Serum Extracellular Vesicles Cargo Approach in Bitches with Mammary Tumors.

This study investigated serum extracellular vesicles (EVs) in bitches with mammary neoplasms, in order to understand their size, shape, and concentration, as well as their association with tumor malignancy. Thirty bitches were categorized into control (n = 10), mammary tumor grades I and II (GI, n = 13), and grade III (GII, n = 7). Serum was separated from blood collected during mastectomy, and EVs were isolated using size exclusion chromatography. The analysis revealed no significant differences in EV concentrations among groups, with similar concentrations for control, GI, and GII. Ninety-one proteins were identified in EV-enriched samples, with six showing varied abundance across groups. Notably, keratin 18 was highly abundant in GI, while sushi domain-containing protein, EvC ciliary subunit 2, and the joining chain of multimeric IgM and IgA were increased in GII. Additionally, protocadherin 17 and albumin were upregulated in both GI and GII. ROC curves identified potential biomarkers for differentiating tumor grades. Enrichment pathway analysis revealed AFP gene upregulation in the GI. Mass spectrometry proteomics data were deposited in Mendeley Data. The study provides valuable insights into serum EV characterization in bitches, suggesting keratin 18 and protocadherin 17 as potential biomarkers for canine mammary neoplasia, with implications for future diagnostic and therapeutic strategies.

The interaction between seminal plasma, sperm, and endometrium in inter- and intra-species breeding in equids.

In brief: In some instances, extra-species breeding in equids is more successful than intraspecies breeding; however, little is known about the immunomodulatory effect of donkey semen and seminal plasma on the mare's endometrium. This study compared the mare uterine inflammatory response during extra- and intraspecies breeding. Abstract: Anecdotal experience suggests horse mares have less post-breeding inflammation and better fertility when bred with donkeys. This study aimed to compare the post-breeding inflammatory response of mares exposed to donkey and horse semen and seminal plasma and evaluate the proteome and metabolome of donkey and horse sperm and seminal plasma. Uterine edema, intrauterine fluid accumulation, polymorphonuclear neutrophils on cytology, and concentrations of progesterone, and pro- and anti-inflammatory cytokines (IL1A, IL1B, IL4, IL6, CXCL8, IL10) were assessed pre- and post infusion of semen and seminal plasma (donkey and horse). The metabolome and proteome were analyzed by LC-MS/MS. Mare cycles bred with horse semen had a greater progesterone concentration than those bred with donkey semen at 8 days post ovulation (P = 0.046). At 6 h post infusion, the inflammatory response due to the donkey semen tended to be lower (P = 0.074). Donkey seminal plasma had anti-inflammatory properties compared to horse semen and seminal plasma, as determined by fewer neutrophils on uterine cytology (P < 0.05). Horse semen resulted in greater concentrations of IL6 and lesser concentrations of IL1B (P < 0.05). PGE1, PGE3, and lactoferrin concentrations were significantly more abundant in donkey sperm and seminal plasma. Prostaglandins play an important role in immunomodulation and might contribute to the response triggered in interspecies breeding. In conclusion, breeding horse mares with donkey semen induces similar post-breeding endometritis as observed with horse semen. Donkey seminal plasma results in a lower post-infusion inflammatory response compared to other combinations in the immediate post-breeding.

Proteomics approach reveals urinary markers for early pregnancy diagnosis in buffaloes.

This study aimed to compare urine proteomics from non- and pregnant buffaloes in order to identify potential biomarkers of early pregnancy. Forty-four females underwent hormonal ovulation synchronization and were randomly divided into two experimental groups: inseminated (n = 30) and non-inseminated (n = 14). The pregnant females were further divided into two groups: pregnant at Day 12 (P12; n = 8) and at Day 18 (P18; n = 8) post-ovulation. The non-pregnant group was also subdivided into two groups: non-pregnant at Day 12 (NP12; n = 7) and at Day 18 (NP18; n = 7). Urine was collected from all females on Days 12 or 18. The samples were processed for proteomics. A total of 798 proteins were reported in the urine considering all groups. The differential proteins play essential roles during pregnancy, acting in cellular transport and metabolism, endometrial remodeling, embryonic protection, and degradation of defective proteins. We suggest that some proteins from our study can be considered biomarkers for early pregnancy diagnosis, since they were increased in pregnant buffaloes. SIGNIFICANCE: Macromolecules have been studied for early pregnancy diagnosis, aiming to increase reproductive efficiency in cattle and buffaloes. Direct methods such as rectal palpation and ultrasonography have been considered late. Thus, this study aimed to compare urine proteomics from non- and pregnant buffaloes to identify potential biomarkers of early pregnancy. The differential proteins found in our study play essential roles during pregnancy, acting in cellular transport and metabolism, endometrial remodeling, embryonic protection, and degradation of defective proteins. We suggest that these proteins can be considered possible biomarkers for early pregnancy diagnosis since they were increased in the pregnant buffaloes.

Can blood progesterone concentration identify non-pregnant buffaloes to support oestrous resynchronization?

This study compared the plasma progesterone concentrations from pregnant and non-pregnant buffaloes to identify non-pregnant females and submit cows earlier to oestrous resynchronization. Forty-four multiparous mix-breed Murrah buffaloes were selected for the study. The cows were subjected to hormonal oestrous synchronization and separated into 4 groups, P12 (pregnant, n = 8) and P18 (n = 8) at 12 and 18 days post-insemination; NP12 (non-pregnant, n = 7) and NP18 (n = 7) at 23 and 29 days after the onset of synchronization, respectively. The embryos and blood were collected, and the plasma was separated for centrifugation and used to determine progesterone concentration. Progesterone concentration was higher in P18 than P12 (p = .02) and NP18 groups (p < .001). The steroid was also increased in the P12 group compared with NP12 (p = .031). There was no difference between NP12 and NP18 (p = .906). We conclude that the plasma progesterone concentration can be an alternative to identify earlier non-pregnant buffaloes, advancing the oestrous resynchronization or natural service to improve productivity.

Proteomics Approach of Rapamycin Anti-Tumoral Effect on Primary and Metastatic Canine Mammary Tumor Cells In Vitro.

Rapamycin is an antifungal drug with antitumor activity and acts inhibiting the mTOR complex. Due to drug antitumor potential, the aim of this study was to evaluate its effect on a preclinical model of primary mammary gland tumors and their metastases from female dogs. Four cell lines from our cell bank, two from primary canine mammary tumors (UNESP-CM1, UNESP-CM60) and two metastases (UNESP-MM1, and UNESP-MM4) were cultured in vitro and investigated for rapamycin IC50. Then, cell lines were treated with rapamycin IC50 dose and mRNA and protein were extracted in treated and non-treated cells to perform AKT, mTOR, PTEN and 4EBP1 gene expression and global proteomics by mass spectrometry. MTT assay demonstrated rapamycin IC50 dose for all different tumor cells between 2 and 10 µM. RT-qPCR from cultured cells, control versus treated group and primary tumor cells versus metastatic tumor cells, did not shown statistical differences. In proteomics were found 273 proteins in all groups, and after data normalization 49 and 92 proteins were used for statistical analysis for comparisons between control versus rapamycin treatment groups, and metastasis versus primary tumor versus metastasis rapamycin versus primary tumor rapamycin, respectively. Considering the two statistical analysis, four proteins, phosphoglycerate mutase, malate dehydrogenase, l-lactate dehydrogenase and nucleolin were found in decreased abundance in the rapamycin group and they are related with cellular metabolic processes and enhanced tumor malignant behavior. Two proteins, dihydrolipoamide dehydrogenase and superoxide dismutase, also related with metabolic processes, were found in higher abundance in rapamycin group and are associated with apoptosis. The results suggested that rapamycin was able to inhibit cell growth of mammary gland tumor and metastatic tumors cells in vitro, however, concentrations needed to reach the IC50 were higher when compared to other studies.

Calcium chloride combined with dimethyl sulphoxide for the chemical sterilization of dogs.

The aim of this study was to evaluate the effect of intratesticular injection of calcium chloride (CaCl2 ) combined with dimethyl sulphoxide (DMSO) as a chemical sterilization in dogs. Twelve dogs were divided into two groups: the treated group (n = 6), in which 15 mg/kg of a 7.5% CaCl2 solution combined with 0.5% DMSO was injected into each testicle (volume range 1.0-4.76 ml); and a control group (n = 6) that received the same volume/kg of 0.9% sodium chloride solution (NaCl). Semen characteristics pre- and post-treatment were evaluated. Serum testosterone concentration was determined before the injection (D-1) and at 15 (D15), 30 (D30) and 60 (D60) days after intratesticular injection. Testicle sizes and local pain were evaluated for 7 consecutive days (D1 to D7) and at D15, D30 and D60 after injection. At D60, testicle histological evaluation was performed after orchiectomy. No pain was observed by testicular palpation, with the exception of one dog in the treated group; this dog then received analgesic therapy. An increase in testicular volume was evident within 24 hr after treatment, followed by gradual reduction for 3 weeks. Five of 6 dogs from the treated group presented azoospermia at D15; the remaining dog presented at D30. There was no significant difference in testosterone concentrations in the treated group during the experimental period. Histological evaluation showed testicular degenerative lesions, especially at the proximal and middle portions. The results indicated that one injection of 7.5% CaCl2 combined with 0.5% DMSO into each testis is a viable alternative for canine sterilization.

Testicular histological evaluation and serum testosterone concentrations of bulls after chemical castration with calcium chloride / Avaliação histológica e das concentrações séricas de testosterona de touros após a castração química com cloreto de cálcio

Orchiectomized bulls have advantages in the meat quality and ease of handling. Chemical castration is an option for surgical castration and the sclerosing agents can be administered into the testicular or epididymis parenchyma. These agents have a lower incidence of complications than surgery, especially when associated with dimethylsulfoxide (DMSO), which has anti-inflammatory action and increases the absorption of other drugs. Thus, this study aimed to evaluate the effect of a single intratesticular injection of calcium chloride solution associated with DMSO for the chemical sterilization of bulls. Twenty-four young adult bulls were utilized, distributed into 3 groups (G20, G30 and G40, n = 8/group), according to the calcium chloride concentration (20, 30 and 40%), in 10mL volume. Serum concentrations of testosterone, body weight, testicular volume and ecotexture, clinical signs and behavior and were evaluated for 45 days. Thus, the animals were orchiectomized and testicles were assessed histologically. There were no changes in body weight, decreased serum testosterone concentrations (except G30), signs of scrotal sensitivity or changes in behavior over the period. However, there was significant increase in testicular volume, especially on the 2nd and 3rd day after treatment, with values returning to the value initials at 15 days. Testicular adherence and firm consistency were observed during orchiectomy. Ultrasound examination revealed a loss of integrity of the median raphe, with cavity formation and an alteration of the testicular echotexture. In the histological evaluation, coagulation necrosis of seminiferous tubules and interstitial cells was observed, mainly in the medial portion in all groups. Some animals presented total absence of tubular formations in all the studied groups, being the effects of greater intensity in the G40. Additionally, pronounced edema was noted in all groups, especially in G40. Inflammatory infiltrate, fibroplasia and neovascularization were found to be predominantly discrete. Based on the conditions used in this study, we conclude that calcium chloride associated with DMSO can be used as a method of chemical sterilization in bovines.(AU)

Bovinos orquiectomizados apresentam vantagens na qualidade da carne e facilidade no manejo. A quimioesterilização é uma opção à castração cirúrgica e os agentes esclerosantes podem ser administrados no parênquima testicular ou epidídimo. Estes produtos possuem menor incidência de complicações, comparados a cirurgia, especialmente quando associados ao dimetilsulfóxido (DMSO), que apresenta ação anti-inflamatória e aumenta a absorção de outros fármacos. Assim, este estudo teve como objetivo avaliar o efeito de uma única injeção intratesticular de solução de cloreto de cálcio associado com 0,5% de DMSO para a esterilização química de bovinos. Vinte e quatro touros adultos jovens foram utilizados, distribuídos em 3 grupos (G20, G30 e G40, n = 8/grupo) de acordo com a concentração de cloreto de cálcio (20, 30 e 40%), em um volume de 10mL. Foram avaliadas as concentrações séricas de testosterona, peso corporal, volume e ecotextura testicular, sinais clínicos e comportamento por 45 dias. A seguir, os animais foram submetidos à orquiectomia e os testículos avaliados histologicamente. Não foram observadas alterações do peso corporal, diminuição das concentrações de testosterona sérica (exceto no G30), sinais de sensibilidade escrotal ou alterações no comportamento no período avaliado. Porém, houve aumento significativo do volume testicular, especialmente nos 2º e 3º dia após o tratamento, com valores retornando aos iniciais aos 15 dias. Aderência e consistência firme dos testículos foram achados observados durante a orquiectomia. O exame ultrassonográfico revelou perda de integridade da rafe mediana, com formação de cavidades e alteração da ecotextura testicular. Na avaliação histológica, verificou-se necrose de coagulação de túbulos seminíferos e células intersticiais acentuada, principalmente, na porção medial em todos os grupos, sendo que em alguns animais havia ausência total das formações tubulares em todos os grupos estudados, sendo os efeitos de maior intensidade no G40. Além disso, edema foi acentuado em todos os grupos, principalmente em G40. Infiltrado inflamatório, fibroplasia e neovascularização foram achados predominantemente discretos. Com base nas condições utilizadas neste estudo, conclui-se que o cloreto de cálcio associado com o DMSO pode ser utilizado como um método de esterilização química em bovinos.(AU)

Evaluation of neonatal vitality and blood glucose, lactate and cortisol concentrations in foals of the Paint Horse breed / Avaliação dos padrões de vitalidade neonatal, glicemia, lactato e cortisol em potros da raça Paint Horse

Clinical evaluations of newborns, as well as defining the therapeutic decision, represent significant challenges to the veterinarian. This study aimed to evaluate neonatal vitality using the modified Apgar score and blood glucose, lactate, and cortisol concentrations in newborn equines. Twenty foals of the Paint Horse breed born by normal delivery were evaluated during the first 48 hours of life. The modified Apgar score was determined at birth and 10 minutes later, with values ranging from 0 to 10. Blood samples for the laboratory tests were collected at birth and at 4, 8, 12, 16, 20, 24, 36, and 48 hours. The mean Apgar score was significantly lower at birth than at 10 minutes (p < 0.001). Blood glucose concentrations increased significantly between birth (98.90 ± 35.97 mg/dL) and 8 hours (127.90 ± 27.15 mg/dL), stabilizing after the first 12 hours (148.95 ± 29.59 mg/dL). Serum lactate concentrations showed a significant decrease across the time points tested, with the highest value occurring at birth (5.48 ± 2.17 mmol/L) and stabilizing after 24 hours (2.76 ± 1.31 mmol/L). Cortisol concentrations significantly decreased (p < 0.01) between 4 (10.46 ± 7.46 µg/dL) and 48 hours (5.21 ± 3.35 µg/dL), with the highest value occurring 4 hours after birth. The common occurrence of laboratory abnormalities in healthy newborn foals reinforces the need to conduct regular clinical examinations and to develop laboratory reference values for different breeds and ages.(AU)

A avaliação clínica dos recém-nascidos, bem como a definição da conduta terapêutica adotada representam expressivos desafios ao Médico Veterinário. O objetivo deste estudo foi avaliar a vitalidade neonatal pelo escore de Apgar modificado, glicemia, lactatemia e cortisol em neonatos equinos. Para tal, foram utilizados 20 potros da raça Paint Horse, nascidos em eutocia, durante as primeiras 48 horas de vida. O escore de Apgar modificado foi realizado ao nascimento e 10 minutos após, atribuindo-se notas de 0 a 10. As colheitas das amostras de sangue, para a realização das análises laboratoriais, ocorreram nos momentos ao nascimento, 4, 8, 12, 20 16, 20, 24, 36 e 48 horas de vida. A média do escore de Apgar ao nascimento foi significativamente menor que aos 10 minutos (p<0,001). A glicemia aumentou significativamente entre o nascimento (98,90 ± 35,97 mg/dL) e às 8 horas (127,90 ± 23 27,15 mg/dL), com estabilização após as primeiras 12 horas (148,95 ± 29,59 mg/dL). A concentração sérica de lactato apresentou decréscimo significativo entre os momentos estudados, sendo o maior valor encontrado ao nascimento (5,48 ± 2,17 mmol/L), com estabilização a partir das 24 horas (2,76 ± 1.31 mmol/L). As concentrações de cortisol, reduziram significativamente (p<0,01), entre as 4 (10,46 ± 7,46 µg/dL) e as 48 horas (5,21 ± 3,35 µg/dL), sendo o maior valor encontrado as 4 horas após o nascimento. A ocorrência comum de anormalidades laboratoriais em potros recém-nascidos, aparentemente saudáveis, reforça a necessidade da realização de exames clínicos regulares e o desenvolvimento de valores laboratoriais de referência, em diferentes raças e idades.(AU)

Aspectos morfológicos da cérvice de ovelhas / Morphological aspects of ovine cervix

The major barrier to accomplishment of artificial insemination in ewes is the anatomy of the cervix combined with low viability and survival of frozen ram semen. Thus, the aim of the present study was to evaluate the morphology of the cervix in ewes. Eighty-one specimens were obtained from a slaughterhouse and the following characteristics were evaluated: type of external cervical os, cervical size, integrity and interdigitation of cervical rings, gross characteristics and size of the ovaries (follicles and corpus luteum), and the time spent to pass an insemination catheter through the lumen of the cervix. The most frequent was the slit type cervical opening and grade II internal ring arrangement. The time spent to pass the insemination catheter through the cervix was 6 minutes and 15 seconds, and the dye was spread throughout the cervical lumen reaching the uterus in most sheep. The average values of cervical opening diameter and cervical length were 0.68cm and 4.4cm respectively. Ovarian follicular activity was found in 75 percent of the ewes. A positive correlation was established between some of the variables. We conclude that cervical opening size is influenced by estrogen, slit type cervical os and grade III cervical ring arrangement; also, the greater length of the cervix was associated with greater difficulty to pass the insemination catheter.

A principal barreira para a aplicação da inseminação artificial transcervical é a anatomia cervical aliada à baixa viabilidade e sobrevida do sêmen ovino congelado. Assim, este experimento teve como objetivo estudar a morfologia da cérvice de ovelhas. Para tal, foram adquiridas, em matadouro, 81 peças do trato reprodutor de ovelhas, nas quais se avaliou a morfologia cervical, segundo as seguintes características: tipo de óstio cervical, mensuração do tamanho da cérvice, integralidade e interdigitação entre os anéis das pregas cervicais, tamanho e características macroscópicas dos ovários (folículos e corpo lúteo) e tempo da passagem do aplicador de sêmen pela cérvice. Foi identificada maior frequência do tipo liso de abertura da cérvice e integralidade e interdigitação dos anéis grau II. O tempo de passagem do aplicador pela cérvice foi em média de seis minutos e 15 segundos, sendo que o corante aplicado se difundiu por todo o canal, atingindo o útero na maioria das ovelhas. A média do diâmetro da abertura cervical foi de 0,68cm e o comprimento cervical de 4,4cm. A atividade folicular ovariana foi encontrada em 75 por cento das fêmeas. Foi possível estabelecer várias correlações entre as variáveis. Conclui-se que o tamanho da abertura cervical sofre influência estrogênica, e o tipo liso de abertura cervical, o grau III de integralidade e interdigitação dos anéis e o maior comprimento da cérvice foram associados à maior dificuldade de passar o cateter no lúmen cervical.