Effects of bovine herpesvirus 1 on thawed bull sperm and evaluation of methods for its removal from experimentally infected semen

TL;DR

This study examines how bovine herpesvirus 1 affects thawed bull sperm and tests methods to remove the virus from infected semen.

Contribution

The study introduces and evaluates two novel methods for removing bovine herpesvirus 1 from infected bull semen.

Findings

BoHV-1 had no direct negative effect on thawed bull sperm quality.

Percoll gradient effectively removed the viral load from infected semen.

Magnetic nanoparticles with anti-BoHV-1 antibodies also reduced the virus without harming sperm motility.

Abstract

The first experiment assessed the effect of bovine herpesvirus 1 (BoHV-1) on thawed bull semen. Semen samples testing negative for BoHV-1 via Nested-PCR were incubated at 37 °C with viral concentrations of 0 (control), 10⁴, 10⁵, and 10⁶ TCID₅₀/mL. Sperm quality parameters were evaluated over an 8-hour period. The second experiment assessed the efficacy of two protocols for reducing the viral load (viral titration) in experimentally infected semen (10⁴ TCID₅₀/mL): Percoll gradient (PG) and magnetic nanoparticles coupled with anti-BoHV-1 antibodies (MNPs). Sperm kinematics were influenced by both bull and time, with bull × time interaction (P < 0.001). Membrane integrity and morphology were influenced only by time (P < 0.01), with no effect of viral infection (P > 0.05). In viral titration, all PG-treated samples tested negative. Samples treated with MNPs also yielded negative results after a 60 min-incubation. It is concluded that BoHV-1 had no direct effect on thawed bull sperm quality and that PG appears to be effective removing the viral load. Furthermore, MNP is a viable alternative for virus control in semen, with no apparent deleterious effects to sperm motility.

Figures

Effect of percoll gradient and incubation with magnetic nanoparticles coupled with anti-BoHV-1 antibodies (MNPs) for 60 min on sperm motility (A) and concentration (B) of thawed bovine semen samples artificially infected with Bovine Herpesvirus 1 (10⁴ TCID₅₀/mL). Different letters above each boxplot indicate significant differences (P < 0.05) between the different treatments for the same parameter

Full text

Introduction

Transmitted among animals through direct or indirect contact, usually via the respiratory tract or the mucosa of the reproductive tract, BoHV-1 causes reproductive disorders in cattle, adversely affecting herd health and economic productivity (Straub 1990). Once infected, animals become lifelong carriers of the virus (Ashbaugh et al. 1997). Moreover, BoHV-1 reduces the outcomes of in vitro embryo production by reducing sperm fertilizing capacity and impairing oocyte competence, thereby disrupting early embryonic formation and development (Vanroose et al. 1999; Alves et al. 2019).

In Brazil, BoHV-1 seroprevalence in high cattle herds, with approximately 70% of animals from properties with a history of abortion testing seropositive (Lima et al. 2011). Despite this high prevalence, current national regulations do not establish specific sanitary requirements for BoHV-1 in bulls allocated to artificial insemination (AI) Centers (BRASIL, 2003). Consistent with this regulatory gap, a study utilizing PCR for viral detection reported BoHV-1 in 44.7% of fresh semen samples from actively breeding bulls and in 21.7% of frozen semen samples from AI centers (Oliveira et al. 2011).

Bielanski et al. (1998) demonstrated that the use of 0.3% trypsin could eliminate BoHV-1 from artificially infected bovine semen (10³ or 10⁴ TCID₅₀/mL). However, this treatment adversely affected sperm quality. Alternative virus removal techniques tested on other species include sperm washing, medium acidification, and incubation with monoclonal antibodies that block viral attachment to target cells (Bielanski 2007; Wrathall et al. 2006).

Considering that viral contamination in semen samples has been a problem in assisted reproduction laboratories (Michou et al. 2011), the high prevalence of BoHV-1 infection in Brazil, and that high-genetic-merit bulls may be BoHV-1 positive, effective strategies to reduce viral load in bovine semen are needed to mitigate reproductive losses. Such protocols could also be applicable for controlling seminal infection in other species. Therefore, this study aimed to: (1) evaluate the influence of BoHV-1 on thawed bull sperm quality and (2) assess the efficiency of a Percoll gradient (PG) and magnetic nanoparticles (MNPs) coupled with anti-BoHV-1 antibodies in reducing viral load in experimentally infected bovine semen.

Materials and methods

Biologicals

Semen batches from three Angus bulls (3 to 5 years old) confirmed negative for BoHV-1 DNA by Nested-PCR, were provided by an AI Center (Alta Genetics, Uberaba, Brazil) and used throughout the experiments. The BoHV-1 Los Angeles strain was replicated in MDBK cell monolayers (37 °C, 5% CO₂), using Low Glucose DMEM (Gibco™, Grand Island, USA) supplemented with 0.4 mg/L streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 1.6 mg/L penicillin (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (FBS, Gibco-BRL, Grand Island, USA). Viral titers were determined using the 50% tissue culture infectious Dose (TCID₅₀) method (Reed and Muench 1938). For the MNPs functionalization, serum was collected from naturally BoHV-1 infected cows exhibiting an anti-BoHV-1 neutralizing antibody titer of 128, as determined by the serum neutralization technique (House and Baker 1971).

Experiment I – effect of BoHV-1 on thawed bull sperm

Three straws from each bull were thawed in a water bath (37 °C, 30 s) with the contents of each straw pooled into microtubes. After one minute of equilibration (designated as Time 0, T0), sperm quality was assessed. Kinetics parameters – including total motility (%), curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s), and average path velocity (VAP, µm/s) – were evaluated using Computer Assisted Sperm Analysis (CASA). Sperm membrane integrity and morphology were assessed using the eosin-nigrosin stain technique. All sperm endpoints were evaluated according to the methodology described by Matias et al. (2021).

Following the initial assessment (T0), the semen sample was equally divided into four aliquots. Then, BoHV-1 was added to three aliquots to achieve final concentrations of 10⁴, 10⁵, and 10⁶ TCID₅₀/mL, while one aliquot remained untreated as a control (0 TCID₅₀/mL). All aliquots were then incubated in a water bath at 37 °C. Sperm analyses, identical to those performed at T0, were repeated after 2, 4, and 8 h of incubation (designated as T2, T4, and T8, respectively).

Experiment II – evaluation of percoll gradient (PG) and magnetic nanoparticles associated with anti-BoHV-1 antibodies (MNPs) to reduce viral load

This study was conducted in duplicate. For each replicate, three straws per bull were thawed (37 °C, 30 s), their contents pooled into a single microtube, and homogenized. As a negative control, an aliquot (200 µL) of non-infected semen was transferred to a microtube and maintained at room temperature (approximately 22 °C) under agitation. Sub-samples were collected at 0, 15, 30, and 60 min for subsequent viral titration.

Artificial infection of the semen was performed by serial dilution of a BoHV-1 stock solution (10^8^ TCID₅₀/mL; thawed at 37 °C for 30 s) in TL-Semen medium (Bioembryo, Bauru, Brazil), to achieve a final concentration of 10⁴ TCID₅₀/mL of semen. This concentration was selected as it reflects the viral load reported in ejaculates from artificially infected bulls (Spradbrow 1968). Following 10 min of agitation at room temperature, one aliquot was collected (designated as positive control, T0) for viral titration, sperm concentration assessment (Neubauer chamber), total motility analysis (CASA), and subsequent application of the PG and MNP treatments. The remaining infected sample was maintained under constant agitation at room temperature. Further aliquots were then collected after 15, 30, and 60 min for viral titration (positive controls T15, T30, and T60, respectively) and for MNP treatment.

A discontinuous PG was prepared with 90% and 45% Percoll (Sigma-Aldrich, St. Louis, MO, USA) diluted in TL-Semen medium. In a 1.5 mL conical-bottom microtube, 500 µL of the 90% Percoll solution was slowly underlaid beneath 500 µL of the 45% Percoll solution. Subsequently, 200 µL of the infected semen was layered on top of the gradient. The tube was centrifuged (7000 g, 5 min), and 50 µL of the resulting sperm pellet was collected for viral titration (PG treatment). Additional aliquots of the pellet were taken for sperm concentration and total motility assessment, as previously described.

Magnetic nanoparticles (MNPs) were prepared by combining 50 µL of Dynabeads™ Protein G nanoparticles (Invitrogen, Carlsbad, CA, USA), 100 µL of immune serum, and 100 µL of TL-Semen medium. The mixture was incubated at room temperature under constant agitation for 10 min to allow antibody coupling. The microtube was then placed on a magnetic stand (Exoflow 700 A-1, System Biosciences, CA, USA) until the nanoparticles aggregated against the tube wall. The supernatant was carefully removed, 1.0 mL of artificially infected semen was added to the coated MNPs, and the suspension was maintained under agitation at room temperature for different intervals (15, 30, and 60 min). At each time point, the microtube was returned to the magnetic stand. Following magnetic separation, an aliquot was collected for viral titration. Additionally, sperm concentration and total motility were assessed in the infected semen sample treated with MNPs for 60 min.

For viral titration, a 50 µL aliquot from each sample (negative control, positive control, PG-treated, and MNP-treated at different intervals) was diluted in TL-Semen medium (50 µL), homogenized, loaded into 0,25 mL straws, and stored in liquid nitrogen until analysis. Viral titrations were performed on MDBK cells cultured in Low Glucose DMEM medium (37 °C, 5% CO₂), by endpoint titration method, with titers expressed as TCID₅₀/mL (Reed and Muench 1938). Specifically, the assay was conducted in a 96-well microtiter plate (USA Scientific, Inc., Ocala, FL). Each well contained 50 µL of Low Glucose DMEM, supplemented with 2% FBS (Gibco™), 1% PSA (Gibco™), 1% L-Glutamine (Gibco™), and 5 × 10⁴ MDBK cells. Following 72 h of incubation, wells were examined for cytopathic effect, with results interpreted by comparison to virus-negative and virus-positive control wells (OIE, 2021).

Statistical analysis

In Experiment I, data normality was assessed using the Shapiro-Wilk test, whereas the homogeneity of variances was determined by Levene’s test. The influence of the variables bull, time, and viral concentration, as well as their interactions, were determined using two-way ANOVA. Differences between means were compared using Tukey’s test. In Experiment II, the effects of PG and MNPs (60 min) treatment on sperm concentration and motility, compared to infected non-treated samples, were assessed using the Mann-Whitney test. For all comparisons, differences with probabilities lower than 5% (P < 0.05) were considered significant.

The efficacy of PG or MNPs (T15, T30, T60) for reducing the viral load in the semen samples was compared using descriptive statistics, considering the effectiveness in achieving negative titration results.

Results

Experiment I

After eight hours of incubation, motility was completely absent across all treatments; consequently, this time point was excluded from statistical analysis. For all sperm kinetics parameters (TM, VSL, VCL, and VAP), a significant effect of both bull and incubation time was observed (P < 0.001), with a significant bull x time interaction (P < 0.001). The reduction on sperm kinetics values was observed already at T2, compared with T0 (P < 0.01). However, viral infection had no effect on any kinetic parameter (P > 0.05). Plasma membrane integrity and the percentage of morphologically normal sperm were influenced by incubation time (P < 0.001), but not by bull or viral concentration (P > 0.05), and no significant interaction was detected. Taking all together, the addition of BoHV-1 at concentrations up to 10⁶ TCID₅₀/mL did not affect any of the sperm quality parameters assessed.

Experiment II

Sperm motility and concentration were first compared between artificially infected semen samples with no treatment and those subjected to viral reduction by either PG or 60 min-incubation with MNP. Incubation time with MNP was limited to 60 min to avoid that incubation itself negatively affects sperm quality, as detected in Experiment I. Compared to the untreated infected control, PG treatment increased sperm motility (P < 0.05), whereas MNP treatment had no effect on this parameter (Fig. 1A). Both MNP and PG treatments reduced sperm concentration relative to the control (P < 0.05), with the PG treatment exhibiting the lowest concentration – Fig. 1B.

Fig. 1. Effect of percoll gradient and incubation with magnetic nanoparticles coupled with anti-BoHV-1 antibodies (MNPs) for 60 min on sperm motility (A) and concentration (B) of thawed bovine semen samples artificially infected with Bovine Herpesvirus 1 (10⁴ TCID₅₀/mL). Different letters above each boxplot indicate significant differences (P < 0.05) between the different treatments for the same parameter

No cytopathic effect was detected in any of the negative control samples during viral titration. In positive control samples – consisting of semen infected with 10⁴ TCID₅₀/mL of BoHV-1 and incubated at room temperature for 0, 15, 30, and 60 min post-thaw – viral titers increased over time, from approximately 10² TCID₅₀/mL to 10³ TCID₅₀/mL after 60 min.

Viral titration results demonstrated that PG treatment rendered 100% of samples negative. In contrast, treatment with MNPs reduced the mean viral titer in a time-dependent manner, achieving 100% negative samples after 60 min of incubation (Table 1).

Table 1. Viral titers for bovine herpesvirus 1 in thawed bovine semen samples artificially infected (10⁴ TCID₅₀/mL), treated with Percoll gradient (PG) or magnetic nanoparticles coupled with anti-BoHV-1 antibodies (MNPs) for different periodsTreatmentViral titer(TCID_50_/mL)Positive samples after titration (%)PGBelow detection0MNP1510^2.3^50MNP3010^1.5^50MNP60Below detection015, 30 and 60 refer to different incubation times (min) of infected samples with MNP

Discussion

This study detected no detrimental effects of BoHV-1 on thawed bovine sperm, even at viral concentrations thousands of times higher than those reported in ejaculates of artificially infected bulls (Spradbrow 1968). These results align with previous reports indicating no impact on sperm quality or fertility in infected bulls (Souza et al. 2018; Montoya-Monsalve et al. 2021). Although Tanghe et al. (2005) suggested that BoHV-1 may interfere with sperm-zona pellucida binding, their study showed no concomitant alterations in the kinetics or acrosomal integrity.

In contrast, El-Mohamady et al. (2020) reported that bulls naturally infected with BoHV-1 exhibited lower sperm motility, concentration, and viability, along with a higher percentage of sperm defects, when compared to non-infected bulls. We hypothesize that these discrepancies with other reports may reflect individual variation on semen quality per se, or the effect of an acute infection, as baseline semen quality prior to BoHV-1 infection was not reported.

The increasing in viral titers in positive control samples during incubation was unexpected. In vitro, BoHV-1 propagated in MDBK cells can increase its titer from 10⁴ TCID₅₀/mL to approximately 10⁵ TCID₅₀/mL after 24 h (Marin et al. 2012). Furthermore, BoHV-1 is not believed to replicate directly within spermatozoa (Van Engelenburg et al. 1993). Therefore, the observed titer likely reflects a temporary reduction in viral infectivity immediately after mixing with semen. This could be attributed to differences in composition between the viral culture medium and the semen extender, or to intrinsic properties of semen, which contains proteolytic enzymes known to influence viral activity (Bielanski et al. 1998), subsequently affecting titration outcomes, as demonstrated for other viruses in human semen (Münch et al. 2007; Chen et al. 2021).

For years, discontinuous density gradients, such as PG, has been used primarily to obtain higher quality sperm (Avery and Greve 1995; Vega-Hidalgo et al. 2022). Nevertheless, the efficacy of PG in eliminating viruses, as demonstrated here for BoHV-1, is consistent with previous studies. Galuppo et al. (2012) reported that PG reduced the viral load of bovine viral diarrhea virus (BVDV) in semen from 10⁶.⁷ to approximately 10¹ TCID₅₀/mL. Similarly, Hanabusa et al. (2000) observed that PG followed by swim-up rendered human immunodeficiency virus 1 (HIV-1) RNA and proviral DNA undetectable in semen, whereas PG alone reduced the rate of positive samples to 8%.

The efficacy of density gradient or sperm washing techniques to reduce viral load in semen samples differ among viruses, probably due to the source of the virus – sperm, blood cells, or seminal plasma (Michou et al. 2011). For BoHV-1, seminal plasma, rather than sperm, is probably the main source of BoHV-1 (Van Oirschot 1995). Since after PG high-quality sperm are separated from seminal plasma, extender, and other background materials, that remains in the upper fraction of the gradient (Henkel and Schill 2003), it can be surmised as an explanation for the present results.

To our knowledge, this is the first demonstration that MNPs coupled with high-titer anti-BoHV-1 serum from naturally infected cattle can effectively reduce or eliminate BoHV-1 in semen. It’s worth noting that using purified polyclonal or monoclonal antibodies, rather than whole serum for MNP coupling, could potentially reduce the incubation time required for viral clearance, as the concentration and specificity of antibodies on the nanoparticle’s surface directly influence separation efficiency (Smith et al. 2011; Haghighi et al. 2020).

The MNPs exhibited no apparent motility impairment to bovine sperm. This finding supports the potential adoption of antibody-conjugated MNPs for controlling other semen contaminants, without compromising sperm quality, thereby expanding biosafety strategies in assisted reproduction (Pérez-Duran et al. 2020). Potential applications could include the removal of environmental contaminants, such as removing nanoplastics (Chen et al. 2024) or the selective depletion of defective sperm cells (Odhiambo et al. 2014).

As limitations, it is important to note that the negative titers for BoHV-1 following PG treatment should be interpreted with caution. The treatment was applied only 10 min post-infection, a time point at which the viral titer in positive controls was substantially lower than after 60 min of incubation. Furthermore, both PG and MNP techniques were tested using small volume of semen and reduced sperm count, limiting to preparing sperm for in vitro fertilization (Blomqvist et al. 2011).

In conclusion, BoHV-1 did not impair the quality of thawed bovine spermatozoa following artificial infection. The Percoll gradient (PG) appears to be effective removing BoHV-1 from semen samples infected with 10⁴ TCID₅₀/mL. Moreover, magnetic nanoparticles coupled with anti-BoHV-1 antibodies exhibited no apparent deleterious effects to sperm motility and represent a promising alternative for reducing viral load in semen.