Maria Isabel Mello Martins

Aim: Feline mammary carcinoma (FMC) shares strong histological and molecular similarities with human breast cancer, facilitating elucidation of pathophysiological tumor mechanisms, identification of prognostic indicators, discovery of biomarkers and therapeutic targets. This study characterized the proteome of two FMC subtypes, aggressive cribiform (CRIB) and less aggressive tubulopapillary (TUB), using formalin-fixed, paraffin-embedded (FFPE) tissue samples to identify aggressive biomarkers.Main methods: Twenty-one FMC samples (CRIB, n = 12, TUB, n = 9) were retrieved from a histological biobank. Following deparaffinization and digestion, proteomes were analyzed by shotgun nLC-MS/MS proteomics, Data were evaluated by principal component analysis (PCA), dendrogram, partial least squares discriminant analysis (PLS-DA), VIP score and Volcano plot (FDR < 0.05). Venn diagrams, String DB and ShinyGO for pathway enrichment.Key findings: A mong 976 identified proteins,113 were differentially abundant between subtypes. PCA showed partial overlap, but 15 key proteins were identified, with 14 elevated in CRIB. CRIB-specific proteins included AHNAK nucleoprotein, glycerol kinase, myosin-2, and an uncharacterized protein, TUB-specific proteins included glucosylceramidase beta 3, β2-glycoprotein-1, SEA domain-containing protein, and EF-hand domain-containing protein. T-complex protein 1 subunit alpha and reticulon-3 (both human breast cancer poor prognosis markers) iwere common to both subtypes. Enriched pathways involved metabolic, immune, and signaling networks.Significance: FFPE-based proteomics effectively identifies subtype-specific biomarkers and pathways, offering translational insights into FMC and human breast cancer.

The skeletal system plays essential roles during development, such as protecting vital organs, supporting muscles, enabling movement, and storing minerals. By the end of gestation, it also assumes a hematopoietic function through bone marrow activity. Despite its importance, data on skeletal development in dogs remain scarce. This study described the formation, chronology, and growth of the skeletal system in canine embryos and fetuses at different gestational stages. Uteri from pregnant bitches were collected during spaying campaigns and cesarean sections at the Veterinary Hospital of UEL. After exposing the embryos and fetuses, crown-rump length (CRL) was measured, and the individuals were divided into three groups: early (up to 20 days), mid (21-40 days), and late (41-60 days), with six individuals per group. Group I embryos underwent histological analysis using hematoxylin and eosin (H&E) staining to identify pre-osseous structures. Skeletons from Groups II and III were stained with Alizarin Red and Alcian Blue and subjected to radiographic examination. From day 27, ossification centers were observed in the frontal bone, humerus, and femur. Over time, new centers emerged in the mandible, ribs, vertebrae, and other long bones. As gestation progressed, these centers expanded toward the epiphyses; although, regions such as the patella and vertebral extremities remained cartilaginous. These findings contributed to the understanding of normal skeletal development in dogs and support future studies on congenital skeletal disorders.

The yolk sac (YS) is a multifunctional and essential structure for early maternal-fetal interactions, playing roles in nutrient supply, organogenesis, immune modulation, and hematopoiesis. Unlike most species, dogs maintain the YS until the end of pregnancy, yet little is known about its functional activity, particularly in later stages. Animal models are crucial for exploring maternal-fetal processes and offering insights into comparative development, including in humans. This study aimed to analyze the YS structure in canine embryos and fetuses at early (up to 20 days, n = 6), middle (21–40 days, n = 6), and late (41–60 days, n = 6) stages. Gestational age was estimated using crown-rump length (CRL) and external anatomy. Samples from the university’s Veterinary Hospital and neutering campaigns in Londrina (Paraná, Brazil) were fixed in 10% formaldehyde for morphological analysis (H&E, PAS, and Picrosirius red) or 4% paraformaldehyde for OCT4 immunostaining (CEUA/UEL protocol number 029.2022). Histologically, the YS showed a trilaminar structure with blood islands in the intermediate layer surrounded by mesenchyme. Within these islands, undifferentiated cellular niches (OCT4+) were identified. During gestation, the YS changed from a thin, smooth, pale-red membrane to a wrinkled structure with narrowed ends and a thickened central region, resembling a "whale tail." The red color, indicative of vascularization, intensified with blood island enlargement. Collagen secretion, particularly type I, progressively increased around the blood islands. Glycoprotein deposits persisted until late gestation, as shown by PAS staining. These findings highlight the structural, secretory, and reserve roles of the canine YS throughout pregnancy.

ProAKAP4 is a protein precursor of AKAP4 present in the principal piece of the flagellum, associated with sperm motility. It has been described in several species, but its concentration has never been measured in sex-sorted sperm. Thus, the study aimed to test the proAKAP4 as a suitable sperm quality biomarker for Bos indicus X-sorted sperm. To achieve the study’s goal, 14 semen straws from different bulls were evaluated, six X-sorted and eight non-sorted. Sperm kinetics and morpho-functional evaluations were carried out, in addition to the concentration of proAKAP4. The characteristics evaluated were compared by the t-test and correlated by the Pearson’s correlation. A difference was found for total motility (P = 0.014), rapid sperm (P = 0.020), and sperm area (P = 0.013) between the non-sorted and X-sorted sperm. The proAKAP4 concentration was higher in the X-sorted sperm (X-sorted sperm: 67.54 ng/106 spermatozoa; non-sorted sperm: 29.76 ng/106 spermatozoa), but it was not correlated to the kinetics or morpho-functional characteristics evaluated. However, the proAKAP4 in non-sorted sperm showed a correlation to sperm total and progressive motility, rapid sperm, and sperm membrane integrity. The study concluded on the unreliability of the proAKAP4 as a biomarker to indicate sperm quality in the X-sorted sperm.

Background: Differentiation of the genital system occurs through a chronological sequence of events involving the determination of chromosomal, gonadal, and phenotypic sex. Disorders of sexual development (DSDs) include chromosomal sex abnormalities, gonadal abnormalities, and phenotypic variations. The purpose of this case report was to demonstrate the clinical signs and diagnosis by histopathology, immunohistochemistry, and karyotyping of a DSD in a French Bulldog (XX, male).Case: A 7-month-old phenotypically female French Bulldog with a history of clitoral hypertrophy and no signs of puberty is reported. The animal presented with a 3 cm micropenis protruding from the vulvar labia, causing discomfort and constant licking. Pelvic radiography confirmed the presence of a penile bone, supporting the differential for a disorder of sexual development (DSD). There were no abnormalities of the vaginal canal or urethral orifice. The animal underwent exploratory celiotomy to remove 2 uterine horns and their respective gonads, followed by clitoridectomy via episiotomy. Sex reversal was confirmed by histology, as seminiferous tubules and bilateral hypoplastic epididymides were identified on ovarian topography. Immunohistochemistry of gonadal tissues was positive with antibodies to progesterone and estrogen receptors, but negative with polyclonal androgen receptors and prostate-specific antigen. Karyotyping revealed XX sex chromosomes.Discussion: This dog was diagnosed as an XX DSD male (DSD 78, testicular XX) due to the presence of abdominal testes, uterus, predominantly female external genitalia with vulva and micropenis, and female sex chromosomes. Six cases of DSD have recently been reported in phenotypically female French Bulldogs due to the presence of clitoral hypertrophy or a micropenis with an atypical location. The increase in cases in this breed may be due to its popularity and increased consanguinity. Animals with clinical suspicion of DSD show signs during puberty and most of these animals tend to have a history of externalized structure in the vulva without signs of urethral and vaginal canal obstruction. In animals with ambiguous genitalia and DSDs, the estrous cycle may be normal, absent, or irregular. Because of the discomfort in the clitoral region, clitoridectomy is the 1 st line of treatment to prevent self-mutilation and future cases of vaginitis, and gonadectomy is also performed to prevent future uterine disorders and gonadal and mammary carcinogenesis. It is not uncommon for XX male animals to be unilateral or bilateral cryptorchids, in this case, both testes were in the abdominal cavity, and the animal had a normal uterus. Several alternative procedures can be performed for a better understanding of DSDs, namely: histopathology; immunohistochemistry or hormone dosage; karyotyping; and molecular analyses. These tests are important to determine the genetic sex of an animal and to confirm its concordance with the phenotypic sex. There is no evidence of interference of specific genes in the phenotypic expression of XX males, suggesting that the factors involved in their occurrence are still unknown. The hereditary nature of this disorder has not been ruled out and there are currently no laboratory tests available to identify carriers. It is recommended that cases of DSD be thoroughly investigated using complementary examinations to ensure accurate categorization of each disorder. Keywords: clitoral hypertrophy, ambiguous genitalia, immunohistochemistry, cytogenetics, sexual dimorphism.

Globalization and habitat destruction pose a significant threat to wildlife felids. Even though conservation banks for genetic materials have been created, the sperm cryopreservation with minimal cell damage is still a great challenge. Thus, this study aimed to compare the effects of two commercial extenders with different concentrations of alternative cryoprotectants on thawed sperm quality of domestic cats. Five adult cats were anaesthetized (using a combination of 40 μg/kg medetomidine associated to 5 mg/kg ketamine), and the semen was collected by electroejaculation (electrical stimulation of 2–3 V). Semen samples were evaluated for sperm characteristics (kinetics, morphology, membrane integrity and morphometry). Subsequently, they were sorted into two aliquots and centrifuged. The aliquots were added to a commercial extender containing 3% glycerol and 2% methylformamide (extender I) or 2% glycerol and 3% methylformamide (extender II), frozen, thawed (37°C/30 s) and reevaluated. Comparatively, the sperm kinetics and membrane integrity of fresh semen were higher (p < .002) than frozen samples in extender I and II. Total and progressive motility were lowest in the thawed samples. However, the subjective analysis indicated high sperm motility, since the kinetics evaluation was impaired by the low cell number in the thawed samples. There were no differences in sperm morphology between the groups. In the sperm morphometric analysis, a significant difference (p = .04) was identified in the length of the intermediate piece in extender II samples compared with fresh and extender I. Thus, it can be concluded that although the concentrations tested did not maintain the kinetic parameters and membrane integrity of spermatozoa after thawing, the extender with a lower concentration of glycerol was less toxic for maintaining the midpiece length.

Cystic endometrial hyperplasia (CEH)–pyometra complex is the most common uterine infection in adult and elderly bitches and can cause renal dysfunction. The aim of this study was to measure and compare urinary creatinine, urea, symmetric dimethylarginine (SDMA), urinary protein–creatinine ratio (UPC), measurement of systolic blood pressure (SBP), and Doppler velocimetry of renal arteries in patients with CEH–pyometra complex before and after an average of 6 months of treatment, evaluating the possibility of the changes persisting. The evaluation was conducted at two moments: M1 (at the diagnosis of CEH–pyometra, n = 36) and M2 (after an average of six months of treatment, n = 16). For the control group, eight bitches with no changes in blood tests or history of conditions underwent Doppler ultrasound evaluation of the renal arteries. At both M1 and M2, we measured creatinine, urea, and serum SDMA, UPC, SBP, and Doppler ultrasound of the renal arteries. Patients were evaluated according to the following groups: azotemic (AZO) and non‐azotemic (NAZO), and open and closed cervix pyometra. The parameters were compared between animals present in both moments presented as M1R (bitches that were in M1 and M2) and M2. Statistical significance was considered when p < .05. The medians found for creatinine in M1 were as follows: 1.15 mg/dL, being 1.8 mg/dL for AZO (12/36) and 0.95 mg/dL for NAZO (24/36); and in M2: 0.85 mg/dL (16/16), being 1.15 mg/dL for AZO (4/16) and 0.8 mg/dL for NAZO (12/36). For urea, in M1 it was 36 mg/dL (32/36), with AZO being 103 mg/dL (11/32) and 33 mg/dL in NAZO (21/32); and in M2 32 mg/dL (16/ 16), being 29 mg/dL for AZO (4/36), and 31 mg/dL for NAZO (3/15). The median SDMA measured in M1R was 17 μg/dL (15/16), with AZO being 31 μg/dL (3/15), and NAZO being 16.5 μg/dL (12/15); and in M2, SDMA was 12 μg/dL (16/16), with AZO being 12.5 μg/dL (4/16), and NAZO being 12 μg/dL (12/16). The median UPC measured in M1 was 1.15 (10/36), with AZO being 0.25 (1/10), and NAZO being 1.38 (9/10); and in M2, it was 0.2 (13/16), being 0.1 in AZO (4/13), and 0.2 (9/16) in NAZO. For SBP, in M1, it was 118 mmHg (30/36), with AZO being 102 mmHg (10/30) and 133 mmHg in NAZO (20/30); and in M2 142.5 mmHg (12/16), being 155 mmHg for AZO (4/12), and 140 mg/dL for NAZO (8/12). When comparing animals with open and closed cervixes, a difference was found between SDMA measurements (p = .001). There was a distinction between PI and RI of the left and right kidneys consecutively (p = .007; p = .033; p = .019; p = .041). Correlations found in M1: SDMA × PI RIM DIR (r = 0.873; p = .002), SDMA × PSV RIM ESQ (r = 0.840; p = .004), SDMA × EDV RIM ESQ (r = 0.675; p = .046). With this study, we conclude a return to normality of renal biomarkers and clinical parameters after six months. Yet, there is a persistence of Doppler velocimetric measurements between the two moments. Thus, this parameter is not suitable for identifying and classifying chronic kidney injury in bitches with pyometra.