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Background: Coronary artery disease (CAD) is a leading cause of death in women. Epicardial adipose tissue (EAT) secretes cytokines to modulate coronary artery function, and the release of fatty acids from EAT serves as a readily available energy source for cardiomyocytes. However, despite having beneficial functions, excessive amounts of EAT can cause the secretion of proinflammatory molecules that increase the instability of atherosclerotic plaques and contribute to CAD progression. Although exercise mitigates CAD, the mechanisms by which exercise impacts EAT are unknown. The Yucatan pig is an excellent translational model for the effects of exercise on cardiac function. Therefore, we sought to determine if chronic aerobic exercise promotes an anti-inflammatory microenvironment in EAT from female Yucatan pigs., Methods: Sexually mature, female Yucatan pigs (n = 7 total) were assigned to sedentary (Sed, n = 3) or exercise (Ex, n = 4) treatments, and coronary arteries were occluded (O) with an ameroid to mimic CAD or remained non-occluded (N). EAT was collected for bulk (n = 7 total) and single nucleus transcriptomic sequencing (n = 2 total, 1 per exercise treatment)., Results: Based on the bulk transcriptomic analysis, exercise upregulated S100 family, G-protein coupled receptor, and CREB signaling in neurons canonical pathways in EAT. The top networks in EAT affected by exercise as measured by bulk RNA sequencing were SRC kinase family, fibroblast growth factor receptor, Jak-Stat, and vascular endothelial growth factor. Single nucleus transcriptomic analysis revealed that exercise increased the interaction between immune, endothelial, and mesenchymal cells in the insulin-like growth factor pathway and between endothelial and other cell types in the platelet endothelial cell adhesion molecule 1 pathway. Sub-clustering revealed nine cell types in EAT, with fibroblast and macrophage populations predominant in O-Ex EAT and T cell populations predominant in N-Ex EAT. Unlike the findings for exercise alone as a treatment, there were not increased interactions between endothelial and mesenchymal cells in O-Ex EAT. Coronary artery occlusion impacted the most genes in T cells and endothelial cells. Genes related to fatty acid metabolism were the most highly upregulated in non-immune cells from O-Ex EAT. Sub-clustering of endothelial cells revealed that N-Ex EAT separated from other treatments., Conclusions: According to bulk transcriptomics, exercise upregulated pathways and networks related to growth factors and immune cell communication. Based on single nucleus transcriptomics, aerobic exercise increased cell-to-cell interaction amongst immune, mesenchymal, and endothelial cells in female EAT. Yet, exercise was minimally effective at reversing alterations in gene expression in endothelial and mesenchymal cells in EAT surrounding occluded arteries. These findings lay the foundation for future work focused on the impact of exercise on cell types in EAT., (© 2024. The Author(s).)

Hypertrophic scarring is a major source of morbidity. Sex hormones are not classically considered modulators of scarring. However, based on increased frequency of hypertrophic scarring in patients on testosterone, we hypothesized that androgenic steroids induce abnormal scarring and developed a preclinical porcine model to explore these effects. Mini-swine underwent castration, received no testosterone (noT) or biweekly testosterone therapy (+T), and underwent excisional wounding. To create a delayed wound healing model, a subset of wounds were re-excised at 2 weeks. Scars from postoperative day 42 (POD42) and delayed wounds (POD28) were harvested 6 weeks after initial wounding for analysis via histology, bulk RNA-seq, and mechanical testing. Histologic analysis of scars from +T animals showed increased mean fibrosis area (16 mm 2 noT, 28 mm 2 +T; p = .007) and thickness (0.246 mm 2 noT, 0.406 mm 2 +T; p < .001) compared to noT. XX+T and XY+T scars had greater tensile burst strength (p = .024 and p = .013, respectively) compared to noT swine. Color deconvolution analysis revealed greater deposition of type I and type III collagen as well as increased collagen type I:III ratio in +T scars. Dermatopathologist histology scoring showed that +T exposure was associated with worse overall scarring (p < .05). Gene ontology analysis found that testosterone exposure was associated with upregulation of cellular metabolism and immune response gene sets, while testosterone upregulated pathways related to keratinization and laminin formation on pathway analysis. In conclusion, we developed a preclinical porcine model to study the effects of the sex hormone testosterone on scarring. Testosterone induces increased scar tissue deposition and appears to increase physical strength of scars via supraphysiologic deposition of collagen and other ECM factors. The increased burst strength seen in both XX and XY animals suggests that hormone administration has a strong influence on scar mechanical properties independent of chromosomal sex. Anti-androgen topical therapies may be a promising future area of research., (© 2024 Federation of American Societies for Experimental Biology.)

The aim of this study was to create a continuous drug delivery system that would yield serum testosterone levels in sexually mature, female pigs consistent with serum testosterone levels of human transgender men. Testosterone enanthate was mixed with medical grade silicone at a ratio of 20 % by weight, placed in silastic tubing to cure at room temperature for 24 h, then removed from the tubing mold and stored at 4 °C until surgical implantation. Testosterone enanthate oxidizes at high temperatures which is why the implants had to be stored cold. Each implant was 9 cm long and contained 0.56 g of testosterone enanthate. A minimum of one implant (0.56 g testosterone enanthate) and a maximum of four implants (2.24 g testosterone enanthate) were placed in the cervical subcutaneous fat of each pig. After implantation, serum testosterone was assessed over 40 days. Silastic testosterone enanthate implants increase serum testosterone and maintain it in a relatively constant state in a dose-dependent manner for ∼ 21-25 days post-implantation. •Effective method for subcutaneous delivery of large quantities of testosterone or testosterone metabolites in compact implants to large animal biomedical model species.•Maintains increased circulating testosterone levels for up to 3, 4 weeks post-implantation in pigs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)

Diet plays a critical role for patients across the cancer continuum. The World Cancer Research Fund International and the American Cancer Society have published evidence supporting the role of nutrition in cancer prevention. We conducted an analysis of the literature on dietary nutrients and cancer to uncover opportunities for future research. The objective of the bibliometric analysis was to describe trends in peer-reviewed publications on dietary components and cancer and to highlight research gaps. PubMed was queried for manuscripts with diet- and cancer-related keywords and Medical Subject Headings (MeSH) terms. Metadata covering 99,784 publications from 6469 journals were analyzed to identify trends since 1970 on diet topics across 19 tumor types. Publications focused largely on breast, colorectal, and liver cancer, with fewer papers linking diet with other cancers such as brain, gallbladder, or ovarian. With respect to "unhealthy" diets, many publications focused on high-fat diets and alcohol consumption. The largest numbers of publications related to "healthy" diets examined the Mediterranean diet and the consumption of fruits and vegetables. These findings highlight the need for additional research focused on under-investigated cancers and dietary components, as well as dietary studies during cancer therapy and post-therapy, which may help to prolong survivorship.

A combination of tiletamine-zolazepam, medetomidine, and azaperone was used to immobilize captive Chacoan peccaries (Catagonus wagneri) for health assessments and biological sample collection at the Centro Chaqueño para la Conservación e Investigación (CCCI) in the Paraguayan Chaco during July in 2017 and 2018. In total, 83 peccaries kept in 0.25-1.50 hectare enclosures were immobilized via dart-administered anesthetic. Mean animal weight was 33.89±3.74 kg (standard deviation; n=77). The mean intramuscular (IM) anesthetic drug and dosages were 0.03±0.00 mg/kg of medetomidine, 0.91±0.10 mg/kg of Zoletil 50 (tiletamine-zolazepam), and 0.30±0.03 mg/kg azaperone. The mean time to recumbency after darting was 6.07±2.65 min. The mean time to reach the anesthetic plane postdarting was 10.00±2.00 min. Muscle relaxation was adequate to allow minor veterinary procedures. A mean dosage of 0.15±0.02 mg/kg of atipamezole was given IM to reverse the medetomidine. Recoveries were smooth and animals were standing by 59.17±30.18 min postreversal. Full recovery and release back to enclosures occurred 90±30 min postreversal. A single dose of this drug combination provided adequate anesthesia for 88% of adult Chacoan peccaries; 12% needed a supplemental dose of tiletamine-zolazepam because of failure to receive the full dose from the anesthetic dart. Sex and age did not impact the dosage required to achieve immobilization. Confinement during recovery from anesthesia is required with this protocol. Aside from mild hypoxemia, no adverse effects from anesthesia were observed. However, oxygen supplementation as a part of this protocol is recommended to support circulatory and respiratory capacity., (© Wildlife Disease Association 2023.)

Steroid hormone analysis is routinely undertaken in the assessment of stress response and reproductive function in the management of both captive and free-ranging wildlife species. Faecal samples have become the preferred sample type for analysis as collection is non-invasive and easily assessable. These investigations are generally aimed at aiding successful translocations, enhanced survival outcomes in captivity and improvement of reproductive rate. Immunoassays are the most common approach in the analysis of hormones, particularly in the case of the southern white rhinoceros (Ceratotherium simum simum). Non-specificity, attributed to structural similarity of steroid metabolites impedes accurate evaluations which can be eliminated by chromatographic techniques which are more specific, selective and provide comprehensive analyses. This study developed and validated three methods using ultra-performance convergence chromatography tandem mass spectrometry for the assessment of classical androgens, progestogens and adrenal steroids, as well as the C11-oxy androgens and C11-oxy progestogens in serum and faeces from white rhinoceros. The limit of detection and quantification were determined for each steroid, parameters such as accuracy (<19.8 % RSD) and precision (<20.2 % RSD) were established with recovery, matrix effect, and process efficiency within acceptable limits. Subsequent analysis of serum and faecal samples from five white rhinoceros identified novel steroids for the first time in this species. In addition to the classical adrenal steroids, the following C11-oxy steroids were detected in faecal samples: 11α-hydroxydihydroprogesterone (168 ng/g), 11α-hydroxyprogesterone (125.9 ng/g), 11β-hydroxyprogesterone (210.2 ng/g) and 11-ketoandrostenedione (3.3-19.6 ng/g) with 11-deoxycortisol being the major glucocorticoid (24.2-67.3 ng/g) together with 21-deoxycortisone (40.7 ng/g) and deoxycorticosterone (7.6-14.6 ng/g). In serum samples 11β-hydroxyandrostenedione (0.35-2.34 ng/mL) and 11β-hydroxytestosterone (0.18-1.62 ng/mL) were the predominant androgens with cortisol (5.8-20.5 ng/mL), the predominant glucocorticoid, while corticosterone, 18-hydroxycorticosterone and aldosterone were also detected. These methods can be applied independently to assess either androgens, progestogens, or adrenal steroid panels or in combination to assess the cohort of gonadal and adrenal steroids in faeces and/or serum, in southern white rhinoceros as well as other wildlife species. Analysis would enable the accurate assessment of reproductive health and stress responses while also distinguishing between stress and distress thus contributing to the conservation of wildlife species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Increased poaching in northern South Africa has necessitated relocation of large numbers of southern white rhinoceros (Ceratotherium simum simum) to the Eastern Cape Province. The climate and grassland ecology of this province differ from that of northern South Africa which may impact the health of this species. This assessment of fecal steroid levels and microbiome in 10 free-ranging southern white rhinoceros in the Eastern Cape will provide insights into white rhinoceros physiology in this biome. Fecal steroid metabolites were analyzed using enzyme immunoassay (EIA) and ultra-performance convergence chromatography tandem mass spectrometry (UPC 2 -MS/MS). Fecal microbial composition was assessed via next generation sequencing. EIAs with antibodies raised against progesterone (P4; mouse monoclonal - CL425 clone), testosterone (T; rabbit polyclonal), corticosterone (B; sheep polyclonal) were utilized. Pregnant females had large quantities of fecal progesterone metabolites (FPMs) detected by CL425 EIA. Pregnant females also had native P4 and 11α-hydroxydihydroprogesterone (11αOHDHP4; 4-pregnen-11α-ol-3,20-dione) detected by UPC 2 -MS/MS but these concentrations were 1000-fold less than the concentrations of FPMs detected by the CL425 EIA. By contrast, non-pregnant females had FPM concentrations detected by CL425 EIA which were similar to native P4 and 11αOHDHP4 concentrations detected by UPC 2 -MS/MS. Mean fecal androgen metabolite (FAM) concentrations detected by the T EIA were similar between males and females. 11-ketoandrostenedione (11KA4) detected by UPC 2 -MS/MS was higher in females than males. However, there was no difference between males and females in the concentration of fecal glucocorticoid metabolites (FGMs) detected by the B EIA. Bacteroidia, followed by Clostridia, was the most abundant classes of fecal microbes. The unfiltered microbiome of females was more diverse than that of males. The core fecal microbiome of young rhinoceros had a higher observed species richness (Shannon diversity index, and Simpson diversity index) than that of old rhinoceros. In the alpha male, immobilization was associated with an increase in FGMs detected by 11-deoxycortisol (S) detected by UPC 2 -MS/MS coupled with decreased abundance of Spirochaetia. We detected substantially different FAM and FPM concentrations from those previously reported for both captive and wild white rhinoceros. Comparison of our UPC 2 -MS/MS and EIA results underscores the fact that most EIAs are highly cross reactive for many steroid metabolites. Our data also demonstrates a distinct effect of stress not only on FGMs but also on the fecal microbiome. This is the first non-invasive assessment of fecal steroid metabolites by UPC 2 -MS/MS and the fecal microbiome in wild white rhinoceros., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

The effects of androgens have been extensively studied in a variety of organs and cell types with an increasing focus on the sexually dimorphic role androgens play not only with respect to cellular functions but also in metabolism. Although the classical mechanism of androgen action is via ligand-dependent binding with the nuclear transcription factor, androgen receptor (AR), cytosolic AR can also activate second messenger signaling pathways. Given that cytosolic AR can signal in this manner, there has been increased interest in the mechanisms by which androgens may control cellular organelle function. This review highlights the effects that androgens have on mitochondrial structure and function with emphasis on biogenesis, fusion/fission, mitophagy, bioenergetics (oxidative phosphorylation), and reactive oxygen species production. There are a number of publications on the effects of androgens in these general areas of mitochondrial function. However, the precise mechanisms by which androgens cause these effects are not known. In addition, given that the nucleus and mitochondria work in tandem to control mitochondrial function and the mitochondria have their own DNA, future research efforts should focus on the direct, mechanistic effects of androgens on mitochondrial function.

Aromatase is a monooxygenase that catalyzes the rate-limiting step of estrogen biosynthesis from androgens. Aromatase inhibitors are widely used for the treatment of patients with hormone receptor-positive breast cancer. However, the effects of aromatase inhibitors on cardiovascular and renal health in females are understudied. Given that estrogen is protective against cardiovascular and kidney diseases, we hypothesized that aromatase inhibition elevates blood pressure and induces kidney injury in female Sprague-Dawley rats. Twelve-week-old female rats were implanted with radiotelemetry transmitters to continuously monitor blood pressure. After baseline blood pressure recording, rats were randomly assigned to treatment with the aromatase inhibitor anastrozole (ASZ) or vehicle (Veh) in drinking water. Twenty days after treatment initiation, rats were shifted from a normal-salt (NS) diet to a high-salt (HS) diet for an additional 40 days. Rats were euthanized 60 days after treatment initiation. Body weight increased in both groups over the study period, but the increase was greater in the ASZ-treated group than in the Veh-treated group. Mean arterial pressure increased in ASZ-treated rats during the NS and HS diet phases but remained unchanged in Veh-treated rats. In addition, urinary excretion of albumin and kidney injury marker-1 and plasma urea were increased in response to aromatase inhibition. Furthermore, histological assessment revealed that ASZ treatment increased morphological evidence of renal tubular injury and proximal tubular brush border loss. In conclusion, chronic aromatase inhibition in vivo with ASZ increases blood pressure and markers of renal proximal tubular injury in female Sprague-Dawley rats, suggesting an important role for aromatization in the maintenance cardiovascular and renal health in females. NEW & NOTEWORTHY Aromatase enzyme catalyzes the rate-limiting step in estrogen biosynthesis. Aromatase inhibitors are clinically used for the treatment of patients with breast cancer; however, the impact of inhibiting aromatization on blood pressure and renal function is incompletely understood. The present findings demonstrate that systemic anastrozole treatment increases blood pressure and renal tubular injury markers in female rats fed a high-salt diet, suggesting an important role for aromatization in preserving cardiovascular and renal health in females.

Transgender men undergoing hormone therapy are at risk for insulin resistance. However, how virilizing testosterone therapy affects serum insulin and peripheral insulin sensitivity in transgender men is unknown. This study assessed the effect of acute, virilizing testosterone on serum insulin concentrations and insulin signaling in liver, skeletal muscle, and white adipose tissue (WAT) of female pigs as a translational model for transgender men. Females received three doses of intramuscular testosterone cypionate (TEST females; 50 mg/day/pig) or corn oil (control) spaced 6 days apart starting on the day of estrus ( D0 ). Fasting blood was collected on D0 , D3 , D5 , D11 , and D13 , and females were euthanized on D13 . On D13 , TEST females had virilizing concentrations of serum testosterone with normal concentrations of serum estradiol. Virilizing serum testosterone concentrations ( D13 ) were associated with decreased serum insulin and C-peptide concentrations. Blood glucose and serum glycerol concentrations were not altered by testosterone. Virilizing concentrations of testosterone downregulated AR and ESR1 in subcutaneous (sc) WAT and upregulated transcript levels of insulin-signaling pathway components in WAT and liver. At the protein level, virilizing testosterone concentrations were associated with increased PI3K 110α in liver and increased insulin receptor (INSR) and phospho(Ser256)-FOXO1 in visceral (v) WAT but decreased phospho(Ser473)-AKT in vWAT and scWAT. These results suggest that acute exposure to virilizing concentrations of testosterone suppresses circulating insulin levels and results in increased abundance of proteins in the insulin-signaling pathway in liver and altered phosphorylation of key proteins in control of insulin sensitivity in WAT. NEW & NOTEWORTHY Acute virilizing doses of testosterone administered to females suppress circulating insulin levels, upregulate components of the insulin-signaling pathway in liver, and suppress insulin signaling in white adipose tissue. These results suggest that insulin resistance in transgender men may be due to suppression of the insulin-signaling pathway and decreased insulin sensitivity in white adipose tissue.

We sought to develop a reversible staining protocol using micro-computed tomography (micro-CT) paired with a radiopaque contrast agent that allows for three-dimensional in situ visualization and characterization of atherosclerotic plaques. Atherosclerotic porcine coronary arteries were dissected from surrounding myocardium and incubated in iohexol at various concentrations and incubation times and then imaged using direct radiography. Line profiles were generated across the artery x-ray to determine effectiveness of the radiopaque contrast agent to penetrate the tissue. Our studies revealed that, to sufficiently delineate tissue constructs, the minimum effective iohexol concentration and incubation time were 240 mgI/mL for 1 hour. Among all groups, 24 hours of de-staining brought radiopacity back to control levels. After iohexol incubation, micro-CT was performed. Our findings demonstrate that extended staining times and a minimum iohexol concentration of 240 mgI/mL are required for effective tissue perfusion, which eliminates the diffusion distribution profile inherent to the ability of the contrast agent to traverse tissue layers.•Iohexol enhances ex vivo micro-CT imaging of atherosclerotic coronary arteries•Iohexol allows for improved tissue segmentation during micro-CT image analysis•Effectiveness of iohexol penetration of the tissue was dependent on concentration and duration of incubation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Background and Aims: Visualization of arterial lesions in situ can enhance understanding of atherosclerosis progression and potentially improve experimental therapies. Conventional histology methods for assessing atherosclerotic lesions are robust but are destructive and may prevent further tissue analysis. The objective of the current study was to evaluate a novel, nondestructive method for visualization and characterization of atherosclerotic lesions as an alternative or complementary to routine histology. Thus, we tested the hypothesis that micro-computed tomography (micro-CT) paired with an iodine-based radiopaque stain would effectively characterize atherosclerotic plaques in a manner comparable to routine histology while maintaining sample integrity and providing whole-volume data., Methods: We examined porcine coronary arteries with varying degrees of atherosclerosis, using micro-CT in the absence and presence of iohexol (240 mgI/ml). Following iohexol washout, routine histological assessment of the samples was performed with hematoxylin and eosin and Masson's trichrome., Results: Iohexol staining generated soft tissue delineation and subsequent atherosclerotic plaque assessment via augmented radiopacity, permitting three-dimensional (3D) reconstruction of these lesions, maintaining in situ architecture. Although plaque distribution and arterial wall tissue layers were discernible, micro-CT was incapable of discriminating cell types comprising the plaque. Calcium phosphate deposition was readily located and visualized in 3D space, independent of iohexol., Conclusions: The results of this study establish micro-CT, combined with a diffusible radiopaque contrast agent, as a powerful imaging modality for visualizing in situ architecture of atherosclerotic plaques. Our findings demonstrate that micro-CT can be used to identify plaque distribution and calcium deposition complementary to routine histological analysis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: We hypothesized that supplementation of nursery and grower pig diets with coconut oil in the absence of antibiotics would yield maintenance of glucose homeostasis, growth performance, and immune function similar to what is achieved with nursery and grower pig diets containing antibiotics. Pigs received the same base treatment diets from d24 (weaning) to d71 of age and had blood and fecal samples collected on d24, d31, d45 and d71 for measurement of whole blood glucose, serum insulin, cortisol and cytokines, and fecal microbiome. Pigs had weekly weights and daily feed consumption measured throughout the study. Animals were euthanized at d71 and subcutaneous fat and ileal contents were collected for assessment for fatty acids and microbiome, respectively. Diet treatments consisted of 2% soybean oil plus antibiotics (ABX; n  = 22), 2% soybean oil without antibiotics (NABX; n  = 22), and 2% coconut oil without antibiotics (COC; n  = 22). Statistical analysis examined the effect of diet within each timepoint using a repeated measures ANOVA., Results: Pigs fed COC diet had decreased serum insulin levels, maintained feed intake, feed conversion and weight gain, and, based on serum cytokines and fecal microbiome, were immunologically similar to ABX-fed pigs. However, NABX-fed pigs performed similarly to the ABX-fed pigs in all parameters except for serum cytokines. Additionally, there was no difference in the incidence of diarrhea between any of the diet treatments., Conclusions: This study demonstrates that dietary antibiotics are not necessary to maintain growth performance in nursery and grower pigs. However, dietary antibiotics appear to modulate circulating cytokine levels. Dietary coconut oil is neither harmful nor helpful to growth performance or immune function in nursery and grower pigs but does modulate serum insulin levels. Therefore, while coconut oil fed at 2% by weight is a suitable substitute for dietary antibiotics, this study suggests that no substitute for dietary antibiotics is needed at all., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2020.)

For unknown reasons, reproductive success varies among zoos in managed red river hogs. In response to urine exposure from novel conspecifics, we hypothesized that males with low libido would exhibit increased concentrations of testosterone metabolites and that acyclic and/or non-breeding females would be induced to cycle or cycle more regularly. Estrous cycle length and progesterone metabolites in same-sex housed females were compared prior to and following exposure to novel red river hog male urine. Male testosterone metabolites and female progesterone metabolites as well as estrous cycle length were compared among: 1) proven-breeder females and males; 2) non-breeding females newly paired with novel males; 3) non-breeding females and males exposed to urine from novel females and males. Fecal samples were collected 3-5 times per week for eight to 12 months, lyophilized, extracted, and assayed for progesterone and testosterone metabolites with validated enzyme immunoassays. Introduction of female urine resulted in an increased number of estrous cycles per female per month, and decreased luteal and increased follicular progesterone metabolites in females. Introduction of male urine resulted in an increase in testosterone metabolites in males. Average progesterone metabolites for pregnant proven-breeder females were more than double that for pregnant females newly paired to novel males. An interaction between season and treatment group, as well as the acyclicity of females from July through November irrespective of treatment group, suggest that season may confound and warrant judicious interpretation of the results. Additionally, females housed with pregnant females were either acyclic or did not carry their pregnancies to term, indicating that reproductive suppression may occur in females. In conclusion, urine may be a cost-effective and efficient means to manipulate estrous cycling in managed red river hogs. Furthermore, careful consideration of the number of females in a managed herd is recommended to avoid reproductive suppression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Reproductive management in zoos requires contraception or physical separation of sexes to ensure captive population viability, but information is sparse on the effects of parity, age, and contraceptive use on lifetime reproductive health in captive Suidae and Tayassuidae species. This retrospective study evaluated reproductive tissues and histories from babirusa (Babyrousa babyrussa), red river hog (Potamochoerus porcus), Visayan warty pig (Sus cebifrons), common warthog (Phacochoerus africanus), Vietnamese pot-bellied pig (Sus scrofa domesticus), domestic cross pig (Sus scrofa), Sunda island pig (Sus celebensis timoriensis), Eurasian boar (Sus scrofa), Bornean bearded pig (Sus barbatus), Ossabaw island hog (Sus scrofa domesticus), Guinea hog (Sus scrofa domesticus), Chacoan peccary (Catagonus wagneri), and collared peccary (Pecari tajacu). Age, parity, litter size, time-since-last-parturition, contraception exposure and type, and lesion prevalence were recorded. Reported chemical contraceptives used in females included porcine zona pellucida vaccine, progestins, GnRH analogues (deslorelin and leuprolide). Average litter size was significantly different between species (p < 0.0001) with the common warthog having the largest average litter size (3.5 ± 0.2 offspring/litter). There was a trend for age to be positively correlated with leiomyoma/sarcomas (r = 0.6135; p = 0.0789). Progestins (medroxyprogesterone acetate, megestrol acetate, depomedroxyprogesterone acetate) were positively correlated (r = 0.8946; p = 0.0161) and GnRH analogues (deslorelin, leuprolide; subcutaneous) were negatively correlated with ovarian cysts (r = 0.9743; p = 0.0010). Across all species, there was a trend for age to be negatively correlated with folliculogenesis (r = -0.6528; p = 0.0566) and parturition gap length to be negatively correlated with follicular cysts (r = -0.8944; p = 0.1). Common warthog, babirusa, and Vietnamese pot-bellied pigs had the greatest diversity of uterine lesions and the highest prevalence of reproductive tract lesions of all species evaluated. Four of the 27 males (14.5%) in the dataset had testicular tumors. All males had prominent testicular interstitial cell populations, which appears to be within normal limits for these species. These data suggest prolonged gaps between pregnancies, age, and contraception are risk factors for reproductive tract lesions in Suidae., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Baseline information about the reproductive physiology of an endangered species is vital to captive breeding programs. This study analyzed reproductive parameters from eleven captive Chacoan peccaries (Catagonus wagneri) (mean age: 10.5 ± 1.2 years old) in Paraguay. After immobilization, testis length, width and firmness (hard to flaccid, 1-3) were assessed, followed by electroejaculation and analysis of semen. Samples were examined for percentage and progressive motility, total cell count, percentage live spermatozoa and morphology. Mean (±SEM) testis volume and firmness were 24.7 ± 1.8 cm 3 and 2.1 ± 0.1, respectively. Mean ejaculate volume was 2.9 ± 0.7 ml with a pH of 7.7 ± 0.3. Few male peccaries had motile spermatozoa (n = 5/9) with a mean percentage and progressive motility of 18.3 ± 8.5% and 0.6 ± 0.3, respectively. The mean percentage of live spermatozoa was 25.1 ± 5.6%. Male peccaries had a low percentage of normal spermatozoa (12.4 ± 2.5%). The mean total count of spermatozoa per ejaculate was also quite low at 1.58 ± 1.01 million total spermatozoa per ejaculate. Spermatozoa defects were predominantly primary (77.7%) with the most common spermatozoa defects being tapered head (19.0 ± 7.4%), diadem/crater (17.7 ± 2.8%), and excess residual cytoplasm (9.6 ± 2.5%). Male age was not correlated with semen parameters (percent live: r=-0.19; motility percentage: r = 0.01; percent normal spermatozoa: r = 0.38; total count: r = 0.29; p>0.05). Evaluation of additional males from this population as well as other captive populations at various time points during the year is warranted., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Forty percent of American women are obese and at risk for type II diabetes, impaired immune function, and altered microbiome diversity, thus impacting overall health. We investigated whether obesity induced by an excess calorie, high fat diet containing hydrogenated fats, fructose, and coconut oil (HFD) altered glucose homeostasis, peripheral immunity, and urogenital microbial dynamics. We hypothesized that HFD would cause hyperglycemia, increase peripheral inflammation, and alter urogenital microbiota to favor bacterial taxonomy associated with inflammation. We utilized female Ossabaw mini-pigs to model a 'thrifty' metabolic phenotype associated with increased white adipose tissue mass. Pigs were fed HFD (~4570 kcal/pig/day) or lean (~2000 kcal/pig/day) diet for a total of 9 estrous cycles (~6 months). To determine the effect of cycle stage on cytokines and the microbiome, animals had samples collected during cycles 7 and 9 on certain days of the cycle: D1, 4, 8, 12, 16, 18. Vaginal swabs or cervical flushes assessed urogenital microbiota. Systemic fatty acids, insulin, glucose, and cytokines were analyzed. Pig weights and morphometric measurements were taken weekly. Obese pigs had increased body weight, length, heart and belly girth but similar glucose concentrations. Obese pigs had decreased cytokine levels (IL-1β, TNF-α, IL-4, IL-10), arachidonic acid and plasma insulin, but increased levels of vaccenic acid. Obese pigs had greater urogenital bacterial diversity, including several taxa known for anti-inflammatory properties. Overall, induction of obesity did not induce inflammation but shifted the microbial communities within the urogenital tract to an anti-inflammatory phenotype. We postulate that the coconut oil in the HFD oil may have supported normal glucose homeostasis and modulated the immune response, possibly through regulation of microbial community dynamics and fatty acid metabolism. This animal model holds promise for the study of how different types of obesity and high fat diets may affect metabolism, immune phenotype, and microbial dynamics.

The measurement of progesterone (P4) and estradiol (E2) is essential for monitoring reproductive cycles and can aid in diagnosing the cause of poor reproductive performance in dairy cattle. Readily available, reproducible, accurate, non-radioactive assays are needed for the assessment of P4 and E2 in bovine serum. The gold standard for hormone assessment, radioimmunoassay (RIA), was compared with enzyme-linked immunoassay (EIA). Serum collected from various points in the estrous cycle was extracted with radiolabeled P4 (ie, 3 H-P4; HE) and without 3 H-P4 (CE) before being used in the assay. For the assessment of P4, there is a great degree of correlation between the RIA and EIA (adjusted R-square = 0.95; Pearson correlation coefficient (PCC) = 0.98, P < 0.001). A difference between the RIA and EIA methods was not detected for E2 concentrations (P = 0.16), but the correlation between techniques was poor (adjusted R-squared = 0.73; PCC = 0.87, P = 0.002). There was no difference in the serum extraction efficiency as measured with 3 H-P4 as opposed to without (P = 0.94). The two methods for the measurement of serum extraction efficiency were highly correlated (adjusted R-square = 0.83; PCC = 0.92, P < 0.001). The concordance correlation coefficient (CCC) showed an excellent agreement between RIA and EIA for P4 determination (0.89) and between HE and CE methods (0.90). Although the 95% limits of agreement of the Bland-Altman plots encompassed 89% (8/9) and 92% (12/13) of the differences between methods for P4 quantification and extraction respectively, the CCC indicated an excellent agreement among them. The CCC between RIA and EIA for E2 quantification was 0.68 which corresponds with a fair agreement; however, the 95% limits of agreement of the Bland-Altman plot encompassed 100% (9/9) of differences between methods. The EIA and CE methods are comparable alternatives to the RIA and HE methods, respectively and can be used to quantify P4 and E2 for bovine serum., (Copyright © 2017 Elsevier Inc. All rights reserved.)

With the increasing knowledge that gender influences normal physiology, much biomedical research has begun to focus on the differential effects of sex on tissue function. Sexual dimorphism in mammals is due to the combined effects of both genetic and hormonal factors. Hormonal factors are mutable particularly in females in whom the estrous cycle dominates the hormonal milieu. Given the severity of the obesity epidemic and the fact that there are differences in the obesity rates in men and women, the role of sex in white adipose tissue function is being recognized as increasingly important. Although sex differences in white adipose tissue distribution are well established, the mechanisms affecting differential function of adipocytes within white adipose tissue in males and females remain largely understudied and poorly understood. One of the largest differences in the endocrine environment in males and females is the concentration of circulating androgens and estrogens. This review examines the effects of androgens and estrogens on lipolysis/lipogenesis, adipocyte differentiation, insulin sensitivity and adipokine production in adipocytes from white adipose tissue with a specific emphasis on the sexual dimorphism of adipocyte function in white adipose tissue during both health and disease., (© 2017 Society for Reproduction and Fertility.)

The discrete effects of obesity on infertility in females remain undefined to date. To investigate obesity-induced ovarian dysfunction, we characterized metabolic parameters, steroidogenesis, and folliculogenesis in obese and lean female Ossabaw mini-pigs. Nineteen nulliparous, sexually mature female Ossabaw pigs were fed a high fat/cholesterol/fructose diet (n=10) or a control diet (n=9) for eight months. After a three-month diet-induction period, pigs remained on their respective diets and had ovarian ultrasound and blood collection conducted during a five-month study period after which ovaries were collected for histology, cell culture, and gene transcript level analysis. Blood was assayed for steroid and protein hormones. Obese pigs developed abdominal obesity and metabolic syndrome, including hyperglycemia, hypertension, insulin resistance and dyslipidemia. Obese pigs had elongated estrous cycles and hyperandrogenemia with decreased LH, increased FSH and luteal phase progesterone, and increased numbers of medium, ovulatory, and cystic follicles. Theca cells of obese, compared to control, pigs displayed androstenedione hypersecretion in response to in vitro treatment with LH, and up-regulated 3-beta-hydroxysteroid dehydrogenase 1 and 17-beta-hydroxysteroid dehydrogenase 4 transcript levels in response to in vitro treatment with LH or LH + insulin. Granulosa cells of obese pigs had increased 3-beta-hydroxysteroid dehydrogenase 1 transcript levels. In summary, obese Ossabaw pigs have increased transcript levels and function of ovarian enzymes in the delta 4 steroidogenic pathway. Alterations in LH, FSH, and progesterone, coupled with theca cell dysfunction, contribute to the hyperandrogenemia and disrupted folliculogenesis patterns observed in obese pigs. The obese Ossabaw mini-pig is a useful animal model in which to study the effects of obesity and metabolic syndrome on ovarian function and steroidogenesis. Ultimately, this animal model may be useful toward the development of therapies to improve fertility in obese and/or hyperandrogenemic females or in which to examine the effects of obesity on the maternal-fetal environment and offspring health.

This study characterizes the effect of an excess-calorie, high-fat, high-cholesterol, high-fructose diet on metabolic parameters and reproductive function in female Ossabaw minipigs. Cycling sows were fed a hypercaloric, high-fat, high-cholesterol, and high-fructose diet (obese, n = 4) or a control diet (control, n = 5) for 13 mo. During the final 4 mo, ovarian ultrasonography was done, blood was collected, and weights and measures were taken. Pigs then underwent ovarian stimulation. Cycle length and androstenedione, total testosterone, progesterone, estradiol, follicle-stimulating hormone, luteinizing hormone, insulin, fructosamine, lipid, and glucose levels were measured. In addition, adipose tissue aromatase gene expression was assessed. As compared with control pigs, obese pigs were hyperglycemic and hyperinsulinemic; had elevated total cholesterol, triglyceride, and leptin levels, and demonstrated abdominal adiposity. Visceral adipose tissue of obese pigs, as compared with control pigs, showed increased aromatase gene expression. Obese pigs had longer estrous cycles, higher serum androstenedione, and higher luteal phase serum luteinizing hormone, compared with control pigs. During the luteal phase, obese pigs had more medium, ovulatory, and cystic ovarian follicles, whereas control pigs had more small ovarian follicles. When fed an excess-calorie, high-fat, high-cholesterol, high-fructose diet, female Ossabaw minipigs develop obesity, metabolic syndrome, and abnormal reproductive function. This animal model may be applicable to studies of the effects of obesity on fertility in women.

Characterization of reproductive seasonality in the African wild dog (Lycaon pictus) could assist reintroduction programs. Male wild dogs (n=14) were assessed quarterly (January, mid-summer; April, late summer; August, late winter; November, early summer) for serum testosterone, body weight, testicular and prostatic volume, preputial gland measurement, and ejaculate characteristics. Bi-monthly fecal samples were collected from male (n=11) and female (n=4) wild dogs for analysis of fecal androgens and progestagens. Fecal androgens were higher in early summer (246.4 ± 14.5 ng/g) than in early winter (218.6 ± 13.4 ng/g). Serum testosterone was higher in mid-summer (1.4 ± 0.3 ng/ml) than in late winter (0.7 ± 0.1 ng/ml). Number of spermatozoa per ejaculate was greatest in late summer (301.4 ± 39.3 × 10(6)). Other semen parameters peaked in mid-summer (pH: 7.4; progressive motility: 85.0 ± 0.1%; live spermatozoa: 81.0 ± 16%; normal morphology: 71.5 ± 8.2%). Total testicular and prostatic volume were greater during summer (testicular: 36.7 ± 4.2 cm(3); prostatic: 12.0 ± 1.9 cm(3)) than winter (testicular: 25.2 ± 1.9 cm(3); prostatic: 5.8 ± 0.8 cm(3)). Preputial pendulance also was greater in summer (7.1 ± 0.5 cm; n=9) than winter (5.9 ± 0.2 cm). Baseline fecal progestagen metabolites were 6.2 ± 2.5 μg/g and peak fecal progestagen metabolites were 14.7 ± 2.8 μg/g. Copulations resulting in pregnancies (n=2) occurred in late summer and gestation was 71 days. Female wild dogs were seasonally monoestrous with mating in summer and winter. In conclusion, wild dogs are reproductively seasonal with improvement in male reproductive variables during summer and a bi-phasic seasonal pattern to female receptivity., (Copyright © 2012 Elsevier Inc. All rights reserved.)