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B-mode Ultrasonography, Doppler, Elastography and Contrast Enhanced Ultrasound (CEUS) of the Reproductive System of Dogs and Correlation to Seminal Quality

Grant number: 22/04907-0
Support Opportunities:Scholarships in Brazil - Doctorate
Effective date (Start): April 01, 2023
Effective date (End): February 28, 2025
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal Investigator:Marcus Antônio Rossi Feliciano
Grantee:Luiz Paulo Nogueira Aires
Host Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil

Abstract

The objective is to perform a study evaluating testicles, epididymis and prostate of dogs using B-Mode ultrasonography, Doppler, Elastography and Contrast-Enhanced Ultrasound (CEUS) in order to correlate the parameters of the ultrasound techniques with the seminal quality in the canine species, establishing sonographic parameters that could predict seminal quality. Thirty dogs will be evaluated, age ranging from 1 to 9 years old weighing up to 25kg. Patients will be divided in groups according to age: G1 - up to 3 years old; G2 - 3 to 6 years old; G3: 6 to 9 years old. In B-Mode, the testes, epididymis and prostate will be evaluated regarding size, volume, contours, shape, echogenicity and echotexture. Doppler evaluation of the testicular artery will be performed in five different portions: proximal supratesticular; middle supratesticular, distal supratesticular, marginal; intratesticular. For the Doppler evaluation of the prostate, the cranial prostatic artery will be evaluated. Parameters of Doppler evaluation will be the spectral morphology of each portion and Doppler velocimetric values: peak systolic velocity (PSV) end diastolic velocity (EDV), mean maximum cycle velocity (TaMax), minimum mean cycle velocity (TaMin), resistivity index (RI) and pulsatility index (PI). The elastographic evaluation will be performed qualitatively, obtaining an elastogram of the relative stiffness of the testes and prostate, as well as quantitatively, obtaining shear velocity values. Testicular stiffness will be assessed in three distinct portions named according to anatomic landmarks: free testicular portion (PTL); mediastinal testicular portion (PTM); epididymal testicular portion (PTE).Elastographic evaluation of the epididymis will be performed on the head, body and tail. For prostatic stiffness, tissue stiffness values will be obtained in different regions: ventral, central and dorsal. The CEUS of the testes and prostate will be performed using the contrast medium SonoVue® (Bracco, São Paulo, SP, Brazil), administered intravenously. They will be evaluated qualitatively and, later, quantitatively, determining the perfusion parameters of the evaluated organs: number of pixels (NP), peak intensity (PI in %), time to peak intensity (Tp in s), time transmission average (TmT) and the area under the curve (AUC). The semen of the animals will be collected through digital manipulation. After collection, evaluations will be carried out regarding their macroscopic (volume, appearance, color) and microscopic aspects. The sperm concentration will be calculated by dilution in a Neubauer chamber (1:20). Computed aided seminal analysis (CASA system) will be performed to obtain data on sperm kinetics: total motility (MT); progressive motility (PM); average path velocity (VAP); straight line velocity (VSL); curvilinear velocity (VCL); amplitude of lateral displacement of the head (ALH); beat frequency (BCF); straightness of the average path (STR); linearity of the curvilinear path (LIN); wobble coefficient (WOB); and rapid cells (RAP). The analysis of sperm morphology will be performed by wet mount preparation in differential interferential contrast microscopy. Furthermore, tests of plasma membrane integrity, acrosomal membrane integrity and mitochondrial membrane potential will be performed through the association of three fluorescent probes (propidium iodide, FITC-PSA and JC-1) and the sperm DNA integrity will be evaluated by acridine orange by epifluorescence microscopy. The variables studied will be submitted to a principal component analysis, trying to identify which variables affect the sperm composition in relation to the data considered normal for dogs and identify two components with the variables that allow to explain at least 70% of the variability. Of these, a partial least squares regression will be performed, trying to relate the set of predictors studied with one or more responses of the sperm parameters, in the form of orthogonal regressio

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