Fig 1.
Testicular artery in three different locations and transducer orientation.
A. Spermatic cord: Supratesticular artery (SA); B. Close to the tail of epididymis: Capsular artery (CA) and, C. Within the parenchyma: Intratesticular artery (IA). Modified image from the book “Ultrasonic imaging and animal reproduction: Color-Doppler ultrasonography,” O.J. Ginther.
Fig 2.
Cross section of the spermatic cord with the three different ultrasound modalities.
A. B-Mode ultrasound (grey scale); B. Colour Doppler ultrasound of the spermatic cord´s vessels; C. Pulse Doppler ultrasound of the supratesticular artery within the spermatic cord. D. Display of the equipment used to measure a cardiac cycle using pulse Doppler. Three Doppler velocities calculated by the ultrasound equipment’s algorithm package.
Table 1.
Doppler parameters assessed in this study.
Fig 3.
Flow cytometry detection of sperm viability and membrane integrity (A) and DNA fragmentation Index (DFI) (B) of stallion sperm.
(A) Representative density plot graphic with the three subpopulations of sperm: Live sperm (unstained spermatozoa), the Yo-Pro-1 positive cells (sperm in the early stages of apoptosis) and spermatozoa stained with Ethidium (dead sperm). (B) Representative histograms of DFI (%) (Sperm Chromatin Structure assay).
Table 2.
Sperm motility and kinematic parameters of fertile and subfertile stallions.
Table 3.
Measurements of Total Testicular Volume and DSOe with B-Mode ultrasonography and parameters of sperm production and testicular efficiency in fertile and subfertile groups.
Table 4.
Blood flow parameters of the supratesticular artery in fertile and subfertile stallions.
Fig 4.
Mean values and standard error of the mean of Doppler parameters measured in the capsular artery in fertile and subfertile stallions.
(A) Doppler Indices: PI (Pulsatility Index: PSV-EDV/TAMV) and RI (Resistive Index: PSV-EDV/PSV). (B) Doppler Velocities: PSV (Peak Systolic Velocity; EDV (the End Diastolic Velocity); and the TAMV (Time Average Maximum Velocity). (C) Total Arterial Blood Flow (TABF: TAMV x A; where A: πr2 is the area of the cross section of the CA). (D) Total Arterial Blood Flow rate (TABF rate: TABF/TTV x 100). TABF and TABF rate were calculated as indicators of testicular perfusion. Values with different superscripts differ (a. b; p < 0.05). (S1 Dataset and S1 File).
Table 5.
Blood flow parameters of the Intratesticular artery in fertile and subfertile stallions.
Fig 5.
The principal component analysis assay (PCA) applied to Doppler parameters to identify fertile and subfertile stallions as described in materials and methods.
Fertile stallions (1) are characterized by high values of TABF ratio, TABF, VDF and TAMV (right lower quadrant). Subfertile stallions (2) are categorized by high values of PI and RI in the supratesticular artery (TC: Testicular Cord) and in the capsular artery (PT) (left upper quadrant). (S1 File).
Fig 6.
Graphical representation of the results obtained by flow cytometry in fertile vs subfertile stallions.
A. Membrane integrity and sperm viability of sperm: Percentage of intact sperm at T0, T24 and T48 h of refrigeration. B Membrane integrity and sperm viability of sperm: Percentage of dead sperm at T0, T24 and T48 h of refrigeration. C. Mitochondrial activity: percentage of active mitochondria on sperm at T0, T24 and T48 h. D. DNA fragmentation Index of sperm at T0, T24 and T28h. a, b; p ≤ 0.05.
Table 6.
DNA fragmentation index (%DFI) of individual fertile (stallion1-7) and subfertile stallions (stallion 8–10) at T0, T24 and T48h of refrigeration.
Table 7.
Correlations obtained by Spearman test of non-parametric values of Doppler parameters and those of flow cytometry at T0, T24 and T48h.
P < 0.05.
Fig 7.
Receiver operating characteristic (ROC) curves for the parameters PSV, EDV, TAMV.
AUC: Area under the Curve.
Fig 8.
Receiver operating characteristic (ROC) curves for the parameters TABF and the diameter of the capsular artery.
AUC: Area under the Curve.
Table 8.
The area under the curve (AUC) of receiver operating characteristic (ROC) curves and Youden’s J statistics were applied to Doppler velocities of the capsular artery and TABF and artery diameters to investigate the value of the proposed variables as indicators of sperm quality.