Cushioned and high-speed centrifugation improve sperm recovery rate but affect the quality of fresh and cryopreserved feline spermatozoa.

The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires a small volume of the insemination dose with a high concentration, not easily reached with the actual frozen technique in this species. Centrifugation is widely used to concentrate spermatozoa for several purposes, but this process is detrimental to spermatozoa. This study verified the effects of conventional and cushioned centrifugation on fresh and cryopreserved feline spermatozoa. To this, semen was collected from 20 toms, grouped in seven pools and diluted. After dilution, the pools were divided into two aliquots, the first used for centrifugation on fresh semen, and the second, after freezing, on cryopreserved semen. Centrifugation regimens were: conventional at 500×g, conventional at 1000×g, and cushioned (iodixanol) at 1000×g. The sperm recovery rate was calculated for the three centrifugation regimens, and sperm kinematics, membrane and acrosome integrity, and plasma membrane stability on viable spermatozoa were assessed as endpoints. The data reported in this study showed that the centrifugation at 500×g resulted in negligible effects on both fresh and cryopreserved spermatozoa, but the lower recovery rate (62.4 ± 3.1 % and 60.2 ± 1.6 %, respectively) underlines the loss of a large proportion of spermatozoa, unfavourable in a species with small total sperm ejaculated. On the other hand, the centrifugation at 1000×g improved the recovery rate (86.9 ± 4.3 % and 89.8 ± 2.4 % in fresh and cryopreserved samples, respectively), but was more deleterious for feline spermatozoa, especially in cryopreserved samples (i.e. total motility of 40.7 ± 5.4 % compared with 57.2 ± 9.8 % in cryopreserved uncentrifuged samples, P < 0.05), resulting in artificial insemination doses of lower quality. The recovery rate in cushioned centrifugation appeared less efficient, likely due to the small volume of feline samples, which makes difficult the separation of sperm pellet and cushioned fluid. Interestingly, in cryopreserved samples centrifuged at 1000×g the number of viable spermatozoa with membrane destabilization (31.3 ± 3.2 %) was greater than uncentrifuged (4.1 ± 0.7 %, P < 0.05) and those centrifuged at 500×g (9.8 ± 1.3 %, P < 0.05), suggesting modifications induced by the cryopreservation amplifies centrifugation sublethal damage on feline spermatozoa. Cushioned centrifugation on cryopreserved samples showed kinematics similar to uncentrifuged samples, but higher viable spermatozoa with membrane destabilization (37.4 ± 3.4 % vs 4.1 ± 0.7 %; P < 0.05). In felines, g-force is crucial for sperm recovery rate during centrifugation, with better results at 1000×g; on the other hand, greater g-forces could have a significant impact on the quality of feline insemination dose, especially in cryopreserved samples.

Ultrasound-Based Technologies for the Evaluation of Testicles in the Dog: Keystones and Breakthroughs.

Ultrasonography is a valuable diagnostic tool extensively used in the andrology of human and domestic animals, including dogs. This review aims to provide an overview of various technologies based on ultrasound, from the basic B-Mode ultrasonography to the more recent advancements, such as contrast-enhanced ultrasonography (CEUS) and ultrasound elastography (UEl), all of which are utilized in the evaluation of canine testicles. The review outlines the principles behind each of these technologies and discusses their application in assessing normal and abnormal testicular conditions. B-mode canine testicular ultrasonography primarily focuses on detecting focal lesions but has limitations in terms of objectivity. Other technologies, including Doppler ultrasonography, B-Flow, and CEUS, allow for the characterization of vascular patterns, which could be further measured using specific applications like spectral Doppler or quantitative CEUS. Additionally, ultrasound elastography enables the assessment of parenchyma stiffness both qualitatively and quantitatively. These ultrasound-based technologies play a crucial role in andrology by providing valuable information for evaluating testicular function and integrity, aiding in the identification of pathological conditions that may impact the health and quality of life of male dogs.

Validation of the volumetric flow cytometry for bovine sperm concentration.

Sperm concentration is a stronghold of the andrological evaluation and the production of insemination doses. The use of haemocytometers, although considered the gold standard, is difficult to apply in field conditions because it is subjective and time-consuming. The present study was designed to validate the volumetric flow cytometry (volFC) in order to estimate bovine sperm concentration, comparing it with the performances of haemocytometer, NucleoCounter, and flow cytometry with the use of fluorospheres. Compared with other methods, volFC appeared less affected by large dilution of the sample, with similar concentrations calculated in the range of dilution 1:200-1:800. Using volFc the population detected on the basis of morphological criteria and fluorescence of DNA better represents the real concentration of sperm in the sample. The volFC showed high repeatability compared with the haemocytometer (coefficient of variation 1.85% and 4.52%, respectively) and stable performances with cryopreserved samples, with negligible effects of the medium components. The present study showed that volFC is as accurate and precise as other techniques to estimate sperm concentration in bovine fresh and frozen semen, but it is less affected by operative conditions, such as sample dilution. The possibility to quantify sperm functional subpopulations by volFC could potentially implement the study of the relationship between sperm attributes and fertility.

Stiffness estimated by strain elastography reflects canine testicular spermatogenesis and histology.

Ultrasound elastography was proposed for the evaluation of testicular focal lesions, but no studies verified the agreement between the whole histological architecture of the testis and the stiffness measured by elastography. The present study explored the use of strain elastography in the evaluation of testis with normal or abnormal spermatogenesis, classified based on epididymal sperm attributes, and the consistency between elastographic parameters and the testicular histological feature. Strain elastography was performed during the routine andrological examination in 22 dogs presented for elective orchiectomy. Epididymal sperm attributes and testicular histology were analyzed after orchiectomy. Based on the epididymal sperm characteristics, testes were classified according to normal or abnormal spermatogenesis, and strain elastographic attributes were compared between groups. Possible correlations between strain elastography and histological features were also explored. Consistent with the literature in humans, testes with abnormal spermatogenesis were stiffer (mean strain elastographic index 3.6 ± 0.6) compared with normal testes (mean strain elastographic index 1.9 ± 0.2; P < 0.01). The strain elastographic index was negatively correlated with the area occupied by seminiferous tubules (Pearson's rho = -0.716; P = 0.0003), the mean diameter (Pearson's rho = -0.742; P = 0.0002), and thickness of the seminiferous tubule (Pearson's rho = -0.728; P = 0.0002). Surprisingly, no correlations were found between the area occupied by connective tissue in histological sections and elastographic attributes, suggesting that the increased stiffness was not related to the increased amount of connective tissue. This study demonstrated that strain elastography could be used to support the andrological examination, but measurements should be acquired in specific regions to be reliable.