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(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography-mass spectrometry (LC-MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition.
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Hypoglycemia is the most common metabolic alteration in the clinical routine of newborn dogs, acting as a predictor of mortality in these patients. The neonatal dog shows hepatic insufficiency and homeostatic mechanisms not yet fully developed, with limited glycogen reserves and limited capacity to perform glycogenolysis and gluconeogenesis. These physiological particularities make newborn dogs particularly susceptible to hypoglycemia when of fasting, even for short periods. Several maternal and neonatal factors may be related to a higher risk of developing hypoglycemia in neonates. This paper reviews glycemic homeostasis, the pathophysiology of neonatal hypoglycemia, the main causes involved and the diagnostic and therapeutic approaches to this condition.
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The aim was to compare the UF proteomics of pregnant and non-pregnant buffalo during early pregnancy. Forty-four females were submitted to hormonal estrus synchronization and randomly divided into two groups: pregnant (n = 30) and non-pregnant (n = 14). The pregnant group was artificially inseminated and divided into a further two groups: P12 (n = 15) and P18 (n = 15). Conceptus and uterine fluid samples were collected during slaughter at, respectively, 12 and 18 days after insemination. Of all the inseminated females, only eight animals in each group were pregnant, which reduced the sample of the groups to P12 (n = 8) and P18 (n = 8). The non-pregnant group was also re-divided into two groups at the end of synchronization: NP12 (n = 7) and NP18 (n = 7). The UF samples were processed for proteomic analysis. The results were submitted to multivariate and univariate analysis. A total of 1068 proteins were found in the uterine fluid in both groups. Our results describe proteins involved in the conceptus elongation and maternal recognition of pregnancy, and their action was associated with cell growth, endometrial remodeling, and modulation of immune and antioxidant protection, mechanisms necessary for embryonic maintenance in the uterine environment. SIGNIFICANCE: Uterine fluid is a substance synthesized and secreted by the endometrium that plays essential roles during pregnancy in ruminants, contributing significantly to embryonic development. Understanding the functions that the proteins present in the UF perform during early pregnancy, a period marked by embryonic implantation, and maternal recognition of pregnancy is of fundamental importance to understanding the mechanisms necessary for the maintenance of pregnancy. The present study characterized and compared the UF proteome at the beginning of pregnancy in pregnant and non-pregnant buffaloes to correlate the functions of the proteins and the stage of development of the conceptus and unravel their processes in maternal recognition of pregnancy. The proteins found were involved in cell growth and endometrial remodeling, in addition to acting in the immunological protection of the conceptus and performing antioxidant actions necessary for establishing a pregnancy.
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Búfalos , Proteômica , Animais , Feminino , Gravidez , Antioxidantes/metabolismo , Búfalos/metabolismo , Endométrio/metabolismo , Secretoma , Útero/metabolismoRESUMO
There is a high perinatal mortality rate in dogs, estimated at 20%, and one of the leading causes of this rate is hypoglycaemia. Therefore, we aimed to evaluate the efficacy of a hypercaloric supplement containing vitamins and amino acids in newborn puppies presenting hypoglycaemia at birth. Ninety-nine pups were divided into four groups: normoglycaemic caesarean section (NORMOCS), hypoglycaemic caesarean section supplemented with the hypercaloric (SUPLCS), hypoglycaemic caesarean section supplemented with glucose (GLICCS) and eutocic delivery (EUT). We evaluated the following parameters glycaemia, Apgar score, neurological reflexes and rectal temperature of neonates at the following moments 5 min (M5), 30 min (M30) and 60 min (M60) after birth. Brachycephalic dogs were 73.3% (22/30) of caesarean sections (c-sections). The puppy's average glycaemia represented about 90% of the maternal glycaemia, while 15.1% (14/99) of the neonates had hypoglycaemia (<90 mg/dL) at M0 and 46.5% (44/99) at M60. Only four neonates had glycaemia below 40 mg/dL at M30 but without showing any clinical signs. The puppy's fasting while waiting for the intraoperative period and the dam's anaesthetic recovery was considered risk factors for hypoglycaemia. There was no difference in mean blood glucose levels or vitality parameters among puppies from the SUPLCS and GLICCS. In conclusion, the hypercaloric supplement can be used as a replacement for glucose in hypoglycaemic puppies and it can also bring nutritional benefits for the puppy. The prepartum glycaemia of the dam is an important parameter to be measured, and the appropriate management of it reduces the chances of the puppies being born with hypoglycaemia.
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This study aimed to describe the secretome of mesenchymal stem cells derived from feline adipose tissue (AD-MSCs) and compare the effects of different culture conditions on AD-MSC proteomics using a shotgun approach. Adipose tissue was collected from 5 female cats and prepared to culture. Conditioned media was collected at third passage, in which the cells were cultured under 4 conditions, normoxia with fetal bovine serum (N + FBS), hypoxia with FBS (H + FBS), normoxia without FBS (N - FBS), and hypoxia without FBS (H - FBS). Then, the secretome was concentrated and prepared for proteomic approaches. Secretomes cultured with FBS-free medium had more than twice identified proteins in comparison with the secretomes cultured with FBS. In contrast, hypoxic conditions did not increase protein amount and affected only a small proteome fraction. Relevant proteins were related to the extracellular matrix promoting environmental modulation, influencing cell signaling pathways, and providing a suitable environment for cell proliferation and maintenance. Moreover, other proteins were also related to cell adhesion, migration and morphogenesis. Culture conditions can influence protein abundance in AD-MSC secretome, and can give also more specificity to cell and cell-free treatments for different diseases.
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Células-Tronco Mesenquimais , Secretoma , Gatos , Animais , Feminino , Proteômica , Tecido Adiposo/metabolismo , Células-Tronco Mesenquimais/metabolismo , Obesidade/metabolismo , Hipóxia/metabolismo , Proliferação de Células , Células Cultivadas , Diferenciação CelularRESUMO
Multiparametric analysis by flow cytometry solves one of the major problems in sperm evaluation, the inability to test multiple attributes simultaneously in a single cell, which would increase the precision to predict fertility potential since several sperm parameters are tested. The association of fluorochromes and compounds conjugated to fluorochromes in multiparametric sperm analysis is well-established in microscopy techniques. However, these techniques are subjective and limit the assessment in small cell numbers, thereby harming analytic accuracy. Therefore, the current study aimed to present new possibilities for assessing the integrity and stability of the sperm plasma membrane, acrosome status, mitochondrial potential, and superoxide anion production in the mitochondrial matrix in only 2 cytometric assays using cytometers equipped with 2 and 3 lasers. For this, human semen samples collected by masturbation and selected by the swim-up technique were divided into 3 treatments: T0 (flash-frozen semen), T50 (flash-frozen semen + fresh semen, V: V), and T100 (fresh semen) for the validation of the multiparametric protocols by flow cytometry. For both protocols, sperm percentage with positive stain for all fluorophores differed significantly between treatments. The determination coefficients presented values close to 1, which validated objective, sensitive, rapid, and reproducible methodologies. Therefore, we concluded that the results reflect the status of analyzed structure, enabling a more accurate diagnosis of male infertility that has become an increasingly prevalent worldwide setback due to exposure to a variety of environmental toxicants.
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Corantes Fluorescentes , Sêmen , Humanos , Masculino , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Espermatozoides , Acrossomo/metabolismo , Motilidade dos Espermatozoides , CriopreservaçãoRESUMO
OBJECTIVES: The aim of this study was to perform neonatal clinical assessments at birth to identify newborn kittens at risk according to type of delivery, thus allowing immediate intervention and increasing their chances of survival. METHODS: This study compared Apgar scores, reflexes and clinical parameters (temperature, weight, blood glucose and peripheral oxygen saturation [SpO2]) between eutocic neonates and those delivered by emergency cesarean section. The animals were evaluated at birth and after 10 and 60 mins. RESULTS: Thirty-two neonates were evaluated, with 19 animals in the eutocic group (EG) and 13 animals in the cesarean group (CG). When comparing groups, CG neonates had significantly lower Apgar scores (P <0.0001), lower SpO2 (P = 0.0535), higher blood glucose (P = 0.0009), reduced reflexes (P <0.0001) and lower respiratory rates (P <0.0001) at birth and after 10 and 60 mins than EG neonates. Apgar scores positively correlated with parameters such as heart rate, reflex score, SpO2 and weight. The mortality rate in evaluated newborns was 15.6% (5/32). The early mortality rate (0-2 days old) was 80% (4/5) and the late mortality rate (3-30 days old) was 20% (1/5). CONCLUSIONS AND RELEVANCE: This study showed lower vitality in cats delivered by emergency cesarean section than in those delivered through eutocic birth. In general, neonates delivered by cesarean section have greater depression and low vitality at birth and may require advanced resuscitation procedures. The evaluations carried out in this study identified newborns with low vitality and those requiring advanced resuscitation, thus allowing immediate intervention. Apgar and reflex scores for feline neonates were suggested. Newborn-specific clinical assessment with these feline vitality scores allows the identification of at-risk neonates. Care immediately after birth increases the chance of survival among these patients.
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Glicemia , Cesárea , Animais , Índice de Apgar , Gatos , Cesárea/métodos , Cesárea/veterinária , Feminino , Recém-Nascido , Saturação de Oxigênio , Gravidez , ReflexoRESUMO
This study aimed to characterize the proteome of spermatozoa and seminal plasma of 4 purebred dogs (Golden Retriever, Great Dane, Bernese Mountain Dog, and Maremmano-Abruzzese Sheepdog). The ejaculate of 13 dogs was collected, and sperm characteristics were subjectively evaluated. Seminal plasma and sperm cells were separated and prepared individually for mass spectrometry. Data were evaluated by univariate and multivariate statistical analysis. A total of 162 proteins were identified, 47 in spermatozoa, 109 in seminal plasma, and 6 in both samples. Serum albumin in spermatozoa and tubulin alpha-3E chain, acrosin binding protein, and tubulin alpha-3 chain in plasma seminal were statistically relevant. Serum albumin and acrosin binding protein improve the sperm capacitation, acrosome reaction, and seminal quality. The tubulin family proteins are related to structural cell organization and flagella movement, and their presence in seminal plasma may be related to sample handling. According to cluster formation, a high association was observed among Bernese Mountain Dog and Great Dane, Golden Retriever, and Maremmano-Abruzzese Sheepdog for sperm proteins. For seminal plasma proteins, Bernese Mountain Dog, Great Dane, and Maremmano-Abruzzese Sheepdog were related. Further studies on breed-specific proteins in the semen of purebred dogs need to be performed to clarify its fertility roles. SIGNIFICANCE: For the first time spermatozoa proteins of dogs are described. The comparison of spermatozoa and seminal plasma proteins of four purebred dogs were performed. These results supporting that differences in semen protein profile of different canine breeds exist, which can improve the biotechnologies of reproduction in this species.
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Acrosina , Proteômica , Acrosina/metabolismo , Animais , Cães , Masculino , Melhoramento Vegetal , Proteômica/métodos , Sêmen/metabolismo , Proteínas de Plasma Seminal/metabolismo , Albumina Sérica/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Seminal plasma has several components that protect the sperm cells and assist in the fertilization process. In contrast, the exact role carried out by seminal plasma during the cooling of canine semen remains controversial. Moreover, concerning the long estrus period, the possibility to store chilled semen at 5°C for more than 72 hours and maintain good sperm quality for additional inseminations could increase fertilization rates. Thus, this study aimed to evaluate the seminal plasma influence on quality and oxidative stress of the extended canine semen stored at 5°C for 7 days. Three ejaculate pools from eight healthy dogs were collected by digital manipulation of the penis. The sperm kinetics, sperm vitality (eosin/nigrosin stain), integrity of plasma and acrosomal membranes, morphology, superoxide and hydrogen peroxide production, mitochondrial potential, lipid peroxidation, and oxygen reactive species production (induced and spontaneous thiobarbituric acid [thiobarbituric acid reactive substances, TBARS] assay) were evaluated every 48 hours (M0, M48, M96, and M168) until 7 days (168 hours) in cooled extended (TRIS egg yolk) semen of dogs at 5°C with (+SP) or without (-SP) autologous seminal plasma. No statistical difference was found for sperm kinetics in cooled samples with +SP and -SP during the experimental time period, except for the progressive motility of +SP samples that was higher at M48 than M96 (p = 0.023). The seminal plasma did not influence any other evaluated sperm characteristics. Finally, our results demonstrated that the presence or lack of seminal plasma during cooling the semen of dogs does not influence sperm quality at 5°C. Moreover, the components of the semen extender may contribute to maintaining good sperm quality and low reactive oxygen species production during the long period of the dog's semen cooling, even after semen centrifugation.
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Preservação do Sêmen , Animais , Cães , Gema de Ovo , Masculino , Espécies Reativas de Oxigênio , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides , EspermatozoidesRESUMO
This study aimed to evaluate the correlation of seminal plasma IGF-I and total proteins from peripubertal bulls with semen characteristics and onset of puberty. Sixteen dairy Gyr bulls were separated into early (n = 8) and regular (n = 8) groups, according to the onset of puberty. Semen was monthly collected by electroejaculation from 14 to 26 months of age, and the onset of puberty was retrospectively determined (17.0 ± 1.6 and 19.2 ± 1.5 months, to early and regular group, respectively). Five time points were evaluated (day -60, day -30, day 0, day 30 and day 60 days of puberty), being day 0 considered as beginning of puberty. Scrotal circumference and body weight were also assessed. Semen characteristics were evaluated, the seminal plasma was separated by centrifugation and total protein and IGF-I concentrations were determined. There was no difference between groups to concentration of the seminal plasma total proteins, but we found an interaction between group and age. Seminal plasma IGF-I concentrations were not different between early and regular groups; thus, the data were combined for analysis. Combined IGF-I concentrations were positively correlated with sperm motility and concentration, age, body weight and scrotal circumference. Negative correlation was found between IGF-I concentration and total sperm defects. Increased IGF-I was observed in day -30 and day 0 of puberty in early and regular groups, respectively. Seminal plasma total proteins are involved in peripubertal modifications and IGF-I from Zebus dairy bulls can influence the seminal characteristics and the growth factor increase occurs concomitantly with body growth but cannot be used to define puberty bulls earlier.
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Bovinos/fisiologia , Fator de Crescimento Insulin-Like I/análise , Proteínas/análise , Sêmen/química , Animais , Peso Corporal , Masculino , Escroto/anatomia & histologia , Maturidade Sexual/fisiologia , Motilidade dos EspermatozoidesRESUMO
The objective of this study was to investigate the effects of seasonality and phase of the estrous cycle on ovarian and oocyte morphology in domestic cats kept in a tropical region. The ovaries from 23 queens were collected after elective ovariohysterectomy at different stages of the estrous cycle and were classified according to the season upon collection (spring/summer vs. fall/winter). Based on the findings, we concluded that queens in tropical regions experience no seasonal influence on oocyte quality, but there is a minimal effect on ovarian morphology, with an increase in ovarian length during proestrus/estrus in the spring/summer period.
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Gatos , Oócitos , Ovário , Clima Tropical , Animais , Gatos/fisiologia , Ciclo Estral , Feminino , Oócitos/fisiologia , Ovário/fisiologia , Estações do AnoRESUMO
Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P < 0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.
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Semen contains several proteins that are important to fertilization and to identify reproductive failures. There are proteins that are specie-specific expressed, although differs among several breeds. This article provides experimental data describing the protein profile of seminal plasma and spermatozoa of four healthy purebred dogs: Golden Retriever (n=3), Bernese Mountain Dog (n=4), Great Dane (n=3), and Maremmano-Abruzzese Sheepdog (n=3), housed at São Paulo state, Brazil. Semen samples were collected by manual stimulation of the penis in a presence of a teaser bitch, when possible. The seminal plasma and sperm cells were separated by centrifugation and prepared for mass spectrometry. The gene ontology annotation of the proteins found is described. This is the first time that proteomic profile of the semen of purebred dogs is described. These data are a valuable resource to improve the biotechnologies of reproduction applied to canid species.
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In human neonates, when the umbilical cord is kept intact postpartum, blood continues to flow to the neonate, but this procedure might be difficult in dogs owing to a shorter umbilical cord and several neonates in a litter. However, it might be possible to detach the placenta and keep the umbilical cord intact, allowing residual blood to flow to the puppies. This study compared the effects of clamping versus no clamping of the umbilical cord in dogs born by cesarean section on neonatal vitality. The puppies were assessed by Apgar and reflex scores. Fifty puppies delivered from 16 bitches were randomly allocated to receive immediate umbilical cord clamping (n=25) or no clamping for at least 3 min after the onset of breathing (n=25). The puppies were assessed during the first 5 min of life and 10 min after the first assessment. The no clamping group showed significantly higher Apgar scores (second assessment, P<0.01) and reflex scores (first and second assessments, P<0.05) than the clamping group, implying higher vitality in the no clamping group. The differences observed between the moments (first versus second assessment) of each group was significant (P<0.01), revealing higher vitality in the second assessment. The results suggest that keeping the umbilical cord intact for at least 3 min after the onset of breathing may contribute to increased vitality in puppies delivered by cesarean section without any negative consequences.
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Animais Recém-Nascidos/fisiologia , Cesárea/veterinária , Cães/cirurgia , Cordão Umbilical , Animais , Índice de Apgar , Cesárea/métodos , Feminino , Ligadura/veterinária , Masculino , Parto , Circulação Placentária , GravidezRESUMO
The objective of this study was to evaluate seminal plasma proteins from cattle and buffalo (Bubalus bubalis), to identify differences between related species. Sixteen buffaloes and 16 cattle between 30 and 60 months of age were used. Semen collection was performed by electroejaculation, followed by macroscopic and microscopic subjective analyses. After analysis, the samples were centrifuged at 800 g for 10 min, and the supernatant (seminal plasma) was recentrifuged at 10,000 g for 30 min at 4°C. The total protein concentration was determined by the Bradford method, and the proteins were digested in solution for mass spectrometry (nLC-MS/MS). Multivariate statistical analysis was used to evaluate the proteomics results by non-hierarchical clustering the considering exponentially modified protein abundance index (emPAI). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used for clustering. Proteomics identified 78 proteins, and multivariate analysis showed 4 that were over-expressed in buffaloes (cystatin C, prosaposin, peptide YY and keratin type II cytoskeletal 5) and 9 in cattle (spermadhesin-1, seminal plasma protein PDC-109, ribonuclease 4, metalloproteinase inhibitor 2, acrosin inhibitor 1, seminal ribonuclease, C-type natriuretic peptide, angiogenin-1 and osteopontin). Among the proteins identified in seminal plasma, the C-type natriuretic peptide and metalloproteinase inhibitors were described for the first time in buffaloes. Some protease inhibitors were found over-expressed in buffaloes, and important proteins in seminal plasma of cattle were not identified or were found at lower expression levels in buffaloes, which can contribute to reproductive performance in this species.
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Búfalos , Bovinos , Proteoma , Proteínas de Plasma Seminal/análise , Animais , Masculino , Sêmen/química , Especificidade da EspécieRESUMO
Sexed sperm in dogs is of interest because of being polytocous, and as a result, the greatest number of offspring of the same sex can improve the market, although few studies assessing sperm sexing have been performed in this species. The present study, therefore, was conducted to evaluate the effects on sperm quality and the effectiveness of three discontinuous density gradients to separate dog sperm containing X and Y chromosomes. Thirty ejaculates from ten adult dogs were collected by digital manipulation of the penis. Cells were separated using gradients of Percoll® and Percoll® associated with Nycodenz® or Ficoll. The cells were evaluated for motility by the CASA system (Computer-Aided Semen Analyzer) and for concentration and recovered sperm concentration (after centrifugation), sperm morphology, plasma and acrosomal membrane integrity, and mitochondrial function pre- and post-centrifugation. The percentage of sperm containing X and Y chromosomes was also evaluated pre- and post-centrifugation by quantitative real-time PCR (qPCR). The use of the Ficoll gradient resulted in the greatest sperm quality after centrifugation; however, no sperm enhancement containing X or Y chromosome occurred with use of any of the methods (Percoll® 54.8 ± 1.9 compared with 45.2 ± 1.9; Percoll® associated with Nycodenz® 53.2 ± 2.0 compared with 46.8 ± 2.0; and Percoll® associated with Ficoll 55.0 ± 1.5 compared with 45.0 ± 1.5 for the percentages of cells containing the X and Y chromosomes, respectively). Thus, it was concluded that the technique of sexing dog sperm using density gradients was not effective for commercial application.
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Centrifugação com Gradiente de Concentração/veterinária , Cães , Pré-Seleção do Sexo/veterinária , Espermatozoides/fisiologia , Animais , Separação Celular/métodos , Masculino , Cromossomos Sexuais , Pré-Seleção do Sexo/métodosRESUMO
The objective of the present study was to describe the proteins from the seminal plasma of buffalo and correlate these proteins with sperm motility. Ejaculates from sixteen Murrah buffalo were used. Semen collection was performed by electroejaculation, and the ejaculate was evaluated by macroscopic (volume) and microscopic analysis (subjective motility and vigor, as well as sperm concentration). After the analysis, the samples were centrifuged (800g for 10â¯min and 10,000 for 30â¯min at 4⯰C), and the supernatant (seminal plasma) was used to determine total protein concentration by the Bradford method. Based on total protein concentration, an aliquot (50⯵g) was taken to conduct protein in-solution digestion for nano-LC-ESI-Q-TOF mass spectrometry analysis. Samples were divided into two groups, minimal (little sperm motility) and greater (typical sperm motility), based on non-hierarchical clustering considering motility and emPAI protein value. The data were analyzed by multivariate statistical analysis using principal component analysis (PCA) and partial analysis of minimum squares discrimination (PLS-DA). Forty-eight proteins were detected in the seminal plasma, and fifteen were common to two groups. There were six proteins that were significantly different between the groups. The main functions of proteins in seminal plasma were catalytic and binding activity. Spermadhesin protein, ribonuclease, 14-3-3 protein zeta/delta and acrosin inhibitor were in greater amounts in seminal plasma from the group with greater sperm motility; prosaposin and peptide YY were in greater amounts in the group with little sperm motility. The proteins detected in the greater motility group were correlated with sperm protection, including protection against oxidative stress, lipid peroxidation, protease inhibition and prevention of premature capacitation and acrosome reaction. In the group with little sperm motility, one of the identified proteins is considered to be an antifertility factor, whereas the function of other identified protein is not definitive. Results from the present study add to the knowledge base about the molecular processes related with sperm motility, and these findings can be used for determining potential markers of semen quality.
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Búfalos/fisiologia , Análise do Sêmen/veterinária , Proteínas de Plasma Seminal/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , Animais , MasculinoRESUMO
The aim of this study was to investigate the impact of prolonged storage at 4⯰C on survival of cat preantral follicles (PAFs) pre- and post-vitrification. Ovaries were obtained from 12 queens and transported at 4⯺C within 2-6â¯h. Parts of the ovaries were stored for an additional 24â¯h or 72â¯h. The ovarian cortex was dissected, analyzed for viability (neutral red - NR) and morphology (histology - HE and ultrastructural analysis by TEM) and vitrified. We used 2â¯mm biopsy punches to obtain equal size pieces as the experimental units. After NR assessment, each sample was fixed and embedded in paraffin for HE staining to determine the number of morphologically intact follicles. Another 2â¯mm piece of ovary was subjected to TEM. NR viability assessment and HE results showed a similar tendency with PAF survival postvitrification even after prolonged cooling at 24â¯h and 72â¯h. With TEM, integrity of mitochondria, plasma and basal membranes as well as the presence of pre-granulose cells of PAFs were documented postvitrification for the control group and 24â¯h prolonged storage group, but not after 72â¯h storage. Our results showed that cat PAFs can survive prolonged storage followed by vitrification. The described set of techniques are applicable towards creating a gamete bank for endangered feline species.
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Criopreservação/veterinária , Folículo Ovariano , Vitrificação , Animais , Gatos , Temperatura Baixa , Feminino , Bancos de TecidosRESUMO
This study aimed to report in detail, the technique and challenges of cloacal massage, to collect and evaluate semen from red-winged tinamou (Rhynchotus rufescens) keep in captivity, performed by only one technician. Sixty-four semen collection attempts, from 16 adult males, during breeding season and 16 attempts form these same 16 males in non-breeding season, were performed. Prior to collection, all animals were conditioned to cloacal massage for 6 weeks and the ejaculates were succeed with viable spermatozoa and then, evaluated for feces, urine and mucus contamination, volume, concentration, sperm vigor, motility, morphological defects and acrosome integrity. Semen collection success rate was 63% in breeding season and 2 (5%) samples were discarded by grade 5 contamination. Only 3 ejaculates from 16 tinamou were obtained in non-breeding season. Sperm concentration and acrosome integrity was higher (p = 0.00) in breeding season, and the percentage of total sperm morphological defects, were high in both in breeding and out breeding season. Overall, we concluded that the red-winged tinamou breeding season, is linked to photoperiod (spring and summer), and at this period time, semen can be obtained by cloacal massage collection satisfactorily, allowing its use in reproduction biotechnologies and sperm cryopreservation.
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Criação de Animais Domésticos , Aves/fisiologia , Estações do Ano , Manejo de Espécimes , Animais , Criopreservação/métodos , Masculino , Sêmen , Preservação do Sêmen/métodos , Contagem de Espermatozoides , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologiaRESUMO
PURPOSE: To describe video-assisted ovariohysterectomy (OHE) with two portals access in adult intact queens. METHODS Fifty-two females cats were used. A 4 mm cannula was positioned in the abdomen through an incision close to the umbilicus (first portal), and a pneumoperitoneum was established. A second portal was positioned in the midline of the pre-pubic region. Females were positioned in right lateral recumbency to locate the left ovarian pedicle, and the uterine horn was held by a transcutaneous suture. The pedicle was cauterized and incised. The procedure was then performed on the contralateral ovary. The ovaries were exteriorized from the abdomen, along with the uterus, through the second access point. The uterine body was exposed, fixed and sectioned, and the abdominal incisions were sutured. RESULTS Surgeries were performed in an average of 41.4±14.2 minutes. The main complications included hypotension (7.7%) and subcutaneous emphysema (7.7%), and 13.5% of the surgeries were converted to laparotomy. CONCLUSION Ovariohysterectomy using a video-assisted technique and two access portals is safe, has minimal risks and is effective for the spaying of queens.