Elsevier

Theriogenology

Volume 86, Issue 4, 1 September 2016, Pages 949-956
Theriogenology

Research article
Extended semen for artificial insemination in swine as a potential transmission mechanism for infectious Chlamydia suis

https://doi.org/10.1016/j.theriogenology.2016.03.018Get rights and content

Abstract

Although typically unnoticed, Chlamydia infections in swine have been shown to be both widespread and may impact production characteristics and reproductive performance in swine. Serum titers suggest Chlamydia infection within boar studs is common, and infected boars are known to shed chlamydia in their ejaculates. Although the transmission of viruses in chilled extended semen (ES) is well established, the inclusion of antibiotics in commercially available extender is generally believed to limit or preclude the transmission of infectious bacteria. The objective of this study was to evaluate the potential of ES used in artificial insemination to support transmission of the obligate intracellular bacteria Chlamydia suis (C suis) under standard industry conditions. First, the effect of C suis on sperm quality during storage was assessed by flow cytometry. Only concentrations above 5 × 105 viable C suis/mL caused significant spermicidal effects which only became evident after 7 days of storage at 17 °C. No significant effect on acrosome reaction was observed using any chlamydial concentration. Next, an in vitro infection model of swine testicular fibroblast cells was established and used to evaluate the effect of chilled storage on C suis viability under variable conditions. Storage in Androhep ES reduced viability by 34.4% at a multiplicity of infection of 1.25, an effect which increased to 53.3% when the multiplicity of infection decreased to 0.1. Interestingly, storage in semen extender alone (SE) or ES with additional antibiotics had no effect on bacterial viability. To rule out a secondary effect on extender resulting from metabolically active sperm, C suis was stored in fresh and expended SE and again no significant effect on bacterial viability was observed. Fluorescent microscopy of C suis in ES shows an association between bacteria and the remaining gel fraction after storage suggesting that the apparent reduction of bacterial viability in the presence of semen is due to adherence to gel fraction. Taken together, the results of this study suggest that C suis remains viable and infectious during chilled storage and is globally unaffected by antibiotics in extender. Thus, ES used in artificial insemination may act as a viable transmission mechanism for C suis in swine.

Introduction

Chlamydia suis, Chlamydophila abortus, Chlamydophila pecorum, and Chlamydophila psittaci are obligate intracellular bacteria that infect swine [1], [2] and may reduce performance of pigs in all stages of the production cycle including reproductive performance [2], [3]. Recently C suis infection of slaughterhouse workers was confirmed [4] suggesting that it has crossed the species barrier and may represent a one-health concern. As with human chlamydia infections, no vaccines for pigs are available but reduced sow-to-piglet transmission rates have been achieved experimentally when sows were treated with probiotics before farrowing [1]. Although antibiotics effectively treat chlamydia infections, their use in animal agriculture continues to draw scrutiny and does not represent an effective long-term solution to prevent infections in a herd. Maintenance of a chlamydia-free herd may best be achieved through stringent biosecurity that restricts movement of animals and production materials. The high number of chlamydia-positive farms in regions where surveillance is performed suggests that transmission is still being achieved despite high biosecurity already in place [5]. One potential avenue for transmission may be through semen which is generally exempt from biosecurity [6]. Instead, semen for artificial insemination (AI) is typically obtained from high health, specific pathogen–free herds, and they are frequently tested for porcine reproductive and respiratory virus (PRRSV) and on occasion other porcine viruses (which do not regularly include C suis or any other bacteria) via polymerase chain reaction to avoid transmission. A number of important swine pathogens have been identified in the semen of infected boars including chlamydia [7], PRRSV [8], circovirus [9], [10], and foot and mouth disease [11]. The following study was conducted to determine if chlamydia negatively impacted sperm viability and if commercial semen stored using industry-standard conditions was a potential mode of transmission for chlamydia [12].

Section snippets

Cell culture and chlamydia propagation

Both swine testicular fibroblast (ST; CRL-1746) and McCoy (CRL-1696) cells were obtained from American Type Culture Collection (Cedarlane, Burlington, Ontario, Canada). ST cells were cultured with minimum essential media (MEM; Sigma–Aldrich, St. Louis, MO, USA) containing 5% fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, CA, USA), and 1X Antibiotic-Antimycotic (Gibco) and McCoy cells were cultured with MEM containing 10% FBS, 1 μg/mL cycloheximide (Sigma–Aldrich) and 10 μg/mL

Results

Dead sperm and acrosome reacted sperm were successfully identified by positive staining for PI and PNA, respectively (Fig. 2A, B). No significant increase in acrosome reaction of sperm was observed after chlamydia was stored for up to 7 days (Fig. 2C). With regard to dead sperm (Fig. 2D), no significant difference in sperm survival relative to control samples was observed at chlamydial concentrations ranging from 5 × 104 to 5 × 105 C suis/mL. However, at a chlamydial concentration of 6.25 × 105

Discussion

In today's swine industry, commercial barns use AI as the preferred method of gilt/sow fertilization. With the ability of boars to produce a large number of insemination doses per week and the widespread distribution of the processed semen, there is tremendous risk that semen contaminated with pathogenic bacteria or viruses can be widely spread [6]. To maintain optimal boar health and minimize adverse effects including infertility and reduced sperm production of the boars, commercial boar studs

Acknowledgments

The authors gratefully acknowledge financial support from the Saskatchewan Health Research Foundation (SHRF) Establishment Grant to F.M. and the Alberta Livestock and Meat Agency Grant (2014R041R) to H.L.W. and J.A.P. G.H. is a recipient of the University of Saskatchewan College of Graduate Studies and Research Graduate Research Fellowship. J.A.P. is the recipient of a fellowship from the Saskatchewan Health Research Foundation and a Natural Sciences and Engineering Research Council of Canada

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    These authors contributed equally to this work.

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