Numerical Pixel Values Research Articles

Ultrasound-based assessment of the expression of inflammatory markers in the rectus femoris muscle of rats.

Ultrasonographic characteristics of skeletal muscles are related to their health status and functional capacity, but they still provide limited information on muscle composition during the inflammatory process. It has been demonstrated that an alteration in muscle composition or structure can have disparate effects on different ranges of ultrasonogram pixel intensities. Therefore, monitoring specific clusters or bands of pixel intensity values could help detect echotextural changes in skeletal muscles associated with neurogenic inflammation. Here we compare two methods of ultrasonographic image analysis, namely, the echointensity (EI) segmentation approach (EI banding method) and detection of selective pixel intensity ranges correlated with the expression of inflammatory regulators using an in-house developed computer algorithm (r-Algo). This study utilized an experimental model of neurogenic inflammation in segmentally linked myotomes (i.e., rectus femoris (RF) muscle) of rats subjected to lumbar facet injury. Our results show that there were no significant differences in RF echotextural variables for different EI bands (with 50- or 25-pixel intervals) between surgery and sham-operated rats, and no significant correlations among individual EI band pixel characteristics and protein expression of inflammatory regulators studied. However, mean numerical pixel values for the pixel intensity ranges identified with the proprietary r-Algo computer program correlated with protein expression of ERK1/2 and substance P (both 86-101-pixel ranges) and CaMKII (86-103-pixel range) in RF, and were greater (p < 0.05) in surgery rats compared with their sham-operated counterparts. Our findings indicate that computer-aided identification of specific pixel intensity ranges was critical for ultrasonographic detection of changes in the expression of inflammatory mediators in neurosegmentally-linked skeletal muscles of rats after facet injury.

A Preliminary Study of the Relationships Between Echotextural Characteristics of the Mammary Gland and Chemical Composition of Milk During Early Lactation in EWES

Abstract This study aimed to examine the ultrasonographic characteristics of the mammary gland in post-partum ewes (1 to 4 weeks after lambing) of varying genotypes for correlations with the chemical composition of milk. Ultrasonograms of mammary glands in coronal (C) and sagittal (S) planes were obtained using 5.0- and 7.5-MHz transducers from four polish mountain (PMt) and six Olkuska (Olk) ewes. Computerized image analyses of all images have been performed to determine pixel intensity (numerical pixel values – NPV) and heterogeneity (pixel standard deviation – PSD) of the mammary gland stroma/parenchyma. In PMT ewes examined with the 5.0-MHz probe, the mean NPV-C correlated negatively with C17:0 (margaric acid) and C17:1 C10 (heptadecenoic acid). Furthermore, NPV-s correlated negatively with C17:0 content of milk. In PMT ewes examined with the 7.5-MHz transducer, the mean NPV-S correlated positively with the crude fat and total solids, and negatively with C18:3 c9, C12, C15 (N-3) (α-linoleic acid) content of milk samples, while PSD-S correlated positively with C16:0 (palmitic acid) and negatively with lactose. In the scans obtained with the 5.0-MHz in olk sheep, the mean NPV-C was directly related to C10:1 (decanoic acid), C14:1 (myristoleic acid), C16:1 iso (iso-hexadecanoic acid), C16:1 C9 (palmitoleic acid), and c18:0 (stearic acid) content; the mean psd-c correlated positively with total solids, c14:1, C15:0, C16:0, C16:1 iso, C16:1 C9, ClA (conjugated linoleic acids), and total saturated fatty acid content, and negatively with C18:1 C11 (N-7) (cis-vaccenic acid), C18:2 C9, C12 (n-6) (linoleic acid), and total polyunsaturated fatty acid content of milk; and the mean psd-s correlated directly with ash and total saturated fatty acid content of the milk. Lastly, the mean NPV-C of images recorded with the 7.5-MHz probe in olk ewes related directly to ash and C12:0 (lauric acid); the mean NPV-S related directly to ash and C16:1 iso; the mean PSD-C correlated positively with ash and total saturated fatty acid (SFA) content, and the mean PSD-S correlated positively with the total SFA. Echotextural attributes of the mammary gland in ewes have the makings of the technique for determining milk content of certain chemical constituents in a breed-specific manner; however, further studies are needed to verify the usefulness of echotextural analyses of the udder to consistently predict complete milk composition.

Udder Ultrasonography of Dairy Cows: Investigating the Relationship between Echotexture, Blood Flow, Somatic Cell Count and Milk Yield during Dry Period and Lactation.

Udder health of dairy cows is related to their productivity and welfare. The period from dry-off to calving and early lactation is crucial. Ultrasonography is a useful and practical tool for the examination of the mammary parenchyma and blood flow. This observational study investigated the relationship between udder echotexture features, blood flow volume (BFVol) in the milk vein, milk somatic cell count (SCC) and daily milk yield (DMY) from late lactation, throughout the dry period and consecutive early lactation. Seventeen repeated measurements were performed on twenty-one Holstein cows. The udder parenchyma was examined with B-mode ultrasonography. Udder echotexture was studied using 15 features: Numerical Pixel Value (NPV), Pixel Standard Deviation (PSD), Skewness, Excess, Contrast, Homogeneity, Correlation, Entropy, Run Percentage, Long-Run Emphasis, Grey Value Distribution, Runlength Distribution, Gradient Mean Value, Gradient Variance and Percentage of Non-zero Gradients. Blood flow in the milk vein was examined with spectral Doppler. Linear mixed-effects models were employed to investigate relationships between BFVol, udder echotexture features, SCC and DMY throughout the study period. Our models showed that a 1 kg increase in DMY was associated with a significant increase of 0.25 L/min in the expected BFVol and that a 1,000,000-cells/mL increase in SCC was associated with a significant BFVol decrease of 0.49 L/min, keeping all other variables constant. Multivariable models showed significant associations between DMY and NPV, between PSD and Long-Run Emphasis, and between SCC and NPV, PSD, Gradient Mean Value, Homogeneity, Gradient Variance and Entropy. In conclusion, udder echotexture and BFVol in the milk vein are related to SCC and milk yield. Ultrasonography can be used for the comprehensive assessment of udder health in support of precision dairy farming.

Luteal Function, Biometrics, and Echotextural Attributes in Santa Inês Ewes Superovulated with Different Total Doses of Porcine Follicle-Stimulating Hormone

Premature regression of corpora lutea (PRCL) may adversely affect the outcome of hormonal ovarian superstimulation in small ruminants, and the total dose of exogenous gonadotropins used may be one of the causes of this condition. There were two major objectives of the present study: (1) to evaluate the effects of different superovulatory doses of porcine follicle-stimulating hormone (pFSH) on the biometry, blood perfusion (Doppler), and echotextural characteristics of luteal structures; and, (2) to determine the usefulness of biometric, vascular, and echotextural luteal variables, as well as measurements of circulating progesterone (P4) concentrations for early detection of PRCL in superovulated Santa Inês ewes. Twenty-seven Santa Inês ewes received an intravaginal P4-releasing device (CIDR) from Days 0 to 8 (Day 0 = random day of the anovulatory period). An IM injection of d-cloprostenol (37.5 μg) was given at the time of the CIDR insertion and withdrawal. On Day 6, all the ewes received 300 IU of eCG IM and were divided into three treatment groups (each n = 9): G100 (100 mg); G133 (133 mg); and G200 (200 mg of pFSH) administered IM every 12 h in eight injections. Transrectal ovarian ultrasonography and jugular blood sampling for serum P4 measurements were performed on Days 11 to 15. On the day of embryo recovery (Day 15), all the ewes underwent diagnostic videolaparoscopy and were classified, based on their luteal characteristics, into three response groups: nCL (ewes with normal CL only); rCL (ewes with regressing CL only); and ewes with both nCL and rCL following the superovulatory regimen. Our present results indicate that the total pFSH doses of 100 mg and 200 mg result in similar ovulatory responses and luteal function/biometrics, although the percentage of donor ewes with nCL was greater (p < 0.05) for G100 compared with the G200 animals. An application of 133 mg of pFSH was associated with diminished luteogenesis. Lastly, circulating P4 concentrations, ultrasonographic estimates of total luteal area, and CL pixel heterogeneity (standard deviation of numerical pixel values) are promising markers of luteal inadequacy in superovulated ewes.

Ultrasonographic assessment of skeletal muscles after experimentally induced neurogenic inflammation (facet injury) in rats.

This study set out to examine ultrasonographic attributes of non-neurosegmentally (pectoral-forelimb) and neurosegmentally linked (hindlimb) myotomes in an experimental model that leads to neurogenic inflammation in segmentally linked myotomes, and to evaluate quantitative correlations among ultrasonographic attributes of the muscles, relative content of various inflammatory mediators, and nociceptive thresholds (hot and mechanical) in rats. Twelve male Wistar Kyoto rats were randomly divided into two equinumerous groups: surgery group, in which the left lumbar (L4-L6) facet joints were compressed for 3 min with modified Kelly forceps under general anesthesia, and sham-operated rats. All ultrasonograms were obtained with the Vevo 2100 Visual Sonic scanner connected to a 24-MHz transducer at four different time points: pre-surgery and 7, 14, and 21 days after surgical procedures. Digital ultrasonographic images of quadriceps femoris, hamstring, and pectoral-brachial muscle groups were analyzed using a polygonal meter region of interest placed on the largest cross-sectional area of the muscles displayed in Image ProPlus® analytical software to compute numerical pixel values and pixel heterogeneity (standard deviation of mean pixel values). On day 21, pain behavior tests (hot plate and von Frey) were performed and then all animals were euthanized. Protein expression of inflammatory mediators in biceps brachii and rectus femoris muscles was measured by Western blot. The most prominent differences in muscle echotextural attributes between the two subsets of rats occurred 14 days post-surgery in pectoral-brachial and quadriceps femoris muscles. The expression of calcitonin-gene-related peptide was directly related to both echotextural variables only in biceps brachii (pixel intensity: r = 0.65, P = 0.02; and heterogeneity: r = 0.66, P = 0.02, respectively). Our findings have revealed the occurrence of echotextural changes in skeletal muscles of rats during myositis; however, the accumulation of inflammatory mediators and the outcomes of sensory tests did not relate to the changes in first-order echotextural characteristics of affected hindlimb muscles.

Correlations between first order echotextural characteristics and chemical composition of pectoralis major muscles in broiler chickens receiving different dietary fat supplements

Abstract This study examined the quantitative relationships among ultrasonographic image attributes and chemical composition of the pectoralis major muscles in broiler chickens that received four different dietary fat supplements (Group SO: soybean oil; Group FO: flaxseed oil; Group SO+FO: soybean oil+flaxseed oil; and Group BT: beef tallow; n=10 birds/group). Ultrasonograms of birds’ pectoral muscles, in a transverse (T) and longitudinal (L) plane, were obtained just before slaughter at 6 weeks of age and were subjected to digital image analyses to determine mean pixel intensity (MPI) and pixel heterogeneity values (standard deviation of numerical pixel values; MPH; a.k.a first order echotextural characteristics). Thirty-eight chemical characteristics of the muscles were determined post-mortem (crude fat, protein, and dry matter as well as fatty acid profiles) and were analyzed for correlations with the echotextural variables. A total of 12 (L-MPI: 7; L-MPH: 4; and T-MPH: 1 correlation), 5 (L-MPI: 2; L-MPH: 2; and T-MPI: 1 correlation), 15 (L-MPI: 10; T-MPI: 4; and T-MPH: 1 correlation) and 8 (L-MPI: 2; L-MPH: 1; and T-MPH: 5 correlations) significant correlations were recorded in Groups SO, FO, SO+FO and BT, respectively. When the data were pooled for all 40 birds studied, significant correlations with echotextural attributes were recorded for eighteen different chemical constituents, with the strongest overall correlation found between crude fat content and T-MPI (r=0.52, P=0.0005). In conclusion, there exists a potential application for ultrasonographic imaging in situ combined with computerized image analysis to estimate certain chemical constituents of pectoralis major muscles in broiler chickens. However, the existence and strength of correlations among ultrasonographic image attributes and muscle composition are affected by the source of dietary fat and relative abundance (“threshold concentrations”) of individual chemical components.

Relationship Between Somatic Cell Counts and Mammary Gland Parenchyma Ultrasonography in Buffaloes.

The aim of the present study was to determine whether the echotextural features of the mammary gland parenchyma in buffaloes during lactation at different somatic cell levels could be used to diagnose mastitis. This study was divided into two parts. In the first experiment, experimental buffaloes (n = 65) with somatic cell counts (SCC) tests (n = 94) in different seasons, including spring (n = 22), summer (n = 24), autumn (n = 37), and winter (n = 11), were used to obtain ultrasonic variables for each quarter of mammary gland that could best explain the corresponding somatic cell level. In the second part of the study, the first part's experimental results were verified by subjecting at least one-quarter udder of eight buffaloes to ultrasonography seven times during mid-July to mid-August for obtaining ultrasonic values at different somatic cell levels. The echo textural characteristics [mean numerical pixel values (NPVs) and pixel heterogeneity (pixel standard deviation, PSD)] were evaluated using 16 ultrasonographic images of each buffalo with Image ProPlus software. The effects of SCC, days in milk (DIM), scanning order (SO), season, as well as the scanning plane and udder quarter (SP + UQ) on both the PSD and NPVs of the mammary gland were significant (p < 0.05). The correlation coefficient between pre-milking sagittal PSD and somatic cell score (SCS) was the highest (r = 0.4224, p < 0.0001) with fitted linear model: y = 0.19445x (dependent variable: SCS, independent variables: pre-milking sagittal PSD; R2 = 0.84, p < 0.0001). In addition, SCC and ultrasonic of udder quarter were followed for 1 month, confirming that pre-milking sagittal PSD of mammary gland value could explain the SCC variation in milk. The current study demonstrated that the ultrasonographic examination of the udder could be one of the complementary tools for diagnosing subclinical mastitis in buffaloes.

Associations between Mammary Gland Echotexture and Milk Composition in Cows.

Simple SummaryTranscutaneous ultrasonography of the four quarters of the cow’s mammary gland with the 5-MHz ultrasound transducer combined with computer-assisted analysis of the resultant grey-scale images have the makings of an inexpensive and rapid technique to determine certain physicochemical properties of the pooled milk. The latter include crude protein, casein and lactose content. The relative ease and practically unlimited frequency with which this method can be used in farm settings makes it an attractive alternative to laboratory testing of milk samples. More studies are needed to determine the suitability of this approach for detecting changes in milk chemical composition in animals with mastitis.Thirty clinically healthy Holstein-Friesian cows underwent twice daily machine milking and ultrasonographic examinations of the udder just prior to and after milking. Digital ultrasonographic images of each udder quarter were subjected to computer-assisted echotextural analyses to obtain mean numerical pixel values (NPVs) and pixel heterogeneity (PSD) of the mammary gland parenchyma. The average milk yield and pH were higher (p < 0.05) in the morning, whereas crude fat, total solids, solids non-fat and citric acid content were higher (p < 0.05) during the evening milking period. Mean NPVs and PSDs of the mammary gland parenchyma were greater (p < 0.05) after than before milking. There were significant correlations among echotextural characteristics of the udder and protein percentage, lactose content and freezing point depression determined in the milk samples collected in the morning and crude protein, casein, lactose and solids non-fat in the evening. Our results can be interpreted to suggest that computerized analysis of the mammary gland ultrasonograms has the makings of a technique for estimating non-fat milk constituents in cows. However, future validating studies are necessary before this method can be employed in commercial settings and research. Moreover, significant inter-quarter differences in udder echogenicity may necessitate further echotextural studies of separate quarters.

Testicular Morphological and Ultrasonographic Characterization of Male Gray Brocket Deers (Mazama gouazoubira) in Different Reproductive Status

Background: Gray brocket deer (Mazama gouazoubira) populations have been declining due to human intervention. Yet, only a few studies have assessed ultrasonographic testicular characteristics in cervids. Considering the relevance of monitoring testicular size, blood flow, and parenchyma, the present study aims to establish baseline information on scrotal circumference, testicular volume, and spectral Doppler parameters, to describe differences among adult male gray brocket deer in different reproductive status, and to correlate ultrasound parameters with testes size measurements.Materials, Methods &amp; Results: Six adult male gray brocket deers were used in the study. Scrotal circumference and testicular volume were measured. B mode ultrasound images of testes (longitudinal and cross-sectional views) and epididymes were subjected to computer-assisted analysis, obtaining the numerical pixel values (NPV) and pixel standard deviation (PSD). Using spectral Doppler, supratesticular artery blood flow velocities (peak systolic velocity - PSV, end diastolic velocity - EDV, time-average maximum velocity - TAMAX, resistivity - RI and pulsatility indices - PI) were obtained. Semen was analyzed through total motility, vigor, and concentration tests. Three animals were normospermic (F+ group) and three were oligo/azoospermic (F- group). Groups were compared using were compared using a one-way ANOVA or Kruskal-Wallis followed by Student-Newman-Keuls (SNK) test. Ultrasound parameters were correlated to testes size parameters using Pearson’s correlation for parametric variables and Spearman’s correlation for non-parametric variables. F+ group presented significantly higher scrotal circumference (14.57 ± 1.19 cm), testicular volume (26.18 ± 4.94 cm3), and testes cross-sectional NPV (69.88 ± 24.00) and PSD (10.78 ± 3.42) than group F- (NPV: 28.26 ± 13.75, PSD: 6.70 ± 1.84). No significant differences were observed between the groups regarding the spectral Doppler ultrasound parameters. Significant correlations were observed between scrotal circumference and longitudinal (r = 0.76) and cross-sectional testes NPV (r = 0.89), and testicular volume was correlated with longitudinal (r = 0.78) and cross-sectional testes NPV (r = 0.91) and with cross-sectional testes PSD (ρ = 0.82).Discussion: Increased testicular echogenicity (higher NPV) has been positively associated with improved testicular growth, cell population expansion, inner and outer seminiferous tubules diameter, spermatids percentages and testis weight. In addition, more heterogenous testes (higher PSD) were associated with higher sperm output. It was suggested that the animals in group F- had compromised testicular development and spermatogenesis. The correlation observed between testes NPV and scrotal circumference was proposed to be associated with seminiferous tubules impairment. The F- group showed lower testicular volume, NPVs and PSDs in cross-sectional testicular images, suggesting higher protein levels and lower lipid contents were present in their parenchyma, influencing in testicular echogenicity and echotexture. No differences in spectral Doppler parameters were observed between the two groups. Also observed in stallions. However, PSV, EDV, TAMAX could be potential infertility indicators in other mammalians. These different results may be due to different locations of the evaluated vessel, species and techniques, age, ambient temperature, pathological conditions, and anaesthesia. Thus, it is suggested that scrotal circumference, testicular volume, and testes NPV are good indicators of male reproductive health in gray brocket deer and may help with better male selection in the species.

Quantitative Echotextural Attributes of Pectoralis Major Muscles in Broiler Chickens: Physicochemical Correlates and Effects of Dietary Fat Source.

Simple SummaryThere is presently no method of predicting meat quality based on a single examination of birds before slaughter. That could potentially be accomplished with a computer-assisted analysis of ultrasonographic images. Hence, this experiment was designed to draw correlations between various physical and chemical properties of chicken pectoral muscles and pixel values (brightness elements) measured in corresponding ultrasonograms (a.k.a. echotexture). Our study revealed that echotextural characteristics of pectoral muscles in live birds were predictive of several important physical parameters (e.g., cutting force, hardness, and chewiness) as well as intramuscular fat and protein content. However, different dietary fats that caused changes in the chemical composition of chicken breast also affected the associations between muscle echotexture and its physicochemical properties. For example, no correlations with the chemical composition of the muscles could be found in birds fed with soybean oil. We concluded that ultrasonographic imaging combined with a computerized image analysis can offer significant benefits to the poultry industry and consumers. It can aid in livestock genetic selection and improvement programs as well as enhance the quality of poultry meat and meat products. However, more confirmatory studies are needed.This study examined the relationships among physicochemical properties and ultrasonographic image attributes of pectoralis major muscles in broiler chickens. Forty male Ross 308 chicks were randomly assigned to four equinumerous fat-supplementation groups (Group SO: soybean oil; Group FO: flax oil; Group SO + FO: soybean oil + flax oil; and Group BF: beef fat). Ultrasonograms of birds’ pectoral muscles were obtained just before slaughter at 6 weeks of age and were subjected to digital image analyses to determine the mean pixel intensity (MPI) and pixel heterogeneity values (standard deviation of numerical pixel values; MPH). A total of 2, 4, 2, and 6 significant correlations were recorded in Groups SO, FO, SO + FO, and BF, respectively; there were no correlations with the chemical composition of the muscles in Groups SO and SO + FO. The strongest correlations were found between muscle lightness (L*) and MPH in Group BF (physical characteristic; r = −0.82, p = 0.003), and between crude fat/protein content and MPI/MPH of pectoral the major muscles in Groups FO/BF (chemical characteristics; r = 0.72, p = 0.02). There exists a potential application of ultrasonographic imaging and computerized image analysis for predicting certain physicochemical properties of pectoralis major muscles in broiler chickens.

Echotextural characteristics of the mammary gland during early lactation in two breeds of sheep varying in milk yields.

The main goal of this preliminary study was to determine and compare ultrasonographic characteristics of the mammary gland in two genotypes of ewes varying in milk productivity at 2, 3 and 4 weeks after lambing. Ultrasonographic images of the udder were obtained using the 5.0- and 7.5-MHz transducers, in axial and coronal planes, in four low milk-yielding Polish Mountain sheep and six high milk-yielding Olkuska ewes. All ultrasonograms were subjected to computerized image analyses using commercially available image analytical software (Image ProPlus ® ; Media Cybernetics Inc., San Diego, CA, USA) to determine numerical pixel values (NPVs) and heterogeneity (pixel standard deviation-PSD) of the mammary gland parenchyma. During the 28-day period post-partum, the Olkuska sheep exceeded (P < 0.05) Polish Mountain ewes in milk productivity (31.6 ± 2.7 l and 25.0 ± 4.2 l, respectively; means ± SEMs) as estimated by the mean weight gains of suckling lambs. In animals examined with the 5.0-MHz transducer, mean NPVs of the mammary gland parenchyma in Olkuska ewes and mean PSD in both genotypes of ewes were lower (P < 0.05) before than after milking. In addition, PSD recorded both before and after milking were lower (P < 0.05) in the Polish Mountain compared with Olkuska breed. Mean PSD values for the mammary gland were less (P < 0.05) before than after milking in Polish Mountain ewes and they were greater (P < 0.05) in Olkuska compared with Polish Mountain ewes examined with the 7.5-MHz probe after milking. It can be concluded that milk quantity, histomorphology of the udder and ultrasound transducer frequency may all impinge on the echotextural characteristics of the mammary parenchyma in different breeds of sheep. Our observations warrant future studies of correlations between milk composition, mammary gland histophysiology and ultrasonographic image attributes of the mammary gland in ruminants.

Elastographic and echotextural characteristics of foetal lungs and liver during the final 5 days of intrauterine development in dogs

Objective was to evaluate the echotexture and characteristics during terminal development of canine foetal respiratory and hepatic systems through elastographic examinations. Fifteen pregnant bitches were evaluated by ultrasonography twice daily, from the 53rd gestational day until whelping, and images obtained from 120 to 0 h before parturition were analysed. Images of foetal lungs and liver were recorded and then used for computer-assisted analyses to determine quantitative attributes. Acoustic Radiation Force Impulse (ARFI) elastographic of internal organs were classified as ‘soft’ (white areas) or ‘hard’ (dark areas) and quantitative analyses determined the mean shear wave velocities (SWV) of foetal lungs and liver. After delivery, canine neonates were clinically evaluated, and their health status was monitored weekly until 60 days post-partum. Sonographic parameters over time were compared by ANOVA and Pearson’s correlations were used to determine associations between SWVs and echotextural variables. Foetal lungs and liver had a homogeneous echotexture and pulmonary parenchyma appeared hyperechoic when compared with that of the liver. Mean numerical pixel values (NPVs) of lungs decreased from 120 to 24 h and subsequently increased until parturition (P = 0.04). Lungs and liver mean (± SD) SWVs (0.98 ± 0.12 and 0.84 ± 0.11 m/s, respectively) didn’t vary (P > 0.05) over time. Fluctuations in pulmonary NPVs indicated there was a pattern corresponding to structural and functional changes that occur during the terminal stage of pre-natal canine development and hence can be a useful diagnostic tool in veterinary. Foetal lung and liver SWVs were relatively consistent and there was no detectable changes during the pre-partum period for this variable or in echotexture.

Changes in testicular size, echotexture, and arterial blood flow associated with the attainment of puberty in Dorper rams raised in a subtropical climate.

There is a paucity of information on the relationships of testicular morphology, echotextural attributes, and blood flow dynamics with pubertal development of rams raised in a subtropical climate. Forty-five Dorper rams (24 rams aged 8-11months and 21 rams aged 12-24months) were examined using a portable ultrasound scanner connected to a 7.5-MHz transducer. Computer-assisted analyses of testicular ultrasonograms utilized commercially available Image ProPlus® analytical software. Spectral Doppler scans of testicular arteries were performed immediately after scrotal (B-mode) ultrasonography to determine peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI=[PSV-EDV]/PSV), and pulsatility index (PI=[SPV-EDV]/mean velocity) of the blood vessels. The length of the testes (9.7±0.3 compared with 9.0±0.2cm) and scrotal circumference (33.3±0.5 compared with 31.8±0.4cm) were greater (p<0.05) but testicular depth (4.5±0.1 compared with 4.9±0.08 cm) was less (p<0.05) in sexually mature compared with peripubertal rams. [Corrections added on 9 Jan 2019 after initial online publication: The testicular size values in the sentence were corrected.] There were no differences (p>0.05) between the two age groups of Dorper rams in blood flow indices of testicular arteries. Mean numerical pixel values (100.5±4.1 compared with 89.2±4.8) and pixel heterogeneity (25.6±0.6 compared with 23.6±0.5) of testicular parenchyma were greater (p<0.05) in peripubertal than in postpubertal rams. Semen volume was negatively correlated with PI of testicular arteries (r=-0.57, p=0.04). In summary, the attainment of sexual maturity in the rams of the present study was associated with significant changes in testicular length and depth, scrotal circumference, and parenchymal echogenicity/hetrogeneity but not in testicular volume and blood perfusion rates. Testicular artery PI can be used to predict the volume of ejaculate in rams.

Validation of a sample preparation system (eFlow) for semen analysis in the AndroVision® CASA-System

Objective was to evaluate the echotexture and characteristics during terminal development of canine foetal respiratory and hepatic systems through elastographic examinations. Fifteen pregnant bitches were evaluated by ultrasonography twice daily, from the 53rd gestational day until whelping, and images obtained from 120 to 0 h before parturition were analysed. Images of foetal lungs and liver were recorded and then used for computer-assisted analyses to determine quantitative attributes. Acoustic Radiation Force Impulse (ARFI) elastographic of internal organs were classified as ‘soft’ (white areas) or ‘hard’ (dark areas) and quantitative analyses determined the mean shear wave velocities (SWV) of foetal lungs and liver. After delivery, canine neonates were clinically evaluated, and their health status was monitored weekly until 60 days post-partum. Sonographic parameters over time were compared by ANOVA and Pearson’s correlations were used to determine associations between SWVs and echotextural variables. Foetal lungs and liver had a homogeneous echotexture and pulmonary parenchyma appeared hyperechoic when compared with that of the liver. Mean numerical pixel values (NPVs) of lungs decreased from 120 to 24 h and subsequently increased until parturition (P = 0.04). Lungs and liver mean (± SD) SWVs (0.98 ± 0.12 and 0.84 ± 0.11 m/s, respectively) didn’t vary (P > 0.05) over time. Fluctuations in pulmonary NPVs indicated there was a pattern corresponding to structural and functional changes that occur during the terminal stage of pre-natal canine development and hence can be a useful diagnostic tool in veterinary. Foetal lung and liver SWVs were relatively consistent and there was no detectable changes during the pre-partum period for this variable or in echotexture.

Qualitative and quantitative ultrasound attributes of maternal-foetal structures in pregnant ewes.

The aim of this study was to examine foetal organs and placental tissue to establish a correlation between the changes in the composition of these structures associated with their maturation and the ultrasonographic characteristics of the images. Twenty-four pregnant ewes were included in the study. Ultrasonography assessments were performed in B-mode, from the ninth gestational week until parturition. The lungs, liver and kidneys of foetuses and placentomes were located in transverse and longitudinal sections to evaluate the echogenicity (hypoechoic, isoechoic, hyperechoic or mixed) and echotexture (homogeneous and heterogeneous) of the tissues of interest. For quantitative evaluation of the ultrasonographic characteristics, it was performed a computerized image analysis using a commercial software (Image ProPlus® ). Mean numerical pixel values (NPVs), pixel heterogeneity (standard deviation of NPVs) and minimum and maximum pixel values were measured by selecting five circular regions of interest in each assessed tissue. All evaluated tissues presented significant variations in the NPVs, except for the liver. Pulmonary NPVmean, NPVmin and NPVmax decreased gradually through gestational weeks. The renal parameters gradually decreased with the advancement of the gestational weeks until the 17th week and later stabilized. The placentome NPVmean, NPVmin and NPVmax decreased gradually over the course of weeks. The hepatic tissue did not show echogenicity and echotexture variations and presented medium echogenicity and homogeneous echotexture throughout the experimental period. It was concluded that pixels numerical evaluation of maternal-foetal tissues was applicable and allowed the identification of quantitative ultrasonographic characteristics showing changes in echogenicity related to gestational age.

Corpus luteum dynamics after ovulation induction with or without previous exposure to progesterone in prepubertal Nellore heifers

The objectives of this study were 1) to monitor corpus luteum (CL) dynamics after two different protocols of ovulation induction in prepubertal Nellore heifers, and 2) to determine differences in luteal function. Fifty-seven heifers (weight 289.61 ± 32.28 kg, BCS 5.66 ± 0.65, age 17.47 ± 0.81 months) were divided into two groups: GP4+GnRH received a progesterone (P4) device of 3rd use for 10 days, followed by the administration of 0.02 mg buserelin acetate (GnRH) 48 h after removal of the device, and GGnRH received only GnRH. The CLs formed were monitored by ultrasonography every 2 days until their functional regression (decrease in the color Doppler signal and serum P4 concentration < 1 ng/mL), determining their diameter and area, numerical pixel value (NPV), pixel heterogeneity, and vascularization percentage. The peak systolic velocity, end diastolic velocity, resistivity index and pulsatility index (PI) of the ovarian artery and serum P4 concentration were also measured. A lifespan of the CL of more than 16 days was classified as normal-function and of less than 16 days as premature regression. The variables were compared between treatments, CL categories (normal-functional, prematurely regressed or non-functional), days of evaluation, and their interactions using the MIXED procedure of the SAS program (p ≤ 0.05). Three animals of each group (6/57 = 11%) did not respond to treatment, corresponding to an ovulation rate of 89%. There was a higher percentage of normal-function CLs in GP4+GnRH (81%) and a higher percentage of non-functional CLs in GGnRH (52%; P4 concentration < 1 ng/mL in all assessments). Normal-function CLs exhibited a greater area, vascularization percentage and P4 concentration than prematurely regressed and non-functional CLs. Lower diameter, area, NPV and P4 concentration were observed for non-functional CLs, but there was no difference in vascularization percentage compared to prematurely regressed CLs. Progesterone concentration was efficient in diagnosing CL function and was positively correlated with CL area (r = 0.62; p < 0.001) and vascularization percentage (r = 0.38; p < 0.001). Diameter and PI were important for the early diagnosis of non-functional and prematurely regressed CLs, respectively. In conclusion, luteal function differed for the first CL that develops after ovulation induction in prepubertal heifers. Ultrasonographic parameters (diameter, area, NPV, vascularization percentage, and PI) can be used to predict CL function.

Relationship between echotextural and histomorphometric characteristics of stallion testes

The goal of this study was to investigate correlations between objective measures of testicular echotexture and histomorphometric attributes related to the histological composition of stallion testes. Fifty-four scrotal testes were obtained from three groups of stallions during routine castrations: colts <1 yr old (n = 18), young stallions 1–5 yrs old (n = 27), mature stallions > 5 yrs old (n = 9). In addition, two scrotal testes with degeneration, 16 retained inguinal and 10 retained abdominal testes were surgically obtained. Cross-sectional and longitudinal ultrasonograms were obtained for each testis. Mean numerical pixel values (NPVs) as well as pixel standard deviations (PSDs) were determined for each image (ImageJ-1.5 software). Histomorphometric attributes of the seminiferous tubules (STs) were derived (three tissue samples per each testis) using image analysis software [relative STs area: RSTA = ST area/total cross-sectional area (TA) x 100%; relative STs lumen: RSTL = ST lumen area/TA x 100%; individual ST area; ISTA; individual ST lumen: ISTL; seminiferous epithelium height: SHE]. Degree of fibrosis was graded semi-quantitatively (0–3) in samples from 17 testes. All measures of testicular echotexture as well as all histomorphometric attributes of STs had highest values when obtained from the scrotal testes of young and mature stallions (P < 0.05). The NPVs and PSDs from the ultrasonographic images of the scrotal testes were significantly correlated with all histomorphometric attributes of STs (P < 0.001). However, there was no correlation between the majority of these measures and attributes if each group of the scrotal testes was analyzed separately. The NPVs from the ultrasonographic images of the retained inguinal testes were correlated with RSTA, RSTL, ISTA, and ISTL, while the NPVs from the retained abdominal testes were not correlated with any of the histomorphometric attributes of the STs. Testes with high degree of fibrosis had variable values of pixel intensity and pixel heterogeneity. Based on the results of this study, we concluded that the pixel intensity and pixel heterogeneity of stallion testes increase during the first year of life and remain stable in young and mature stallions. These changes occur in parallel to the development of the seminiferous tubules. Testicular echogenicity in stallions does not seem to reflect degree of testicular fibrosis. Retained abdominal testes have lower and less heterogeneous echogenicity than scrotal testes from stallions that are more than one year old. While pixel analysis cannot replace biopsy in assessing testicular histomorphology in young and mature stallions, testicular echogenicity is a good indicator of peri-pubertal growth and expansion of the seminiferous tubules.

Prospective ultrasonographic and endocrine predictors of spermatogenic onset in ram lambs

The objective of this study was to examine testicular ultrasonographic characteristics and endocrine profiles in prepubescent ram lambs for correlations with the age at first detection of elongated spermatids (ESt age). Bi-weekly ultrasound examinations and weekly testicular biopsies began at 10 weeks of age or at the time that testicular volume reached 15cm3, and continued until 1–2 weeks after Est's were first detected by histological examination of testicular biopsies in twenty-two spring-born Rideau Arcott×Polled Dorset lambs. Computer-assisted analysis of testicular ultrasonograms was performed using commercially available image analytical software. Blood samples were drawn before each ultrasonographic examination and were used for measurements of free triiodothyronine (fT3) and thyroxine (fT4), and follicle-stimulating hormone (FSH) concentrations. The mean (±SEM) age at first detection of ESts was 15.9±0.5 weeks. Testicular volumes recorded between 10 and 12 weeks of age correlated inversely with the ESt age (r=−0.44 to −0.50, P≤0.05). Statistically significant correlations were recorded between the ESt age and numerical pixel values of testicular parenchyma at 10 (r=−0.48, P=0.05) and 15 (r=0.52, P=0.05) weeks of age, and between the ESt age and testicular pixel heterogeneity in ram lambs aged 14.5 weeks (r=0.60, P=0.007). Lastly, circulating FSH concentrations at 10 weeks (r=−0.43, P=0.05), serum fT3 concentrations at 13 weeks (r=0.44, P=0.04) and fT4 concentrations at 11.5 weeks of age (r=0.48, P=0.03) were all correlated with the ESt age. The present results show that testicular volume has the most stable relationship with pubertal onset; however, testicular echotexture as well as circulating concentrations of FSH and free fractions of thyroid hormones at specific ages may be indicative of more intricate developmental events heralding puberty.

186 QUANTITATIVE ECHOGENICITY AND ECHOTEXTURE ANALYSIS OF THE ACCESSORY SEX GLANDS OF PUBERTAL AND MATURE DORPER RAMS

This study aimed to analyse the ultrasonographic attributes of vesicular, prostate, and bulbourethral glands in pubertal and mature Dorper rams. Forty-five rams were used in the same day (pubertal: 8–11 months, n = 24; mature: ≥12 months, n = 21). The B-mode ultrasounds examinations were performed using MyLab 30Vet equipment (Esaote, Naples, Italy) connected to transrectal linear transducer (frequency of 7.5 MHz). The echogenicity [(mean numerical pixel values (NPV)] and pixel heterogeneity (standard deviation of NPV) of accessory sex glands parenchymas was determined by computerised image analysis using Image ProPlus® software (Media Cybernetics Inc., Rockville, MD, USA). For the pairs organs a mean for each parameter were calculated. Data were analysed by ANOVA with Tukey test (mean ± s.d.; P &lt; 0.05), after the normality and homoscedasticity of residuals were checked with Shapiro-Wilk test and Levene test, respectively, when necessary log-transformation was applied. All statistical procedures was performed with R® software. The echogenicity of parenchymas were different (P &lt; 0.05) between pubertal and mature rams for vesicular glands (181.51 ± 20.80 v. 164.83 ± 26.79) and bulbourethral glands (166.93 ± 16.93 v. 141.80 ± 29.15); however, there was no difference (P &gt; 0.05) for prostate glands (99.39 ± 36.34 v. 87.47 ± 34.24). The pixel heterogeneity did not differ (P &gt; 0.05) between pubertal and mature rams (29.06 ± 2.67 v. 28.09 ± 3.95, 32.14 ± 8.20 v. 30.28 ± 4.71, and 27.82 ± 4.53 v. 28.74 ± 4.29) for vesicular, prostate, and bulbourethral glands, respectively. In conclusion, the sexual maturity only influenced the echogenicity of vesicular and bulbourethral parenchymas of Dorper rams.

Retinoic acid treatment alters germ cell heterogeneity and testicular echotexture in prepubescent ram lambs.

Testicular echotextural attributes are closely associated with spermatogenic development; however, precise characterisation of specific germ cell types is difficult due to tremendous germ cell heterogeneity. Recently, retinoic acid (RA) administration in neonatal mice was found to induce highly synchronised spermatogenesis as adults. A RA-treatment protocol was tested in 17 ram lambs treated with or without RA at 8 weeks of age, with scrotal ultrasonography and blood samples collected until castration 24h or 2.5 weeks later. At 8.2 weeks of age, the nuclear:seminiferous tubule (ST) area was higher in the treated compared with the control group. Serum testosterone concentrations and numerical pixel values (NPVs) of the testicular parenchyma reached a peak at 9 weeks of age in both groups of ram lambs studied. At 10.5 weeks of age, the percentage of ST cross-sections with different germ cells as the most mature germ cell type was lower and the inter-tubular heterogeneity and NPVs were also lower in the treated compared with the control animals. RA manipulation of spermatogenesis in prepubertal ram lambs may provide a suitable model for further investigation of the echotextural characteristics of specific germ cell types and critical developmental events.