Short-term consumption of the mycotoxin zearalenone by pubertal gilts causes persistent changes in the histoarchitecture of reproductive tissues.

Consumption of zearalenone (ZEN) detrimentally affects tissues and systems throughout the body, and these deleterious effects are especially pronounced in swine. The objectives of this project were to determine the effects of short-term consumption of ZEN (at concentrations that could be found on-farm) on growth, carcass weight, liver weight, and reproductive tissues of pubertal gilts, and to determine if the effects are transient or persistent. Cross-bred gilts (107.25 ± 2.69 kg) were randomly assigned to one of three feed treatments: 1) solvent only for 21 d (CON; n = 10), 2) ZEN for 7 d followed by 14 d of solvent (ZEN-7; 6 mg/d; n = 10), and 3) ZEN for 21 d (ZEN-21; 6 mg/d; n = 10). Body weights were collected at the beginning and end of the experiment (189.1 ± 0.8 and 211.1 ± 0.8 d of age, respectively). Carcass weights and tissues were collected at harvest. There were no treatment-based differences in growth, carcass, liver, or reproductive tissue weights. Histological analyses revealed differences based on treatment and the interaction between treatment and luteal status. The thickness of the ampullary muscularis declined with ZEN exposure (P < 0.05), while the isthmic epithelial cell height (P < 0.01) and uterine endometrial thickness (P < 0.02) increased. Interestingly, the thickness of the isthmic muscularis, uterine myometrium, and epithelial cell height only differed in the presence of a corpus luteum. Uterine epithelial cell height in the luteal phase was lowest in ZEN-7 pigs (P < 0.01). The isthmic muscularis in the luteal phase was thinner in pigs from both ZEN treatments (P < 0.01). Conversely, the luteal-stage myometrium was thicker in pigs from both ZEN treatments (P < 0.01). The discovery of these tissue-based differences during the luteal phase is particularly concerning since this corresponds with the time when embryos would be affected by the functional competency of the oviduct and uterus. The results of this work demonstrate that short-term consumption of ZEN produces microscopic, but not macroscopic alterations in reproductive organs which are likely to have negative effects on their subsequent function and that these differences persist even after ZEN consumption ceases. Taken together, these results indicate that it is insufficient to rely solely on outwardly visible symptoms as indicators of zearalenone exposure, as detrimental effects on reproductive tissues were found in the absence of phenotypic and morphologic changes.

The mycotoxin zearalenone is a common contaminant of livestock feed. The consumption of zearalenone is particularly problematic for pigs as they are very sensitive to its effects. This study evaluated the effects of zearalenone on growth, carcass weight, liver weight, and reproductive tissues in young female pigs. Thirty pigs were split across three treatment groups. The control group was given standard feed (no zearalenone added) for 21 d, the second group received zearalenone-treated feed for 7 d followed by 14 d of standard feed, and the third group received zearalenone-treated feed for the full 21 d. Pigs receiving the treated feed exhibited no visible symptoms associated with zearalenone consumption. There were also no treatment-related differences in growth, carcass weight, liver weight, or reproductive tract weight. Histological analyses of both the oviduct and uterus revealed changes in tissue thickness that could indicate potential impairments in reproductive organ function. Changes in tissue layer thickness were especially prominent in the luteal phase. This interaction between the treatment and the presence of a corpus luteum is noteworthy because tract function during the luteal phase is imperative for fertilization and early embryonic development.

Survey of zearalenone and type-B trichothecene mycotoxins in swine feed in the USA.

New information is needed regarding the types and concentrations of mycotoxins in swine feed. We hypothesized that (1) the mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), nivalenol (NIV), and zearalenone (ZEN) vary among swine ingredient and feed types, and (2) the inclusion of specific ingredients is associated with mycotoxin contamination in complete feed. A total of 707 samples were collected from cooperators in 14 states between June 2018 and January 2020 then analyzed for DON, 3-AcDON, 15-AcDON, NIV, and ZEN contamination using gas chromatography-mass spectrometry (GC-MS). Ninety-four percent (663/707) of samples contained DON, 33% (230/707) of samples contained 3-AcDON, 57% (404/707) of samples contained 15-AcDON, 1% (6/707) of samples contained NIV, and 47% (335/707) of samples contained ZEN. Seventy-three percent (514/707) of samples contained multiple mycotoxins. Resulting DON concentrations were below the national advisory limits for all sample types, and no advisory limits are imposed for the other mycotoxins studied. Increased incorporation of distiller's dried grains with solubles (DDGS) was associated with increased DON in complete feed (R2 = 0.82).

Effects of short-term moderate ZEN consumption on uterosacral ligament elasticity in pubertal gilts.

Zearalenone (ZEN) is a potent estrogenic toxin in swine, contributing to economic losses in herds via reproductive consequences such as pelvic organ prolapse (POP). To better understand the relationship between ZEN-consumption and reproductive symptoms, an animal feeding study with pubertal gilts was designed. The gilts were exposed to three different treatments: solvent-only feed for 21 days (n = 10), ZEN-spiked feed for 7 days followed by solvent-only feed for 14 days (n = 10), and ZEN-spiked feed for 21 days (n = 10). The gilts did not display any ZEN-related symptoms throughout any of the treatments. At the end of the trial the elastic properties of the USLs from participating gilts were evaluated along two loading directions: main direction (MD) and perpendicular direction (PD). The elastic properties included average stresses at 2% and 4% strains, and secant moduli. Overall the elastic properties of the USLs did not vary across treatment groups or between loading directions. In the MD, average stress increased from 32.96 ± 4.43 kPa at 2% strain to 63.21 ± 9.69 kPa at 4% strain, with a secant modulus of 1.52 ± 0.27 MPa. In the PD, average stress increased from 40.82 ± 4.22 kPa at 2% strain to 83.38 ± 9.17 kPa at 4% strain, with a secant modulus of 2.13 ± 0.31 MPa. Continued research into the relationship between ZEN consumption and reproductive symptoms such as POP is necessary in order to mitigate their deleterious effects in herds.

Quantification of zearalenone and α-zearalenol in swine liver and reproductive tissues using GC-MS.

The mycotoxin zearalenone (ZEN) is a common contaminant of swine feed which has been related to a wide range of reproductive anomalies in swine, such as pelvic organ prolapse, anestrous, and pseudopregnancy. New information is needed to understand how ZEN and related metabolites accumulate in swine reproductive tissues. We conducted a feeding study to track ZEN and the metabolite α-zearalenol (α-ZEL) in swine liver and reproductive tissues. Thirty pubertal gilts were randomly assigned one of three treatments, with ten pigs in each treatment group: (1) base feed with solvent for 21 days, (2) ZEN-spiked feed for seven days followed by base feed with solvent for 14 days, and (3) ZEN-spiked feed for 21 days. At the end of the trial, liver, anterior vagina, posterior vagina, cervix, uterus, ovaries, and broad ligament were collected from pigs. ZEN was found in the anterior vagina, posterior vagina, cervix, and ovaries, with significantly higher concentrations in the cervix relative to other reproductive tissues. ZEN and α-ZEL were found in liver tissue from pigs in each treatment group. Our results show that ZEN accumulates more in the cervix than other reproductive tissues. The presence of ZEN in reproductive tissues may be indicative of ZEN-related reproductive symptoms. Future work could examine how ZEN concentrations vary in reproductive tissues as a factor of the pigs age, weight, sex, or parity, to establish parameters that make pig more sensitive to ZEN.

Biaxial Stress Relaxation of Vaginal Tissue in Pubertal Gilts.

Pelvic organ prolapse (POP) is a condition characterized by displacement of the vagina from its normal anatomical position leading to symptoms such as incontinence, physical discomfort, and poor self-image. Conservative treatment has shown limited success and surgical procedures, including the use of mesh, often lead to severe complications. To improve the current treatment methods for prolapse, the viscoelastic properties of vaginal tissue need to be characterized. We determined the biaxial stress relaxation response of vaginal tissue isolated from healthy pubertal gilts. Square specimens (n = 20) with sides aligned along the longitudinal directions (LD) and circumferential direction (CD) of the vagina were biaxially displaced up to 5 N. The specimens were then kept at the displacements corresponding to 5 N for 20 min in both the LD and CD, and the corresponding strains were measured using digital image correlation (DIC). The stresses in the LD and CD were found to decrease by 49.91 ± 5.81% and 46.22 ± 5.54% after 20 min, respectively. The strain in the LD and CD increased slightly from 0.080 ± 0.054 to 0.091 ± 0.064 and 0.050 ± 0.039 to 0.058 ± 0.047, respectively, but these changes were not significant (p > 0.01). By using the Peleg model, the initial decay rate and the asymptotic stress during stress relaxation were found to be significantly higher in the LD than in the CD (p≪0.001), suggesting higher stress relaxation in the LD. These findings may have implications for improving current surgical mesh, mechanical devices, and physical therapy used for prolapse treatment.

Evaluation of GeneMarker® HTS for improved alignment of mtDNA MPS data, haplotype determination, and heteroplasmy assessment.

Existing software has not allowed for effective alignment of mitochondrial (mt) DNA sequence data generated using a massively parallel sequencing (MPS) approach, combined with the ability to perform a detailed assessment of the data. The regions of sequence that are typically difficult to align are homopolymeric stretches, isolated patterns of SNPs (single nucleotide polymorphisms), and INDELs (insertions/deletions). A custom software solution, GeneMarker® HTS, was developed and evaluated to address these limitations, and to provide a user-friendly interface for forensic practitioners and others interested in mtDNA analysis of MPS data. GeneMarker® HTS generates an exportable consensus mtDNA sequence that produces phylogenetically correct SNP and INDEL calls using a customizable motif-based alignment algorithm. Sequence data from 500 individuals, with various alignment asymmetries and levels of heteroplasmy, were used to assess the software. Accuracy in producing mtDNA haplotypes, the ability to correctly identify low-level heteroplasmic sequence variants, and the user-based features of the software were evaluated. Analyzed sequences yielded correct mtDNA haplotypes, and heteroplasmic variants were properly identified with minimal manual interpretation. The software offers numerous user-defined parameters for filtering the data that address the interests of researchers and practitioners, and provides multiple options for viewing and navigating through the data.