Porcine embryo development and inactivation of microorganisms after ultraviolet-C irradiation at 228 nm

Abstract

It is important to prevent contamination inside the incubator as a method of preventing microbial infections during the embryo culture. In the present study, we examined the effects of ultraviolet-C (UV–C) irradiation, used for microorganism inactivation, on embryo development and the growth of bacteria, including Escherichia coli and Staphylococcus aureus, and the fungus Cladosporium cladosporioides. In the embryo irradiation experiment, we examined the effects of the plastic lid of the culture dish, irradiation distances (10, 20, and 25 cm), and different irradiation wavelengths (228 and 260 nm) during embryo culture for 7 days on the development and quality of porcine in vitro-fertilized embryos. None of the embryos cultured in dishes without plastic lids developed into blastocysts after irradiation with 228 nm UV-C. When porcine embryos were cultured in a culture dish with lids, the 228 nm UV-C irradiation decreased blastocyst formation rates of the embryos but not their quality, irrespective of the UV-C irradiation distance. Moreover, irradiation with 260 nm UV-C, even with plastic lids, had more detrimental effects on embryo development than irradiation with 228 nm UV-C. Investigation of the inactivating effects of UV-C irradiation at 228 nm and 260 nm on the growth of the bacteria and fungus showed that 260 nm UV-C reduced the viability to a greater extent than 228 nm UV-C. Moreover, the disinfection efficacy for the bacteria increased when the irradiation duration increased and the distance decreased. In conclusion, porcine embryos can develop into blastocysts without loss of quality even after continuous long-duration irradiation (7 days) with 228 nm UV-C, which can inactivate the growth of bacteria and the tested fungus; however, the development rate of the embryo is reduced.

Introduction

Pig models are often favored over rodent models due to their clinical relevance and high similarity to human physiology and anatomy, in both genome sequence as well as anatomy and physiology [1]. Embryo production in vitro is an important tool for the generation of valuable pigs, but most ART laboratories use culture media containing antibiotics to minimize the risk of microbial growth, microorganisms occasionally colonize culture dishes containing oocytes and embryos. The exact frequency of these microbial infections is unknown but a review of previous studies indicated that it ranges from 0.35% [2] to 0.69% [3].

It is important to prevent contamination inside the incubator as a method of preventing microbial infections during the embryo culture. Chemical disinfectants, such as ethanol, hypochlorite, chlorhexidine, and ultraviolet (UV) irradiation, have good antimicrobial properties [[4], [5], [6]]. However, the application of chemical disinfectants is prohibited by device manufacturers and typically requires manual execution, limiting their potential applications [7,8]. Ultraviolet radiation has three different regions depending on the wavelength: UV-A (315–400 nm), UV-B (280–315 nm), and UV-C (<280 nm) [9]. Of these, UV-C is used in most laboratory bactericidal studies [10]. Moreover, extensive studies on microorganism inactivation using UV light emitting diodes (LEDs) at wavelengths of 254–280 nm have been performed thus far [[11], [12], [13]]. Most studies have focused primarily on microbial indicators and very few that target in vitro culture of embryos have been conducted [14,15].

The spectral range of 200–230 nm, referred to as far-UV-C, is assumed to be as effective as the 254 nm UV-C irradiation of the widely used mercury vapor lamps, with a much lower risk to humans [16]. Unfortunately, suitable radiation sources are still difficult to obtain, and many properties of far-UV-C radiation have not yet been fully investigated.

Many microbes, including bacteria, viruses, fungi, and spores, are affected by UV-C light [17]. UV light causes damage through the genome of microbes, indirectly impeding transcription and replication and eventually inactivating them [[18], [19], [20], [21]]. The degree of inactivation is proportional to the UV dose received, which in turn is a consequence of its intensity and duration [22], causing less UV-C to reach the target from light sources farther away. Thus, when the distance doubles, only a quarter of the UV-C remains [22,23]. A previous report has demonstrated that exposure of oocytes to UV irradiation for 10 s did not affect the viability of nuclear transfer embryos and the production of live calves [24]. However, exposure to UV light for more than 30 s led to a loss in membrane integrity, decreased methionine incorporation, altered protein synthesis patterns in bovine oocytes [25], as well as stunted in vitro-fertilized porcine oocytes and damaged mitochondria DNA [15]. On the other hand, limitations of UV-C irradiation related to the penetration of light into an object due to parameters, such as organic matter, can affect the transmittance of the media and restrict its efficacy to the surface of the object [22,26]. For example, organic materials absorb the penetration and block the reflection of UV-C, which is why surfaces should be cleaned manually to remove organic substances before decontamination [27].

The primary objective of this study was to investigate the development and quality of porcine in vitro fertilized embryos after UV-C irradiation at 228 nm and 260 nm, which was used to prevent contamination inside the incubator during embryo culture. The second objective was to compare the effects of UV-C irradiation at 228 and 260 nm with different irradiation durations and distances in the inactivation of representative gram-negative and gram-positive bacteria and a representative fungus, to evaluate the efficacy of UV-C irradiation in preventing microbial contamination inside the incubator during embryo culture.