Twelve natural estrogens in urines of swine and cattle: Concentration profiles and importance of eight less-studied.

Although four major natural estrogens (i.e., estrone (E1), 17ß-estradiol (E2), estriol (E3) and 17α-estradiol (αE2)) have been commonly found in livestock urine, this study reports the occurrence of eight other less-studied natural estrogens in urine of swine and cattle, i.e. 2-hydroxyestone (2OHE1), 4-hydroxyestrone (4OHE1), 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 16-epiestriol (16epiE3), 16α-hydroxyestrone (16αE1), 16-ketoestradiol (16ketoE2), and 17epiestriol (17epiE3). Results showed that each estrogen was found in at least one urine sample, and 6 of 8 the less-studied estrogens were present at frequencies of ≥90% in boars, ≥70% in sows, and ≥50% in dairy cattle. Five of eight the less-studied estrogens were present at frequencies of ≥33.3% in four beef cattle and one bull. On a concentration basis, the 8 less-studied natural estrogens represented 73.2%, 85.2%, 39.9%, 47.7%, 26.9%, 56.0% and 44.1% of total concentrations of the twelve natural estrogens when combining data from all animals. Similar results were observed based on estrogen equivalence, which indicated these newly detected eight less-studied natural estrogens were not negligible. This work is the first to figure out the importance of these less-studied natural estrogens in livestock urine, and their potential environmental risks associated with discharge of livestock wastewater should be urgently assessed in a holistic manner.

Anti-androgenic compounds in breast milk and cryptorchidism among Norwegian boys in the HUMIS birth cohort.

The prevalence of cryptorchidism has increased over the past decades, yet its origins remain poorly understood. Testis descent is dependent on androgens and likely affected by endocrine disrupting compounds (EDCs), targeting the androgen receptor (AR). We investigated the association between anti-androgenic activity, not derived from natural hormones, in maternal breast milk and impaired testis descent among boys. We performed a case-control study based on 199 breast milk samples from 94 mothers of cryptorchid boys and 105 random non-cryptorchid boys participating in the Norwegian HUMIS (Human Milk Study) cohort. For each participant, apolar, and polar fractions were extracted, and combined to reconstitute a mixture. Anti-androgenic activity was measured in all three fractions using the human cell-based in vitro anti-AR CALUX® assay and expressed in µg of flutamide equivalent, a well-known antiandrogen. Results from fraction analyses were compared among boys with cryptorchidism and controls using multiple logistic regression, controlling for appropriate confounders identified using a directed acyclic graph. Children's daily exposure to anti-androgenic EDCs through breastfeeding was estimated to 78 µg flutamide eq./kg of body weigh/day. The activity was higher in the polar fraction (1.48 ± 1.37 µg flutamide eq./g of milk) mainly representing non-persistent chemicals, in contrast to other fractions. However, the activity in the polar extracts was decreased when in mixtures with the apolar fraction, indicating synergistic interactions. No significant difference in the activity was observed according to cryptorchid status for polar, apolar or mixed breast milk fractions. The study showed anti-androgenic activity in nearly all human milk samples, and at levels higher than the advisory threshold. However, no significant association was observed between cryptorchidism and antiandrogenic activity measured in either polar, apolar, or mixture fractions derived from breast milk.

Neural correlates of contributions of interpreting training to second language semantic integration.

How interpreting training may enhance semantic integration in general language processing (instead of language processing during interpreting) was not clear, and its neural correlates remained unexplored. Two groups of university students with different amounts of interpreting training experience (more-IE and less-IE) performed a second language (L2) reading task, in which the target sentence ended with three types of lateralized words: expected words, words of related semantic violation (related violation), and words of unrelated semantic violation (unrelated violation). N400 responses elicited by the final words were used to index semantic integration. Two major findings suggested potential contributions of interpreting training to L2 semantic integration. First, the more-IE group exhibited smaller N400 amplitudes than the less-IE group for expected words, and no group differences were obtained for the two violations. Second, N400 amplitudes for related violations were generally smaller than those for unrelated violations in the right visual field, and this difference was more fully captured (more broadly distributed over the scalp) in the more-IE group than in the less-IE group. Therefore, smaller N400 amplitudes for expected words but not for violations, and broader scalp distribution of the differences between related and unrelated violations constitute the neural correlates for the potential contributions of interpreting training to general L2 semantic integration. The results also suggest possible connections between different types of language experience.

An integrated metabolomics and proteogenomics approach reveals molecular alterations following carbamazepine exposure in the male mussel Mytilus galloprovincialis.

Carbamazepine is one of the most abundant pharmaceutical active compounds detected in aquatic systems. Based on laboratory exposures, carbamazepine has been proven to adversely affect aquatic organisms. However, the underlying molecular events remain poorly understood. This study aims to investigate the molecular mechanisms potentially associated with toxicological effects of carbamazepine on the mussel Mytilus galloprovincialis exposed for 3 days at realistic concentrations encountered in coastal environments (80 ng/L and 8 µg/L). An integrated metabolomics and proteogenomics approach, including data fusion strategy, was applied to gain more insight in molecular events and cellular processes triggered by carbamazepine exposure. Consistent metabolic and protein signatures revealed a metabolic rewiring and cellular stress at both concentrations (e.g. intensification of protein synthesis, transport and catabolism processes, disruption of lipid and amino acid metabolisms). These highlighted molecular signatures point to the induction of autophagy, closely related with carbamazepine mechanism of action, as well as a destabilization of the lysosomal membranes and an enzymatic overactivity of the peroxisomes. Induction of programmed cell death was highlighted by the modulation of apoptotic cognate proteins. The proposed integrative omics data analysis was shown to be highly relevant to identify the modulations of the two molecular levels, i.e. metabolites and proteins. Multi-omics approach is able to explain the resulting complex biological system, and document stronger toxicological pieces of evidence on pharmaceutical active compounds at environmental concentrations in sentinel organisms.

Contribution of prenatal endocrine-disrupting chemical exposure to genital anomalies in males: The pooled results from current evidence.

The etiology of hypospadias and cryptorchidism, which are the two most common genital anomalies in males, has not been elucidated. Although prenatal exposure to endocrine-disrupting chemicals (EDCs) may increase the risks of hypospadias and cryptorchidism, the associations have not been confirmed. Therefore, we performed a meta-analysis to establish the relationships between prenatal exposure to EDCs and male genital anomalies. A systematic search of PubMed, EMbase, and Cochrane Library CENTRAL for relevant published studies providing quantitative data on the associations between prenatal EDCs exposure and hypospadias/cryptorchidism in humans was conducted. In total, sixteen case-controlled studies were included. Prenatal exposure to overall EDCs was associated with an increased risk of hypospadias in males (OR, 1.34, 95 % CI 1.12 to 1.60). Although there was no statistically significant association between overall EDCs exposure and cryptorchidism (OR, 1.11, 95 % CI 0.99 to 1.24), exposure to phenol substances was associated with an increased risk of cryptorchidism (OR, 1.81, 95 % CI, 1.12 to 2.93). Using the GRADE tool, we found the overall evidence to be of moderate certainty. In conclusion, the current evidence suggests prenatal EDCs exposure may increase the risk of hypospadias in males.

Exposure of men and lactating women to environmental phenols, phthalates, and DINCH.

Phthalates and 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH), bisphenols (BPs), parabens (PBs), and triclosan (TCS) are high-production-volume chemicals of pseudo-persistence that are concerning for the environment and human health. This study aims to assess the exposure to 10 phthalates, DINCH, and environmental phenols (3 BPs, 7 PBs, and TCS) of Slovenian men (n = 548) and lactating primiparous women (n = 536). We observed urinary concentrations comparable to studies from other countries and significant differences among the sub-populations. In our study, men had significantly higher levels of phthalates, DINCH, and BPs, whereas the concentrations of PBs in urine were significantly higher in women. The most significant determinant of exposure was the area of residence and the year of sampling (2008-2014) that mirrors trends in the market. Participants from urban or industrialized sampling locations had higher levels of almost all monitored analytes compared to rural locations. In an attempt to assess the risk of the population, hazard quotient (HQ) values were calculated for individual compounds and the chemical mixture. Individual analytes do not seem to pose a risk to the studied population at current exposure levels, whereas the HQ value of the chemical mixture is near the threshold of 1 which would indicate a higher risk. We conclude that greater emphasis on the risk resulting from cumulative exposure to chemical mixtures and additional studies are needed to estimate the exposure of susceptible populations, such as children.

Mechanistic revealing of reproductive behavior impairment in male guppy (Poecilia reticulata) induced by environmentally realistic 2,2'-dithiobis-pyridine exposure.

Although (PS)2, the primary degradation product of emerging antifouling biocides metal pyrithiones (MePTs), can disrupt the reproductive behavior of fish at an environmentally relevant ng/L level, the underlying mechanism is still largely unknown. This study exposed sexually mature male guppy (Poecilia reticulata) to 20, 200, and 2000 ng/L (PS)2 to explore the compromised effect of (PS)2 on reproductive behavior through a realistic competing scenario. The results showed that (PS)2 suppressed male guppies' sexual interest to stimulus females, reduced their competitive behavior frequencies toward rival males, and decreased their mating time and frequency. (PS)2 exposure did not affect male guppies' secondary sexual characteristics or induce estrogenic activity. Whole-brain transcriptome sequencing identified 1070 differentially expressed genes (DEGs) with 872 up-regulated genes, which were functionally enriched into Gene Ontology terms pertaining to extracellular matrix (ECM) and extracellular region. KEGG enrichment for the DEGs uncovered that the activations of ECM-receptor interaction and focal adhesion pathways could be the underlying molecular mechanism implicated in the (PS)2 induced reproductive behavior impairment. This work would deliver a substantial contribution to the understanding of the ecological safety of MePTs biocides.

High lead level in the Alps in XIXth century, learning from the analysis of 138 historical hair stands.

Analysis of hair is known to provide useful information about environmental and toxic exposures. Very little historical use has been made of this type of investigation. Here we study 138 human hair samples from 19th century in France. In order to examine the potential association between contamination and historical health impacts, we characterized contamination by 33 elements in a set of hair strands sampled during the last quarter of the 19th century in the Savoy region of France. After a selected washing step on 138 hair strands conserved at the Museum National d'Histoire Naturelle in Paris (France), we assessed the presence of inorganics by ICP/MS, and lead level was higher than values reported in literature. We then compared concentrations and distributions between women and men, sampling locations and crossing gender and geographical origin. Hair lead level was high throughout Savoy at the end of the 19th century: significantly higher for people living in towns or industrial valleys, and lower for those of countryside and mountains areas. Environmental and economic changes (industrialization and urbanization with water adduction and leaded paints), living habits (kitchenware, cosmetics, wine, and tobacco), and local features (mines exploitation, railroad development, and industrialized narrow valleys) could be envisaged for explaining the level of lead contamination. In the same period, the two main industrial valleys of Savoy (Maurienne and Tarentaise) had high rates of endemic goiter and cretinism and among the highest hair lead levels. Other lines of evidence will need to be explore to investigate a possible link between historical Pb exposure and goiter in the study area.

Homeostasis effects of fermented Maillard reaction products by Lactobacillus gasseri 4M13 in dextran sulfate sodium-induced colitis mice.

BACKGROUND: The incidence of inflammatory bowel disease (IBD) continues to increase worldwide. Multiple factors, including diet, loss of the intestinal barrier function, and imbalance of the immune system can cause IBD. A balanced diet is important for maintaining a healthy bowel and preventing IBD from occurring. The effects of probiotic Lactobacillus gasseri-fermented Maillard reaction products (MRPs) prepared by reacting whey protein with galactose on anti-inflammation and intestinal homeostasis were investigated in this study, which compared MPRs and probiotics separately. RESULTS: In an animal colitis model induced by 2% dextran sulfate sodium (DSS), FWG administration alleviated colon length loss and maintained intestinal immune system homeostasis as reflected by down-regulated interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α output, and metallopeptidase-9, and epithelial barrier balance as reflected by up-regulated occludin, E-cadherin, and zonula occludens-1 production in the colon. Furthermore, the expression of splenic cytokines such as IL-6, TNF-α, and IL-10 was up-regulated in the FWG-treated mice in a comparable amount to the control group to ensure the balance of immune responses. CONCLUSION: This study showed that the use of FWG protects the intestines from colitis caused by DSS and maintains immune balance. FWG increased antioxidant enzyme activity, increased intestinal permeability, and regulated the balance of pro- and anti-inflammatory cytokines in the intestines and spleen. Continued intake of FWG can alleviate IBD symptoms through the preservation of mucosal immune responses, epithelial junction and homeostasis through the regulated splenic cytokines. © 2021 Society of Chemical Industry.

Monitoring residue levels and dietary risk assessment of thiamethoxam and its metabolite clothianidin for Chinese consumption of Chinese kale.

BACKGROUND: Thiamethoxam is widely used to control pests in Chinese kale, popularly consumed leafy vegetables. The potential risk to the environment and human health has aroused much public concern. Therefore, it is important to investigate the degradation behavior, residue distribution and dietary risk assessment of thiamethoxam in Chinese kale. RESULTS: A sensitive analytical method for determination of thiamethoxam and its metabolite clothianidin residue in Chinese kale was established and validated through a quick, easy, cheap, effective, rugged, and safe (QuEChERS) technique with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The recoveries were 85.4-101.2% for thiamethoxam and 79.5-108.1% for clothianidin, with the relative standard deviations (RSDs) of 0.9-10.2% and 1.8-6.0%, respectively. For the dissipation kinetics, the data showed that thiamethoxam in Chinese kale was degraded with the half-lives of 4.1 to 4.5 days. In the terminal residue experiments, the residues of thiamethoxam were 0.017-0.357 mg kg-1 after application 2-3 times with a preharvest interval (PHI) of 7 days under the designed dosages. The chronic and acute dietary exposure assessment risk quotient (RQ) values of thiamethoxam in Chinese kale for different Chinese consumers were 0.08-0.19% and 0.05-0.12%, respectively, and those of clothianidin were 0.01-0.04% and 0.02-0.04%, respectively, all of the RQ values were lower than 100%. CONCLUSION: Thiamethoxam in Chinese kale was rapidly degraded following first-order kinetics models. The dietary risk of thiamethoxam and clothianidin through Chinese kale was negligible to consumers. The results from this study are important reference for Chinese governments to developing criteria for the safe and rational use of thiamethoxam, setting maximum residue levels (MRLs), monitoring the quality safety of agricultural products and protecting consumer health. © 2021 Society of Chemical Industry.

A record of juvenile Paragonimus skrjabini miyazakii from the urinary bladder of the Japanese toad, Bufo japonicus formosus.

We found juveniles of Paragonimus in the urinary bladder of a Japanese toad (Bufo japonicus formosus) captured in Kyoto. These were molecularly identified as Paragonimus skrjabini miyazakii Kamo, Nishida, Hatsushika et Tomimura 1961. This is the first report of P. s. miyazakii found in anuran hosts in Japan, indicating that anurans can be paratenic hosts of P. s. miyazakii, as is also the case for Paragonimus skrjabini skrjabini in China. This finding suggests that definitive hosts of P. s. miyazakii can be infected by eating not only crabs or mammal paratenic hosts, but also anurans.

Deep learning takes the pain out of back breaking work - Automatic vertebral segmentation and attenuation measurement for osteoporosis.

BACKGROUND: Osteoporosis is an underdiagnosed and undertreated disease worldwide. Recent studies have highlighted the use of simple vertebral trabecular attenuation values for opportunistic osteoporosis screening. Meanwhile, machine learning has been used to accurately segment large parts of the human skeleton. PURPOSE: To evaluate a fully automated deep learning-based method for lumbar vertebral segmentation and measurement of vertebral volumetric trabecular attenuation values. MATERIAL AND METHODS: A deep learning-based method for automated segmentation of bones was retrospectively applied to non-contrast CT scans of 1008 patients (mean age 57 years, 472 female, 536 male). Each vertebral segmentation was automatically reduced by 7 mm in all directions in order to avoid cortical bone. The mean and median volumetric attenuation values from Th12 to L4 were obtained and plotted against patient age and sex. L1 values were further analyzed to facilitate comparison with previous studies. RESULTS: The mean L1 attenuation values decreased linearly with age by -2.2 HU per year (age > 30, 95% CI: -2.4, -2.0, R2 = 0.3544). The mean L1 attenuation value of the entire population cohort was 140 HU ± 54. CONCLUSIONS: With results closely matching those of previous studies, we believe that our fully automated deep learning-based method can be used to obtain lumbar volumetric trabecular attenuation values which can be used for opportunistic screening of osteoporosis in patients undergoing CT scans for other reasons.

Crayfish co-introduced symbiotic ostracod found on native crab in Japan: The first record of epibiont ostracod found a new host.

Ankylocythere sinuosa (Rioja, 1942), a symbiotic ostracod native to North America, was found from the Japanese mitten crab Eriocheir japonica (De Haan, 1835), a species native to Japan, collected from a pond in Shizuoka City, Shizuoka Prefecture, central Japan. Introduced North American crayfish Procambarus clarkii (Girard, 1852), which is a host of A. sinuosa in their native range, inhabits ponds sympatrically with Japanese mitten crabs, and it is thought that the ostracods transferred from the exotic crayfish to the native crabs. In recent years, along with the artificial transportation of crayfish around the world, their symbiotic ostracods also have been found on the body surfaces of exotic crayfish in Europe and Japan. However, no studies have confirmed the infestation of exotic ostracods on native crustaceans in the field. A wide range of developmental stages of A. sinuosa from juveniles to adults were found in Japanese mitten crabs, and mating individuals were also found. This strongly suggests that they can reproduce on the body surface of Japanese mitten crabs. In the future, it will be necessary to strengthen measures against alien species to prevent these exotic symbionts from infestating native ecosystems, and we also need to investigate the exact impact of this symbiont on Japanese mitten crabs.

Improvement of low-acyl gellan gum on gelation and microstructural properties of protein hydrolysates from male gonad of scallop (Patinopecten yessoensis).

This study aimed to examine the gelation and microstructural properties of scallop male gonad hydrolysates (SMGHs) in the presence of low-acyl gellan gum (GG) at different mass ratios. The rheological results showed that both elastic modulus and thermal stability of SMGHs were significantly improved by the addition of GG. Meanwhile, the relaxation time T23 was significantly reduced in SMGHs/GG by low-field nuclear magnetic resonance, indicating a strong interaction between SMGHs and GG. Fourier transform infrared spectroscopy indicated the blueshift of amide I and II peaks in SMGHs/GG further demonstrated the electrostatic interaction between SMGHs and GG. The network structure of SMGHs/GG binary complexes was more compact and the surface was smoother than that of SMGHs by cryo-scanning electron microscopy. Furthermore, increasing the content of GG in the SMGHs/GG binary complex significantly reinforced the gel strength and promoted the gelation process.

New cases of human dirofilariosis on the Balkan Peninsula - "Masked intruders" uncovered by a surgeon.

Dirofilaria spp. nematodes are accidental parasites of humans causing mild to serious, superficial or visceral infections. Superficial dirofilariosis is rather common in Europe and is typically manifested as subcutaneous form. Herein we report 46 new cases of human dirofilariosis (19 patients with subcutaneous, 18 patients with ocular, 4 patients wih genital, 2 patients with submucosal, 2 patients with pulmonary and 1 patient with intramuscular form of infection) that were recorded from the beginning of 2015 to May 2021 on the Balkan Peninsula with a goal to update the prevalence of this parasitosis and point out potential problems in diagnosis and treatment. Besides, given the high possibility of misinterpretation as tumor, our second aim was to encourage the inclusion of this pathogen in the differential diagnosis of subcutaneous nodules. Although quite common forms, subcutaneous and ocular dirofilariosis can be very often misdiagnosed in clinical practice due to the absence of specific clinical manifestations. Therefore, raising awareness of clinicians about this zoonosis is needed as well as closer collaboration between physicians and veterinarians.

Elimination of Giardia duodenalis BIV in vivo using natural extracts in microbiome and dietary supplements.

In this study, a combination therapy of several natural products was evaluated in vivo in the Giardia duodenalis infection model. G. duodenalis infected mice were treated as follows: distilled water (infected control C+), BIOintestil® (BIO; natural products of Cymbopogon martinii and Zingiber officinale), MicrobiomeX® (MBX; extract of Citrus sinensis and Citrus paradisi), MBX + BIO, Camellia sinensis tea (CPR; black tea). These natural compounds were administered in a dose of 100 mg/day and were compared to G. duodenalis-infected mice treated with albendazole (ALB; 50 mg/Kg/day) and metronidazole (MET; 500 mg/Kg/day), the conventional therapies used to this day. One group remained un-infected and untreated as our control group (C-). Treatment started 8 days after infection, and after 5 days of treatment (7 days for MET), all animals were followed for 15 days. We continuously checked for the presence of G. duodenalis by Faust method, in association with detection of the parasite by PCR from feces, as well for the presence of trophozoites in the intestinal mucosa after sacrifice. Animals treated with MBX, BIO and MBX + BIO presented an undetectable parasitic load until the 15th day of monitoring, while animals treated with CPR, MET and ALB continued to release cysts. Animals in the MBX, MBX + BIO, ALB groups consumed lower feed, MBX, CPR, MET had greater weight and MBX, MBX + BIO, BIO, CPR, C- consumed more water when compared to infected-group control. MBX and BIO alone or associated eliminated G. duodenalis without apparent adverse effects and animals of these groups showed better clinical performance in relation to those with high parasitic load. MET, ALB and CPR only decreased the number of cysts, indicating limitations and therapeutic failure.

Calcified lymph nodes in the setting of head and neck squamous cell carcinoma: A predictor of HPV positivity?

PURPOSE: Human papillomavirus (HPV) is an important cause of head and neck squamous cell carcinoma (HNSCC) and accounts for a large majority of new cases. The purpose of this study is to determine whether there is an association between nodal calcification and HPV positivity in the setting of metastatic HNSCC. METHODS: Consecutive patients with HNSCC who underwent CT were retrospectively identified. Patients were then divided into two groups: those with HPV-positive HNSCC and those with HPV-negative HNSCC. Demographic, clinical, and CT data were compared between the two groups to determine factors associated with HPV-positive HNSCC. RESULTS: A total of 179 patients with HNSCC were included in the final analyses, 104 (58%) of whom had HPV-positive tumors. Univariate analyses demonstrated that those with HPV-positive HNSCC were more likely to have calcified lymph nodes (p = 0.044). Analyses also confirmed previously known associations with male gender (p = 0.001), primary oropharyngeal tumors (p < 0.001), and cystic lymph nodes (<0.001). The HPV-positive HNSCC group was also less likely to have necrotic lymph nodes (p < 0.001). CONCLUSION: In addition to known clinical and imaging factors associated with HPV-positive metastatic HNSCC, such as male gender, oropharyngeal primary location, and cystic lymph nodes, the presence of calcifications within cervical lymph nodes, although infrequent, provides an additional useful feature to predict HPV positivity in HNSCC. Additionally, if calcified lymph nodes are present, then a primary oropharyngeal tumor site should be considered.

Short exposure to nitenpyram pesticide induces effects on reproduction, development and metabolic gene expression profiles in Drosophila melanogaster (Diptera: Drosophilidae).

Although the toxicity of neonicotinoid insecticides has been demonstrated in several studies, the information on metabolism, behavior, and health risk remains limited and has raised concerns about its potential toxicity. Thus, in this study we assessed the effects of nitenpyram using different sublethal concentrations (one-third and one-tenth of the acute LC50 values) on various developmental and metabolic parameters from gene expression regulation in Drosophila melanogaster (model system used worldwide in ecotoxicological studies). As a result, nitenpyram sublethal concentrations prolonged the developmental time for both pupation and eclosion. Additionally, nitenpyram sublethal concentrations significantly decreased the lifespan, pupation rate, eclosion rate, and production of eggs of D. melanogaster. Moreover, the mRNA expression of genes relevant for development and metabolism was significantly elevated after exposure. Mixed function oxidase enzymes (Cyp12d1), (Cyp9f2), and (Cyp4ae1), hemocyte proliferation (RyR), and immune response (IM4) genes were upregulated, whereas lifespan (Atg7), male mating behavior (Ple), female fertility (Ddc), and lipid metabolism (Sxe2) genes were downregulated. These findings support a solid basis for further research to determine the hazardous effects of nitenpyram on health and the environment.

First reproductive signs of inbreeding depression in Southern California male mountain lions (Puma concolor).

Long-term studies of mountain lions (Puma concolor) in Southern California have documented persistent small population sizes and the lowest genetic variation of any mountain lion population, except for the Federally endangered mountain lion subspecies, the Florida panther (Puma concolor coryi). There is overwhelming molecular evidence supporting inbreeding and low genetic diversity in these Southern California populations but there is a lack of phenotypical evidence of inbreeding depression. The primary goal of this study was to assess male mountain lions, in Southern California, for teratospermia (>60% abnormal sperm production), one of the first signs of inbreeding depression in mountain lions that are associated with decreased reproduction and population decline. From December 2019 to December 2020, we surveyed mountain lions during live captures, after mortality events, and in images collected from camera traps in the following populations: Santa Monica Mountains, Santa Susana Mountains, Santa Ana Mountains, and the Eastern Peninsular Range. Mountain lions were sampled for known physical abnormalities associated with inbreeding depression such as teratospermia, cryptorchidism, and distal tail kinks. For teratospermia, we extracted testes from five males post-mortem to assess sperm morphology. Epididymal sperm evaluations revealed all males were teratospermic. Across all samples, on average, 93% of observed spermatozoa were abnormal. We physically examined 32 mountain lions (males and females) for distal tail kinks, and we observed one individual affected. We examined 15 male mountain lions for cryptorchidism, and we observed one unilaterally cryptorchid male and one male with testes that differed significantly in size, likely reflecting asynchronous migration of the testes during puberty. Further, we identified three other animals in camera-trap images that had distal tail kinks, for a total of four. In conclusion, from December 2019 to December 2020, we identified nine individuals exhibiting physical signs of inbreeding depression. These reproductive and physical signs of inbreeding depression in Southern California mountain lions increase the urgency of conservation efforts in the region.

Hydroxytyrosol effectively improves the quality of pig sperm at 17 °C.

Artificial insemination (AI) is a proven breeding technology which has been widely used in pig reproduction. Low temperature can cause very serious damage to pig sperm below 15 °C and the situation is even more serious at lower temperature. Besides, the preservation of pig sperm is mainly carried out at 17 °C because of its outstanding performance in pig reproduction. However, the accumulation of a large amount of reactive oxygen species (ROS) during the preservation process is the main reason for the deterioration of sperm quality. In our research, by adding different concentrations of hydroxytyrosol to the diluent during the storage of pig sperm at 17 °C, we compared them with the traditional diluent to study the sperm motility, the cumulative amount of ROS, the extent of sperm membrane damage, the sperm acrosome integrity, the sperm DNA damage and the activity of antioxidant enzymes (CAT, T-AOC, SOD, GSH-PX, MDA) to evaluate the effect of hydroxytyrosol on the sperm quality during storage. We used proteomics sequencing technology to monitor difference in sperm protein between the control samples and the addition of 120 µmol/L hydroxytyrosol samples (optimum concentration) after three days storage. Ultimately, we selected the control samples and the addition of 120 µmol/L hydroxytyrosol samples to test the effect of AI. The results of our research showed that during storage of pig sperm at 17 °C, the sperm quality and antioxidant capacity of the hydroxytyrosol-treated samples significantly improved (HT 120 µmol/L) (P < 0.05). Proteomics sequencing analysis proved that the addition of 120 µmol/L hydroxytyrosol treatment samples had potential value in improving sperm quality. The significant increase in sow pregnancy rate and piglet birth weight proved that hydroxytyrosol had important practical value in pig reproduction. Based on our results, we demonstrated that the addition of hydroxytyrosol to the diluent could improve the quality of pig sperm and the efficiency of AI.