoculation (dpi)
Stallions IDs L136
L137
L138
L139
L140
L141
L142
7 5 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 21 28 35 42
0 0 0 0 0 0 0 0 0 0 þþ þþ þþ 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 þþ þþ þþþ þþþ þþþ þþþ þþþ þþ þþ þþ 0 0 0 0
0 0 0 0 0 0 0 0 0 þ þþþ þþþ þþþ þþþ þþ þþ þþ þþ 0 0 0 0 0
0 0 0 0 0 0 0 þ þ þ þþ þþ þþ þþ þþ þ þ 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 þþ þþþ þþþ þþþ þþþ þþþ þþþ þþ þ 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0, edema absent; þ, mild edema; þþ, moderate edema; þþþ, severe edema.
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Fig. 2. Smoothed average response in semen quality parameters after experimental inoculation of stallions with the KY84 strain of EAV. (A) Total motile spermatozoa (%TMOT); (B) progressive motile spermatozoa (%PMOT); (C) total number of spermatozoa (TNS); (D) curvilinear velocity (VCL); (E) live spermatozoa (%LS); and (F) morphologically normal spermatozoa (%MNS). The shaded area represents the 95% confidence interval.
fluid at 1 and 30 dpi. All stallions seroconverted to EAV starting from 8 dpi and maintained high serum neutralizing antibody titers until at least 42 dpi (1:64 to >1:512). 3.2. Evaluation of semen for virus shedding After experimental inoculation of the stallions with the KY84 strain of EAV, semen samples from each stallion were
tested for EAV shedding by VI in cell culture until 198 dpi. All stallions shed EAV in their semen starting at 5 dpi and continuing to shed at least until 107 dpi (3.5 months) (Fig. 1). Infectivity titers in their semen varied from 1 101 to 1.88 107 PFU/mL during the acute phase of infection. After the acute phase of infection, all but two of the seven stallions continued to shed very high titers of virus in their semen until 107 dpi. The exceptions were stallions L138
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Table 2 (A) Average onset and recovery times (days postinoculation [dpi]) of semen quality parameters after experimental inoculation of stallions (n ¼ 7) with the KY84 strain of EAV; (B) average onset and recovery times of abnormalities in spermatozoa. (A) Semen parameters Semen quality parameters
Total motility
Progressive motility
Total number of spermatozoa
Percentage of morphologic normal spermatozoa
Percentage of live spermatozoa
Onset (dpi) Recovery (dpi)
15 65
15 65
44 62
9 76
13 23
(B) Morphologic abnormalities in spermatozoa Defects in spermatozoa
Head
Detached head
Acrosome
Mid-piece
Tail
Proximal droplet
Distal droplet
Onset (dpi) Recovery (dpi)
23 69
20 65
58 72
13 23
41 48
13 27
9 13
Note that this table is based on an ANOVA test comparing the average response with baseline and does not indicate that all stallions had biologically relevant declines in these parameters during these time periods.
and L137, which shed low titers of virus equivalent to 6 101 and 8 101 PFU/mL, respectively, at 107 dpi. Those stallions were no longer semen shedders of virus at 128 and 149 dpi, respectively. Stallion L142 stopped shedding virus sometime between 170 and 198 dpi. Four of seven stallions (57%) continued to shed high titers of virus at least until 198 dpi (L136, L139, L140, and L141). 3.3. Evaluation of sperm motion characteristics and total number of spermatozoa A total of 201 sequential semen samples (29 per stallion from stallions L136–L141, and 27 from stallion L142) were collected over a period of 12 weeks, including 21 samples (three per stallion) before the experimental inoculation of stallions with the KY84 strain of EAV. Only one stallion (L142) failed to ejaculate at 1 and 30 dpi. Three semen samples from each stallion collected before experimental inoculation (7, 5, and 2 days) were used to establish the normal semen characteristics (baseline values) for TNS per ejaculate, percentage of TMOT and PMOT, VCL, percentage of LS, and percentage of MNS. Before inoculation, the mean baseline values for TNS, TMOT, PMOT, VCL, LS, and MNS were 5.87 109 spermatozoa; 75.6%; 66.1%; 141.6 mm/s, 90%; and 53.5%, respectively. Decrease in at least one of the parameters analyzed for semen quality was observed in all stallions after inoculation. On average, semen quality of stallions inoculated with the KY84 strain of EAV was reduced between 9 and 76 dpi and then returned to baseline (Fig. 2). With the exception of one stallion (L141), TMOT, PMOT, VCL, TNS, and the percentage of LS followed a similar trend in the other six stallions. Stallion L141 showed a drop only in MNS after challenge with virus, but never showed a decrease in the other semen parameters. The alteration in the number of MNS followed a similar trend in all seven stallions, including L141. The percentage of TMOT and PMOT, VCL, and TNS across all stallions was averaged for each collection and compared with the baseline average before virus inoculation (Fig. 2). There was more than 50% reduction in TMOT and PMOT of sperm between 30 and 51 dpi. On average, semen motility decreased (TMOT and PMOT) between 15 and 65 dpi and returned to preinoculation values around 65 dpi. Equine
arteritis virus infection of the stallions also had an effect on the VCL of spermatozoa, with decreased mean value observed between 23 and 34 dpi. Total number of spermatozoa reduced on average between 44 and 62 dpi. Two stallions, L139 and L142, had azoospermia at 34, 37, 41, 48, 51, and 55 dpi, and 44, 48, and 51 dpi, respectively. The TNS count returned to baseline values at around 62 dpi. Average onset and recovery times (dpi) of semen quality parameters after experimental inoculation of stallions are shown on Table 2. 3.4. Membrane integrity The percentages of spermatozoa with intact plasmatic membrane (LS) across all stallions were averaged for each collection and compared with the baseline average before inoculation with the KY84 strain of EAV. Before inoculation, all stallions had at least 81% LS and the mean baseline value for LS was 90% (Fig. 2). Changes in membrane integrity observed after experimental inoculation of the stallions followed a similar trend in all stallions, with the exception of stallion L141, as mentioned previously. The percentage of LS decreased on average between 13 and 23 dpi. 3.5. Evaluation of sperm morphology The percentages of MNS across all stallions were averaged for each collection and compared with the baseline average before inoculation with the KY84 strain of EAV (Fig. 2). All stallions had at least 50% MNS before experimental inoculation with the exception L136 (48%) and L141 (41%). Morphologic abnormalities in spermatozoa followed a similar trend in all seven stallions, including L141, with an overall reduction in the percentage of MNS observed from 9 to 76 dpi. The abnormalities in spermatozoa across all stallions were averaged for each collection and compared with the baseline average before inoculation (Fig. 3). Baseline values for all sperm abnormalities were seen on a frequency lower than 10%, with the exception of mid-piece defects, which were observed at a high frequency (25.10%). An increase in a wide variety of morphologic alterations in spermatozoa was observed after inoculation of the stallions and individual abnormalities were often higher than 10%. The
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Fig. 3. Smoothed average abnormalities (%) in spermatozoa after experimental inoculation of stallions with the KY84 strain of EAV. (A) Detached spermatozoa; (B) head abnormalities; (C) proximal droplets; (D) distal droplets; (E) mid-piece abnormalities; (F) acrosome abnormalities; and (G) tail abnormalities.
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Fig. 4. Representative picture of morphologic abnormalities observed in spermatozoa after experimental inoculation of stallions with KY84 strain of EAV. (A) Normal spermatozoa; (B) detached spermatozoa; (C) mid-piece abnormality (bent); (D) tail abnormalities (bent and coiled); (E) proximal droplet; (F) distal droplet; (G) head abnormality; (H) acrosome abnormality; and (I–L) multiple abnormalities in the same spermatozoon. Ten percent formalin-fixed nonstained semen samples viewed by differential interference contrast microscopy.
abnormalities that mostly increased after the inoculation of the stallions were detached heads, followed by head and proximal droplet defects, tail, mid-piece, and finally acrosome. Although there was an increase in acrosome and tail defects, those abnormalities were never detected at a frequency higher than 10%. A decrease in the percentage of distal droplets was seen between 20 and 58 dpi. During the period of the most dramatic decrease (23–58 dpi) in the percentage of MNS individual sperm frequently had three or more abnormalities, although only the most proximal was counted (Fig. 4). In summary, after inoculation of the stallions with the KY84 strain of EAV, all stallions had a significant decrease in the number of MNS accompanied by a consequent increase in the number of abnormalities in spermatozoa. On average, alteration in semen morphology occurred between 9 and 76 dpi. Average onset and recovery times (dpi) of morphologic abnormalities in spermatozoa after experimental inoculation of stallions are shown in Table 2. 3.6. Effects of fever, edema, and virus on semen quality The timing of the changes in semen quality indicates that fever and edema play a direct role in loss of semen quality, whereas the mere presence of virus does not
(Fig. 5). The onset of high temperatures and scrotal edema in the acute phase of the infection are consistently followed by a drop in semen quality over the next 2 months (roughly the duration of spermatogenesis). Virus concentrations, on the other hand, remain high, well after the semen quality parameters have returned to baseline. These observational conclusions were borne out in formal analyses using linear mixed models (Fig. 6). These models estimated the effect of a 1 SD change in fever, scrotal edema and virus exposure on various semen quality parameters, given that the other two exposures remained constant. Thus, a 1 SD change in temperature over the course of the sperm cycle is estimated to result in a 16% drop in normal morphology, an 11% decline in TMOT, a 14% decline in PMOT, a 4% decrease in VCL, and a 3% decrease in the number of LS. The equivalent change in edema is estimated to result in a 14% drop in normal morphology, a 10% decline in TMOT, a 12% decline in PMOT, a 6% decrease in VCL, and a 2% decrease in the number of LS. The estimated effects of changes in viral concentration, on the other hand, were insignificant for all the aforementioned semen quality parameters. The only significant association involving viral concentration involves TNS; a 1 SD increase in exposure to viral concentration over the course of spermatogenesis is estimated to result in a 7% decrease in TNS. The explanation
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Fig. 5. Overlay of smoothed average responses for body temperature, scrotal edema, and semen virus titers along with semen quality parameters. All variables have been put on a common axis representing the number of standard deviations away from baseline.
for this can be seen in Figure 5, in which TNS returns to baseline much later than the other parameters, and thus more closely matches the timing of viral exposure. 4. Discussion Using an objective assessment of semen quality (CASA), this study revealed that semen parameters are negatively affected after experimental inoculation of stallions with the KY84 strain of EAV. These results agree with the findings of Neu et al. [15] that deterioration in semen quality after experimental inoculation of stallions with the same strain of EAV appears to be temporary, and that the duration of reduced semen quality corresponded approximately with the duration of spermatogenesis in the horse [21]. In the study of Neu et al. [15], it was not clear if the negative effect on semen parameters was because of the direct effect of the presence of the virus in semen. The loss of stallions from that study at intervals made statistically significant results difficult to obtain. In addition, the use of a subjective method of semen analysis could have allowed the results to be susceptible to bias. Our findings indicate that edema and fever exert independent deleterious effects on semen quality, whereas viral concentrations, which remained high long after semen quality returned to baseline, appear to exert little to no direct effect. After challenge, all stallions shed virus in the semen starting from 5 until at least 107 dpi. Thus, the presence of virus in semen was constant throughout the experimental observation period. High body temperature and scrotal edema caused by infection were not
constant, however, and were observed sometime between 1 and 15 dpi during the acute phase of the disease. After 6 to 13 days of exposure to the clinical consequences of fever and/or scrotal edema, deleterious effects on spermatogenesis were noticed. Yet, it is not possible to entirely rule out the direct effect of the virus infection on semen quality given that, in a previous study, the KY84 strain of EAV was isolated from the testis of stallions euthanized up to 20 dpi [9]. Nonetheless, it is unlikely that virus, rather than the combined effect of fever and scrotal edema, was responsible for the detrimental effect seen in semen quality, because microscopic lesions observed in the reproductive tract of stallions during the acute phase of EAV infection were a result of viral replication and lysis of endothelial cells, and changes in germ cells were never reported [9]. In contrast, histopathologic changes in germ cells of testis exposed to heat were detected in different species [22]. It is well known that testicular temperature, 3 C to 5 C cooler than the body, is required in most mammals for normal spermatogenesis to occur [21,22]. Previous studies assessing the effects of increased testicular temperature in different species such as mice [23], rams [24], boars [25], bulls [26], and stallions [27,28] have shown that transient and chronic exposure of the testis to elevated temperatures can disrupt spermatogenesis. When testicular temperature is elevated because of conditions such as fever, testicular trauma, inflammation, and edema, the thermoregulatory mechanism necessary to cool the testis and allow for normal spermatogenesis is disrupted [29]. Under these conditions, metabolism and oxygen demand increase at a
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Fig. 6. Linear mixed models for the effect of body temperature (Temp), virus titer (Log Virus), and scrotal edema (Edema) on sperm quality measurements over a period of 67 days before semen collection. (A) Total motile spermatozoa (%TMOT); (B) progressive motile spermatozoa (%PMOT); (C) total number of spermatozoa (TNS); (D) curvilinear velocity (VCL); (E) live spermatozoa (%LS); and (F) morphologically normal spermatozoa (% MNS).
greater rate than blood flow from the testicular artery to the testes, and testicular hypoxia occurs with consequent detrimental effects on sperm production and quality [21,22]. In this study, with the exception of one stallion (L141) that showed a drop only in the number of MNS, semen of the other six stallions experimentally infected with EAV showed a significant decrease in all parameters evaluated for semen quality (TMOT and PMOT; TNS; LS; MNS; VCL) between 9 and 76 dpi. Stallion L141 had fever but not scrotal edema during the acute phase of the disease. Perhaps, the fever in this animal was not sufficient to elevate the intrascrotal temperature to a degree needed to interfere with motility, TNS, LS, and VCL, but it was sufficient to decrease the percentage of MNS. Neu et al. [15] observed a similar outcome in a group of stallions experimentally infected with the KY84 strain of EAV, which experienced a decrease in the percentage of MNS but not in motility, and suggested that mechanisms important in the formation or maintenance of normal sperm structure may be the most sensitive to an increase in temperature. Spermatogenesis in stallions takes 57 days [21], and this is followed by the epididymal transit of spermatozoa, a process that lasts 8 to 11 days [30]. Therefore, spermatozoa evaluated
anytime between 9 and 76 dpi were at different stages of development at the time of exposure to adverse conditions (1–15 dpi). It is possible that various stages of developing spermatozoa (spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and mature sperm cells) were sensitive to the effects of high body temperature and scrotal edema observed during the acute phase of EAV infection. However, most of the morphologic defects were observed at around 41 dpi. Taking into consideration the length of spermatogenesis, the lifespans of primary (19 days) and secondary spermatocytes (0.7 days), spermatids with round nuclei (8.7 days), and elongated spermatids (10.1 days), as well as knowing that epididymal transit is approximately 9 days [31], the appearance of morphologically abnormal spermatozoa mostly at 41 dpi suggests that the cells that were more dramatically affected at the time of testicular injuries were primary and secondary spermatocytes. These results are similar to the results found by Freidman et al. [27], in which primary and secondary spermatocytes were the germ cells most adversely affected after increased testicular temperature resulting from 24 and 48 hours insulation of stallions’ testis. In the assessment of morphologic abnormalities, spermatozoa were assigned only to one morphologic category
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and the most proximal defect was prioritized over the distal defects. The defects seen in spermatozoa were varied and all were increased after experimental inoculation of the stallions with virus, with the exception of the percentage of distal droplets, which was decreased. The spermatozoa with distal droplets decreased significantly between 23 and 58 dpi; however, this decrease did not necessarily mean that their frequency had dropped, but rather, that another abnormality had increased. The period in which distal droplet abnormalities were significantly decreased coincided with the period of the most dramatic decrease (23–58 dpi) in the percentage of MNS, when an increase in the multiple defects in the spermatozoa was seen. The most dramatic increase was seen in spermatozoa with detached heads with an 11-fold increase occurring at 41 dpi, compared with baseline, followed by head abnormalities with a sixfold increase at 51 dpi. Detached heads and head defects are among those abnormalities that appear to have a deleterious effect on fertility, and although proximal droplets were also observed at a high frequency after experimental inoculation of the stallions, the droplets seem to have a minor effect on fertility [32]. It is worth noting that our use of onset and recovery is based on hypothesis tests regarding changes from baseline and are only intended to convey a rough sense of the overall timing of changes in semen quality. They are neither guided by scientific judgment concerning levels of change thought to be biologically meaningful nor do they convey the variability between apparent onset and recovery times for four stallions. For example, although edema was observed on average from 6 to 15 dpi, two stallions never experienced edema. 4.1. Conclusions Fever and scrotal edema are the most likely cause of deterioration of semen quality after experimental inoculation of stallions with the KY84 strain of EVA. This reduction in quality was great enough to cause temporary subfertility in the stallions. In addition to the risk of acutely EAVinfected stallions becoming long-term carriers and shedding virus in semen for years, the deterioration in semen quality of acutely EAV-infected stallions can cause considerable economic loss associated with the temporary removal of these animals from breeding activity for as long as 3 to 4 months, until semen quality returns to normal. Acknowledgments This work was supported by the Hildegard Rosa Shapiro Endowed Equine Research Fund endowment fund at the Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky and the Agriculture and Food Research Initiative competitive grant number 2013-68004-20360 from the USDA-National Institute of Food and Agriculture. The authors would like to thank the staff at the University of Kentucky’s Maine Chance Farm for their excellent care of the horses and Dr. Elizabeth Woodward at the Maxwell H. Gluck Equine Research Center for helping with the collection of clinical samples.
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