Spada, Stefano (2024) Clinical application of multiparametric imaging in the evaluation of the canine reproductive tract. [Tesi di dottorato]

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Abstract

Canine infertility has posed considerable financial challenges to the expanding breeding industry, especially in the past decades. Assessing fertility in dogs may be challenging due to the absence of standardized tests for both males and females. A comprehensive clinical approach to evaluate canine fertility should include an accurate physical and ultrasonographic examination of the reproductive tract, hormonal tests and semen analysis. While B-mode ultrasound (US) is an excellent tool at detecting reproductive conditions, a multiparametric ultrasonographic approach is recommended due to the limited specificity of two-dimensional ultrasound in the differentiation of pathologic conditions or in the monitoring of physiologic reproductive processes. Contrast-enhanced ultrasound (CEUS) emerged as a promising technique involving an intravenous contrast-agent capable of assessing the microvascularization of an organ, resulting a suitable method in distinguishing benign from malignant lesions giving superior information compared to colour and power Doppler ultrasound. The primary focus of the current thesis is to evaluate the application of a multiparametric ultrasonographic approach for both male and female reproductive conditions, including CEUS. Prostatic neoplasia (PN), though uncommon in canines, tends to be more prevalent in castrated dogs. The assessment of prostate malignancies may be challenging due to low specificity of B-mode ultrasonographic signs and to the few information available in literature. CEUS revealed to be a suitable method to differentiate benign from malignant lesions in prostate of intact dogs. The first study of the current thesis was to assess prostatic perfusion in castrated dogs using CEUS. CEUS was performed in 64 neutered dogs, using a 5–7.5 MHz linear transducer with coded harmonic capability, dedicated analytical software, and a second-generation contrast agent, SonoVue. B-mode evaluation was performed to assess mean prostate volume and, subsequently a CEUS examination was undertaken. It was possible to detect the vascular prostatic flow in all specimens and quantitative parameters such as peak perfusion intensity (PPI) and time to peak (TTP) were measured using a dedicated software to analyse contrast enhancement (Qontrast). The parameters were compared with the one from previous studies revealing a higher PPI when compared to intact dogs. CEUS showed promising results indicating potential diagnostic applications for PN diagnosis in neutered dogs. The parameters derived from the present studies exhibited a high standard deviation among specimens, possibly indicative of the considerable variability in size, age, age at castration, and time elapsed since castration. Therefore, another study was undertaken to assess potential variations in prostatic vascularization detected using CEUS in dogs. Image analysis was performed by using a digital software (ImageJ), able in objectivize measurement of tissue characteristics, favouring the detection of changes in echogenicity or vascular blood supply which may not be distinguished by the human eye. Twenty-three neutered dogs underwent B-mode and CEUS of the prostate using the same methodology as the previous study. Videoclips were analysed with both software (Qontrast and ImageJ) and the following parameters were calculated: PPI, TTP and Vascularization Ratio (VR). Correlation tests revealed higher vascularization in younger compared with older dogs (p <0.05) and in smaller compared with larger dogs (p <0.05). Time elapsed since orchiectomy (p >0.05) had no effect on prostatic perfusion. CEUS and the post-processing analysis tool ImageJ allowed analysis of vascular perfusion in all dogs and showed the potential to improve the diagnostic possibilities for andrological examination. The lack of correlation with time elapsed since castration may contribute to the high variability observed in the specimens. Consequently, a preliminary study was conducted to describe vascular regression after at least 3 months since castration, aiming to determine whether vascular regression persists beyond this timeframe. Ten neutered dogs were evaluated twice six years apart using the same techniques (B-mode and CEUS) to detect morphologic and vascular involution of the prostate after castration. The prostate exhibited similarity in terms of morphology and echotexture on B-mode US, minimally decreasing in volume over time. Prostate perfusion significantly decreased in all dogs between T0 and T1, with a PPI reduction from 54.9 to 29.6 % and an increase in TTP from 26.3 to 47 s. These preliminary data suggest that prostatic involution after neutering is not a short-term process and may be of clinical relevance. Another section of the present thesis was conducted in female dogs, aiming to address the knowledge gap concerning uterine and ovarian changes during oestrus using a multiparametric ultrasonographic approach. A study employing high definition (HD) US was conducted to assess the reproductive tract of female dogs during follicular development. Eight females were examined at five different time points during the follicular phase, determined through vaginal cytology and blood progesterone concentrations. US examinations were performed using the ACUSON S2000/SIEMENS device equipped with a multifrequency HD transducer (5.5-18 MHz). The ovarian structures were evaluated and measured throughout the oestrus cycle. A correlation was observed between uterine thickness increase and the rise in ovarian and follicular dimensions. The HD ultrasonography technique, with its excellent image resolution, favoured a more precise characterization of the reproductive structures in female dogs and the changes occurring during the follicular phase of the estrous cycle. In the context of a multiparametric approach to the detection of ovulation in dogs, an additional study was conducted, integrating pulsed wave Doppler and CEUS in the evaluation of follicular development. Eight healthy bitches were enrolled in the study and were evaluated by using B-mode, Pulsed wave Doppler and CEUS at five different timepoints (T1 – T5) of the estrous cycle established by vaginal cytology and serum progesterone concentration. Ovarian vascular blood supply evaluated with Pulsed wave Doppler and CEUS evaluation increased across all the timepoints. Quantitative CEUS analysis revealed a statistical difference in PPI (%), MTT (s) and AUC (%) in particular at T5. CEUS evaluation of the ovaries was feasible and demonstrated a marked increase in perfusion parameters in the late postovulatory period, demonstrating its applicability in the assessment of canine corpora lutea development. Another focal point of the current thesis centered on placental hemodynamics, aiming to determine quantitative and qualitative parameters for pregnant brachycephalic bitches. Moreover, it wants to describe placental vascularization and perfusion in females with fetal abnormalities close to delivery. Previous studies found an increase incidence of fetal anomalies in brachicephalic dogs, but it is not known if placental function could play a role in the occurrence of these conditions. The study involved forty-four healthy fetuses from 22 brachycephalic bitches and 9 fetuses with gestational abnormalities (anasarca and hydrocephalus) from 8 brachycephalic bitches. Evaluations were conducted at days 25 (M1), 45 (M2), and 58 (M3) of gestation in normal pregnancies, whereas fetuses with gestational abnormalities were evaluated at the last time point. Biometric values of the fetuses were determined through B-mode, and vascular indices by Doppler fluxometry of the umbilical artery, whereas qualitative assessment of contrast filling and quantitative parameters of placental perfusion were performed using CEUS. Contrast distribution remained uniformly homogeneous in placental tissues and CEUS filling parameters exhibited consistency throughout the evaluated periods (P < 0.05). In fetuses with hydrops, Doppler values were similar to those obtained in healthy subjects (P > 0.05), while CEUS evaluation revealed a heterogeneous distribution with lower intensity of placental tissue filling and a delay in perfusion time (P < 0.05) exhibiting a diagnostic accuracy of 75%. CEUS showed demonstrated its applicability in detecting failures in placental vascular filling (tissue dysfunction) in fetuses with anasarca and hydrocephalus. The concluding section of the present thesis was dedicated to exploring innovative approaches to enhance canine fertility and semen quality, with a specific focus on the use of antioxidants. Artificial insemination is a routinely performed method in canine breeding programme and semen evaluation results crucial for the successful achievement of a pregnancy. Insemination can be performed by using fresh, chilled or frozen semen. However, semen storage should be performed with the presence of antioxidants, which helps in preventing damaging processes to spermatozoa. The aim of these studies was to evaluate the effect of two antioxidants, Maca and Crocin, in the supplementation of semen extender on quality-related canine semen parameters during cooling and freezing. The first experiment aimed to evaluate the effect of Maca on frozen-thawed sperm quality in canine semen. Ejaculates from ten dogs were frozen in the absence (control group) or the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca and were evaluated immediately after thawing and after 1h and 2 h at 37 C° for sperm viability, motility, DNA fragmentation and lipid peroxidation. Canine sperm cells frozen with an extender supplemented with Maca exhibited higher total motility. Moreover, the supplementation of Maca was responsible for a surge in hyperactivation and WOB of sperm cells after one hour at 37°C. What emerges from this study is the protective role of Maca against lipid membrane peroxidation of canine spermatozoa, which is a primary marker of oxidative stress. The second experiment evaluated the effect of Crocin supplementation extender at three different concentrations (C0,5, C1 e C2) on quality-related canine semen parameters after cooling. Ejaculates from ten dogs were evaluated for sperm viability, sperm motility, membrane integrity and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage at 4 C°. the addition of 0.5 mM crocin in the extender significantly increased the proportion of spermatozoa with intact membranes at both 4 and 7 days compared to the control group and despite similar values of total motility and progressive motility most of the sperm kinetic parameters improved in C0.5 group compared to the control after 4 days of storage. The present findings suggested that the enrichment of the extender with the crocin improves to a certain improved canine semen quality, particularly after 4 days of storage at 4 °C.

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