Objective:To characterize the total and inorganic serum iodine concentrations of canines with varying thyroid disease status. Methods:Leftover serum samples submitted to the Michigan State University Veterinary Diagnostic Laboratory for diagnostic thyroid panel screening between January and June 2023 were utilized for this retrospective study. Thyroglobulin autoantibody was used as a marker for autoimmune thyroiditis. Inorganic and total iodine were measured via an inductively coupled plasma-mass spectrometer. An estimated reference interval was calculated for inorganic and total iodine using results from euthyroid dogs. Results:Iodine concentrations were measured in dogs with autoimmune thyroiditis (n = 101) and hypothyroidism (97) and euthyroid dogs (86). Serum iodine concentrations ranged widely but were not increased in dogs with autoimmune thyroiditis or hypothyroidism in comparison to euthyroid animals. Concentrations of both total and inorganic iodine were statistically different between dogs suffering from autoimmune thyroiditis and those with hypothyroidism. Conclusions:Elevated serum iodine is not associated with autoimmune thyroiditis or hypothyroidism in dogs. Clinical Relevance:The concentration of iodine alone in canine serum is not a marker for thyroid disease in dogs.

Introduction: Women diagnosed with cancer often undergo aggressive chemotherapy that can impair fertility and lead to long-term ovarian damage, significantly affecting their quality of life. Cyclophosphamide (CPA), a chemotherapeutic agent known for its gonadotoxic effects, has been shown to reduce ovarian follicle reserves, thereby contributing to the development of primary ovarian insufficiency in both humans and animal models. This study sought to identify the molecules and intracellular signaling pathways associated with CPA's effects on ovarian tissue of rats bearing mammary tumors. Methods: To address the objective of the study, transcriptomic (RNA-Seq) and proteomic (2D-DIGE/MS) methodologies were applied. The study was conducted on rats with N-methyl-N-nitrosourea (MNU)-induced mammary neoplasia, randomly assigned to control or cyclophosphamide (CPA)-treated groups. CPA was administered intraperitoneally (50 mg/kg on day 3, then 10 mg/kg weekly until day 31). Animals were euthanized on day 34, and ovaries were collected for RNA-Seq and 2D-DIGE/MS analyses. Results: Our results demonstrated that the crucial mechanism of CPA action during follicular depletion in the ovary may be linked to CPA-induced immune cell responses. Moreover, we found that CPA may trigger apoptosis or ferroptosis of follicular cells, ultimately leading to ovarian dysfunction. Conclusion: The obtained results highlight the importance of mechanisms contributing to ovarian toxicity from cancer chemotherapy, paving the way for developing targeted strategies for ovarian protection. Further functional experiments are needed to identify substances that could effectively preserve the fertility of female cancer survivors.

Endocrine diagnostics currently depend on the ability to measure low and high concentrations of diagnostic hormones using immunoassays. This often is challenging in species other than humans, dogs, cats, and horses due to lack of validated assays and reference intervals. There are strategies to approach endocrine testing in zoo, wildlife, and zoologic companion animals but caution is needed in interpreting results. Newer techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) may be more useful for all species, although technical hurdles remain for this method too.

Hypothyroidism in dogs is a common diagnosis with some breeds being more prone to the condition. Autoimmune thyroiditis has an inherited component. Breeders wish to reduce the incidence by any means possible. Currently, the only opportunity lies in phenotypic testing of thyroid functionality. This retrospective study evaluated thyroid hormone and thyroglobulin autoantibodies (TgAA) analyses in dogs assessed multiple times to determine if the outcome changed over time. Data were extracted from the Orthopedic Foundation for Animals (OFA) database for 8,309 dogs which had been evaluated two or more times and the initial classification was compared to a final classification. More than 90% of dogs evaluated as normal for thyroid function remained normal in follow-up assessments. The greatest change was seen for dogs initially evaluated as equivocal; this was followed by a compensative autoimmune thyroiditis diagnosis being revised to normal, though 50% of the latter evaluation remained classified as compensative autoimmune thyroiditis. This suggests the presence of low levels of autoantibodies may be transient and that a dog presenting with autoantibodies should be reevaluated to confirm the development of autoimmune thyroiditis.

Infertility due to ovarian toxicity is a common side effect of cancer treatment in premenopausal women. Tamoxifen (TAM) is a selective estrogen receptor modulator that prevented radiation- and chemotherapy-induced ovarian failure in preclinical studies. In the current study, we examined the potential regulatory role of long noncoding RNAs (lncRNAs) in the mechanism of action of TAM in the ovaries of tumor-bearing rats receiving cyclophosphamide (CPA) as cancer therapy. We identified 166 lncRNAs, among which 49 were demonstrated to be differentially expressed (DELs) in the ovaries of rats receiving TAM and CPA compared to those receiving only CPA. A total of 24 DELs were upregulated and 25 downregulated by tamoxifen. The identified DELs shared the characteristics of noncoding RNAs described in other reproductive tissues. Eleven of the identified DELs displayed divergent modes of action, regulating target transcripts via both cis- and trans-acting pathways. Functional enrichment analysis revealed that, among target genes ascribed to the identified DELs, the majority were involved in apoptosis, cell adhesion, immune response, and ovarian aging. The presented data suggest that the molecular mechanisms behind tamoxifen’s protective effects in the ovaries may involve lncRNA-dependent regulation of critical signaling pathways related to inhibition of follicular transition and ovarian aging, along with the suppression of apoptosis and regulation of cell adhesion. Employing a tumor-bearing animal model undergoing chemotherapy, which accurately reflects the conditions of mammary cancer, reinforces the obtained results. Given that tamoxifen remains a key player in the management and prevention of breast cancer, understanding its ovarian-specific actions in cancer patients is crucial and requires detailed functional studies to clarify the underlying molecular mechanisms.

BACKGROUND:Hypercalcemia has been associated with hypergastrinemia in humans. Hypergastrinemia could be responsible for gastrointestinal (GI) signs in dogs with primary hyperparathyroidism (PHPT).HYPOTHESIS/OBJECTIVES:(a) Determine whether hypergastrinemia occurs in dogs with PHPT, (b) assess for potential correlations among ionized calcium (iCa), parathyroid hormone (PTH), and serum gastrin concentrations, and (c) determine whether gastrin concentrations decrease after management of PHPT.ANIMALS:Phase 1: 151 client-owned dogs at the time of PHPT diagnosis, Phase 2: 24 dogs that underwent treatment for PHPT.METHODS:Dogs with azotemia, concurrent disease, or those receiving acid suppressants were excluded. Twenty-four treated dogs had baseline and repeat quantification of serum gastrin, PTH, and iCa concentrations 4 weeks after treatment. The effect of treatment on gastrin, iCa, and PTH concentrations was assessed using Wilcoxon signed rank sum tests. Fisher exact testing was used to compare the proportion of dogs with hypergastrinemia in dogs with and without GI signs.RESULTS:Twenty-seven of 151 PHPT dogs (17.9%) had increased pre-treatment serum gastrin concentrations (median, 45.0 ng/L; interquartile range [IQR], 20.0 ng/L). Gastrin concentrations were not correlated with iCa (P = .92) or PTH (P = .60). Treatment of PHPT decreased PTH (P < .001) and iCa concentrations (P < .001), but not gastrin concentrations (P = .15). The proportion of dogs with hypergastrinemia with and without GI signs did not differ (P = 1.00).CONCLUSIONS AND CLINICAL IMPORTANCE:Mild increases in serum gastrin concentrations may be seen in dogs with PHPT, but this finding is independent of the presence of GI signs.

OBJECTIVE:The objective of this study was to follow long-term changes in the concentration of thyroid hormones in dogs with subclinical thyroiditis.SAMPLES:Samples were obtained from 125 dogs with subclinical thyroiditis. The study population included 70 female and 55 male dogs. The mean testing interval was 3.9 years from initial testing (SD, 2.3 years; range, 1 to 9 years).METHODS:Dogs with subclinical thyroiditis were identified retrospectively using results from the Orthopedic Foundation for Animals Canine Thyroid Profile performed by the Endocrinology Section of the Michigan State University Veterinary Diagnostic Lab. Owners were invited to submit follow-up serum samples with their veterinarian along with a medical history form, including subsequent treatments.RESULTS:At the time of retesting, 30% of the dogs had progressed to hypothyroidism and/or were treated with thyroxine. Fifty percent maintained positive or equivocal thyroglobulin autoantibody (TgAA) results while remaining euthyroid. Fourteen percent of the dogs became TgAA negative and remained euthyroid. In 6% of the cases tested, proper medical histories were not available, and a final classification could not be determined.CLINICAL RELEVANCE:These results indicate that most dogs with elevated thyroglobulin autoantibodies either exhibit persistent autoimmune thyroiditis with continued risk of hypothyroidism or progress to hypothyroidism when monitored for more than 1 year. Thyroid function in dogs with subclinical thyroiditis should be monitored every 12 months or if there is change in the clinical presentation.

Abstract Disclosure: R.J. Egbert: None. R.C. Fowkes: None. S.J. Niessen: None. B.K. Petroff: None. Spontaneous models of endocrinopathies are powerful tools to understand disease etiology. Endocrine diseases in companion animals are particularly useful to study, as they share their environment with humans and typically present with clinical disease that phenocopies human symptoms. One such disease in domestic cats, hypersomatotropism (HST, feline acromegaly), is a disorder of excessive growth hormone and increased IGF-1 (Insulin like Growth Factor 1), usually caused by hyperplasia or neuroendocrine tumors of the pars distalis of the pituitary gland. This retrospective study used serum IGF-1 results to investigate population characteristics for domestic cats with HST in the United States and Canada. Laboratory records from 1Jan06 to 31Dec22 were reviewed and animals with a circulating IGF-1 hormone concentration of 190 nmol/L (1,450 ng/mL) or greater were presumed to have acromegaly. This review identified 4,620 such cats with a mean age (n=4,351) of 11.2 years (median: 11.2, range: .5-20.3yr) and mean weight (n=2,086) of 6.03 Kg (median: 5.91, range: 2.27-15.0 Kg). Males outnumbered females in the database, which included 3,048 males and 1,181 females. Domestic Short/Medium/Longhair cats were the most prevalent breed (n=3,563; 77%), followed by Maine Coon cats (n=120; 2.6%) and Siamese cats (n=111, 2.4%). Just under half of the animals (n=2,037, 44%) had a history of diabetes mellitus on the submission form. Most submissions were from the US (n=4,347, 94%) with the most hypersomatotropic cats from California (n=841, 18.2%), New York (n=604, 13.1%), Massachusetts (n=402, 8.7%), Colorado (225, 4.9%), and Texas (200, 4.3%). The mean serum IGF-1 concentration of these animals was 358 nmol/mL (median: 345, range 190-761 nmol/mL). For comparison, an age and sex matched control group of 95 animals with residual samples previously submitted for unrelated testing was selected. The mean IGF-1 concentration for the control group was 97 nmol/mL (median: 85, range 0-311 nmol/mL). In summary, HST is diagnosed most frequently in older male cats in urbanized North America, and potentially displays some breed disposition, suggesting both a genetic and environmental component to the disease etiology. Presentation: 6/2/2024

BACKGROUND Adherence to traditional 24-h fasting periods for serum gastrin concentration in dogs can be challenging and may delay the institution of therapies for suspected hypergastrinemia. Peer-reviewed publications regarding serum gastrin reference intervals (RI) are lacking. Hypercalcemia is associated with hypergastrinemia in people; limited data exist in dogs. OBJECTIVE The objective of the study was to generate a RI for a 12-h fasted serum gastrin concentration in dogs and to investigate whether correlations exist with age, weight, sex, and total calcium concentration. METHODS Fifty-five healthy adult dogs (>1 year of age). The screening included: medical history, physical examination, CBC (15 dogs), and serum chemistry (55 dogs). Gastrin was measured via a commercial radioimmunoassay. The RI for 12-h fasted serum gastrin concentration was calculated according to the recommendations of the American Society for Veterinary Clinical Pathology. Additionally, data were evaluated for correlation with selected variables. RESULTS The RI for serum gastrin following a 12-h fasting period was 15.1-78.9 ng/L with 90% confidence intervals for the lower and upper limits of 14.0-22.9 and 68.3-83.0 ng/L, respectively. A generalized linear model did not detect significant relationships between gastrin and age (P = 0.48), sex (P = 0.30), weight (P = 0.93), or total calcium concentration (P = 0.84). CONCLUSIONS A 12-h fasted serum gastrin concentration RI has been established. Given the limited range of serum calcium concentrations in our healthy study population, additional investigations are needed to determine the effects of hypercalcemia on serum gastrin concentrations in dogs and for any potential clinical consequences thereof.

Background Premenopausal women diagnosed with breast cancer often face aggressive chemotherapy resulting in infertility. Tamoxifen (TAM) is a selective estrogen receptor modulator that was previously suggested as a protective agent against chemotherapy-induced ovarian failure. In the current study, we examined mechanisms of the protective action of TAM in the ovaries of tumor-bearing rats treated with the chemotherapy drug cyclophosphamide (CPA). Results TAM prevented CPA-induced loss of ovarian follicular reserves. The protective TAM effect in the rat ovary partially resulted from decreased apoptosis. In addition, transcriptomic and proteomic screening also implicated the importance of DNA repair pathways as well as cell adhesion and extracellular matrix remodeling in the protective ovarian actions of TAM. Conclusions Tamoxifen shielded the ovary from the side effects of chemotherapy without lessening the tumoricidal actions of mammary cancer treatment. Graphical Abstract

Summary of Assessment of Ki-67 Staining Problems. Supplementary Table 1: Summary of Values, Changes, and Statistical Results for Ki-67 Assessments. Supplementary Table 2: Summary of Analyses of Different Subsets of Subjects.

Tamoxifen (TAM) is a drug commonly used in patients with breast cancer. The anticancer effect of TAM occurs via its ability to antagonize estrogen-dependent growth of mammary epithelial cells. Previously, we demonstrated that TAM prevented the chemotherapy-induced loss of ovarian follicular reserves in both cancer-free rats and rats with cancer. Such follicular loss is a main cause of infertility in young women treated for cancer. The current study was undertaken to discover the molecules and intracellular pathways involved in the action of TAM in the ovaries of rats with mammary tumors. To meet this goal we used transcriptomic (RNA-Seq) and proteomic (2D-DIGE/MS) approaches. TAM inhibited the expression of genes and lncRNAs involved in ovarian steroidogenesis. Moreover, TAM altered the expression of genes related to primordial follicle activation or arrest. In addition, proteomic screening indicated the importance of basic metabolic processes in the ovarian actions of TAM. Although simple extrapolation of these data to humans is not possible, the results of this study emphasize the need to explore the ability of TAM to affect ovarian function in women undergoing cancer treatment.

Abstract Background The relationship between the development of SHPT and ionized magnesium (iMg) concentrations in blood of dogs with chronic gastrointestinal (GI) disease and abnormally low 25(OH)D is undefined. Objectives Evaluate relationships between ionized magnesium (iMg), PTH, ionized calcium (iCa), and 25(OH)D in dogs with chronic enteropathy (CE) with or without protein‐losing enteropathy (PLE) and abnormal 25(OH)D. Determine whether dogs with CE or PLE, decreased 25(OH)D and SHPT have differences in iMg, iCa, or 25(OH)D when compared to dogs that do not have SHPT. Animals Fifty dogs with CE +/− PLE and abnormally low serum 25(OH)D. Methods Retrospective search of submissions database at a veterinary diagnostic laboratory for vitamin D profiles submitted in years 2017 to 2020. Cases were excluded if supplemented with Ca, Mg, or vitamin D. Spearman correlation was performed to evaluate relationships between iMg, PTH, 25(OH)D, and iCa. Ionized Mg, iCa, and 25(OH)D concentrations were compared between dogs with SHPT and those with normal PTH concentrations. Results Concentrations of iMg were weakly negatively correlated with PTH (rho, −.31; P = .03), and weakly positively correlated with serum 25(OH)D (rho, .34, P = .02) and iCa (rho, .42, P = .003). Ionized magnesium concentrations were lower in dogs with abnormally low 25(OH)D and SHPT compared to dogs with abnormally low 25(OH)D and normal parathyroid hormone concentrations (P = .01). Conclusions and Clinical Importance Hypomagnesemia might contribute to alterations in iCa and parathyroid hormone in dogs with CE +/− PLE and abnormally low 25(OH)D.

Supplementary Figures and Tables

The relationship between the development of SHPT and ionized magnesium (iMg) concentrations in blood of dogs with chronic gastrointestinal (GI) disease and abnormally low 25(OH)D is undefined. Evaluate relationships between ionized magnesium (iMg), PTH, ionized calcium (iCa), and 25(OH)D in dogs with chronic enteropathy (CE) with or without protein-losing enteropathy (PLE) and abnormal 25(OH)D. Determine whether dogs with CE or PLE, decreased 25(OH)D and SHPT have differences in iMg, iCa, or 25(OH)D when compared to dogs that do not have SHPT. Fifty dogs with CE +/− PLE and abnormally low serum 25(OH)D. Retrospective search of submissions database at a veterinary diagnostic laboratory for vitamin D profiles submitted in years 2017 to 2020. Cases were excluded if supplemented with Ca, Mg, or vitamin D. Spearman correlation was performed to evaluate relationships between iMg, PTH, 25(OH)D, and iCa. Ionized Mg, iCa, and 25(OH)D concentrations were compared between dogs with SHPT and those with normal PTH concentrations. Concentrations of iMg were weakly negatively correlated with PTH (rho, −.31; P  = .03), and weakly positively correlated with serum 25(OH)D (rho, .34, P  = .02) and iCa (rho, .42, P  = .003). Ionized magnesium concentrations were lower in dogs with abnormally low 25(OH)D and SHPT compared to dogs with abnormally low 25(OH)D and normal parathyroid hormone concentrations ( P = . 01). Hypomagnesemia might contribute to alterations in iCa and parathyroid hormone in dogs with CE +/− PLE and abnormally low 25(OH)D.

High neonatal mortality among red pandas (Ailurus fulgens) challenges the long-term sustainability of the Species Survival Plan population. Congenital hypothyroidism (CH) is a rare condition in domestic animals, typically due to an inherited genetic defect. Nongoitrous CH was presumptively diagnosed in 75% (n = 6/8) of red panda neonates from four successive litters, with a common sire and two closely related dams. Antemortem diagnosis of CH was made in three cubs (n = 3/6) based on elevated thyroid stimulating hormone and decreased free thyroxine and total thyroxine levels. Affected cubs also had suggestive clinical signs, which included delayed growth with cretinous dwarf appearance, atonic bladder, delayed gastrointestinal motility, hypercholesterolemia, and hypocalcemia. With sodium levothyroxine therapy, two of the three cubs developed into normal adult red pandas in terms of body size, appearance, and behavior. On necropsy cubs (n = 4) were small with varying degrees of cretin dwarf appearance and hypoplastic thyroids with reduced to no colloid in follicles. These cases demonstrate the importance of collecting thyroid tissue, (or proximal trachea/larynx if gross visualization not possible), in neonates for histopathology. Further investigation into the role of thyroid disease in neonatal red panda mortality is warranted.

Male factors, including those of autoimmune origin, contribute to approximately 50% of infertility cases in humans. However, the mechanisms underlying autoimmune male infertility are poorly understood. Deficiency in autoimmune regulator (AIRE) impairs central immune tolerance because of diminished expression of self-antigens in the thymus. Humans with AIRE mutations and mice with engineered ablation of Aire develop multiorgan autoimmunity and infertility. To determine the immune targets contributing to infertility in male Aire-deficient (-/-) mice, Aire-/- or wild-type (WT) males were paired with WT females. Aire-/- males exhibited dramatically reduced mating frequency and fertility, hypogonadism, and reduced serum testosterone. Approximately 15% of mice exhibited lymphocytic infiltration into the testis, accompanied by atrophy, azoospermia, and reduced numbers of mitotically active germ cells; the remaining mice showed normal testicular morphology, sperm counts, and motility. However, spermatozoa from all Aire-/- mice were defective in their ability to fertilize WT oocytes in vitro. Lymphocytic infiltration into the epididymis, seminal vesicle, and prostate gland was evident. Aire-/- male mice generated autoreactive antibodies in an age-dependent manner against sperm, testis, epididymis, prostate gland, and seminal vesicle. Finally, expression of Aire was evident in the seminiferous epithelium in an age-dependent manner, as well as in the prostate gland. These findings suggest that Aire-dependent central tolerance plays a critical role in maintaining male fertility by stemming autoimmunity against multiple reproductive targets.

Abstract We conducted a multiinstitutional, placebo-controlled phase IIB trial of the lignan secoisolariciresinol diglucoside (SDG) found in flaxseed. Benign breast tissue was acquired by random periareolar fine needle aspiration (RPFNA) from premenopausal women at increased risk for breast cancer. Those with hyperplasia and ≥2% Ki-67 positive cells were eligible for randomization 2:1 to 50 mg SDG/day (Brevail) versus placebo for 12 months with repeat bio-specimen acquisition. The primary endpoint was difference in change in Ki-67 between randomization groups. A total of 180 women were randomized, with 152 ultimately evaluable for the primary endpoint. Median baseline Ki-67 was 4.1% with no difference between arms. Median Ki-67 change was −1.8% in the SDG arm (P = 0.001) and −1.2% for placebo (P = 0.034); with no significant difference between arms. As menstrual cycle phase affects proliferation, secondary analysis was performed for 117 women who by progesterone levels were in the same phase of the menstrual cycle at baseline and off-study tissue sampling. The significant Ki-67 decrease persisted for SDG (median = −2.2%; P = 0.002) but not placebo (median = −1.0%). qRT-PCR was performed on 77 pairs of tissue specimens. Twenty-two had significant ERα gene expression changes (<0.5 or >2.0) with 7 of 10 increases in placebo and 10 of 12 decreases for SDG (P = 0.028), and a difference between arms (P = 0.017). Adverse event incidence was similar in both groups, with no evidence that 50 mg/day SDG is harmful. Although the proliferation biomarker analysis showed no difference between the treatment group and the placebo, the trial demonstrated use of SDG is tolerable and safe.