Aplicación de la ultrasonografía en la evaluación andrológica del semental ovino

Abstract

Andrological evaluation in rams is a necessary practice in semen donor males in breeding and genetic improvement centers and in farm rams to know and optimize their reproductive capacities and, ultimately, to increase fertility rates. In this Doctoral Thesis, several experiments were carried out in this context, which gave rise to three scientific articles. In the first paper, the method for calculating testicular volume was optimized by choosing the most accurate tool for obtaining testicular measurements and proposing a new formula based on these measurements. In addition, the relationship between testicular volume and sperm production was studied. For this purpose, the testes were measured in vivo and post mortem by comparing classic and electronic calipers (integrated in the ultrasonography machine) and these measurements were used to calculate the testicular volume, which was related to the accurate testicular volume calculated by water displacement. Ultrasonographic measurements showed lower standard deviations and allowed proposing a new formula (Testicular volume = testicular depth x testicular width x testicular length x 0.61) which was the only one that did not show statistically significant differences with the accurate testicular volume. In addition, and over a period of 4 years, rams from an artificial insemination center were grouped into three groups depending on the frequency of semen collection and the time of the year, all in the context of the usual operation of the center. In these males, total testicular volume (TTV), serum testosterone concentration (T) and total sperm production (SPERM) from two consecutive ejaculates on the same day were measured and the correlations between these parameters were established. Sperm production showed strong positive and significant correlations with total testicular volume (r = 0.587, non-breeding season -NBS-; r = 0.773, breeding season, 10 ejaculates/week -BS10-) and with testosterone (r = 0.684, breeding season, 4 ejaculates/week -BS4-) and, based on this, three formulas were established to predict sperm production in relation to the season and semen collection frequency; SPERM (x109 spz) in NBS = 0.02226 x TTV (mL) – 2.977; SPERM (x109 spz) in BS10 = 0.01858 x TTV (mL) – 2.873; SPERM (x109 spz) in BS4 = 0.80850 x T (ng/mL) + 2.819. In the second paper, the measurement of testicular blood flow in the ram was standardized by studying the histological, morphological and ultrasonographic characteristics of the testicular artery in three different zones: proximal, middle and distal. Arterial diameter and hemodynamics including peak systolic (PSV) and end-diastolic velocities (EDV), resistive index (RI) and pulsatility index (PI), total artery blood flow (TABF) and its ratio (TABF rate) were measured. The artery was characterized on ultrasound imaging by high tortuosity, tending to less sinuosity as it approaches the testis. In addition, a significant increase in arterial diameter, PSV, RI and PI was observed in the proximal supratesticular region and a progressive increase in EDV, TABF and TABF rate as the artery advanced towards the testicle. On the other hand, histological studies were performed in the same areas for the morphometric study of the different anatomical structures of the arterial wall, as well as an immunohistochemical analysis to know the expression of α-smooth muscle actin (α-SMA) and vimentin. Histomorphological studies showed a larger diameter of the vasa vasorum in the tunica adventitia and of the arterial wall in the proximal zone, whereas the outer arterial and inner luminal diameters were larger in the distal zone. Finally, the expression of α-SMA and vimentin, markers of tunica media smooth muscle cells, was higher in the proximal zone compared with the middle and distal zones, implying greater vascular wall thickness at that level. Between the two points with more consistent and representative (middle and distal), and although there are differences between them, due to the technical difficulty of measuring flow at that distal point (impossibility of measurement in some animals) and in order to design a more applicable, reproducible and fast protocol in the field, the middle supratesticular zone is recommended. In the third paper, ultrasonography was applied in the evaluation of testicular function and the correlations between ultrasonography measurements and seminal quantity and quality parameters using different semen collection frequencies as a practical scenario were calculated. For this, rams were subjected to three consecutives semen collection frequencies, of increasing intensity, each lasting one month (abstinence -AF-, standard -SF-, intensive -IF-). At the end of each period, testicular volume, parenchyma echotexture and testicular artery hemodynamics were evaluated. Likewise, on refrigerated semen samples (15 °C, 6h), production, motility, viability, apoptosis and mitochondrial activity were evaluated. In addition, correlations between ultrasonographic and semen parameters were also established. Our results showed a significant increase in echotexture and testicular artery hemodynamics (PSV, RI, PI, TABF) when semen collection frequency was intensified. In contrast, sperm motility and sperm functionality decreased significantly. Regarding the correlations between parameters, moderate and positive correlations were found between ultrasonography parameters (echotexture and Doppler parameters) and sperm motility parameters in SF. In the second part of the work, a fertility study was carried out in the field in which the days of sexual abstinence prior to the experimental semen collection for elaboration of doses for cervical inseminations of previously synchronized ewes were considered. In this study, the highest fertility rates were obtained from males that had 2 to 5 days of abstinence between semen collections. In summary, andrological evaluation, through a multiparametric study that includes a complete ultrasonographic evaluation by measuring testicular volume with the ovine-specific ItraULE formula and testicular hemodynamics in the middle supratesticular area, in addition to semen evaluation, is a very useful tool to categorize rams according to their semen production and to maximize their reproductive performance in order to increase their fertility and therefore the productivity in the farms.