Evaluation of testicular echotexture with Ecotext as a diagnostic method of testicular dysfunction in stallions.

This study aimed to assess if Ecotext, a new software for evaluation of testicular echotexture, is a good method for diagnosis of stallions with testicular dysfunction (TD). Relationships between Ecotext parameters and sperm motility and production, testicular volume, and testicular blood flow were also studied. Ecotext provides a total of six echotexture parameters: Ecotext 1 (black pixels), 2 (white pixels) and 3 (grey pixels), and another 3 parameters related to hypoechogenic areas: Ecotext tubular density (ETD), Ecotext tubular diameter (ETd), and Ecotext tubular area (ETA). Stallions (n = 33) were assessed using proven diagnostic techniques (spermiogram, B-mode and Pulse Doppler ultrasound), and subsequent analysis with Ecotext. Animals were classified as "control stallions" (n:21, acceptable semen quality), and "stallions with TD" (n:12, poor semen quality (TM < 60%, PM < 45% and total nº of sperm with PM < 2000 × 106 spz), that were subdivided into "induced TD group" (immunized, anti-GnRH vaccine) and "acquired TD group". The acquired TD group showed differences in all Ecotext parameters in relation to controls (Ecotext 1:0.11 ± 0.17 vs 2.82 ± 2.52, Ecotext 2:1584.0 ± 575.8 vs 388 ± 368.2, Ecotext 3:134.2 ± 9.26; ETA: 2.14 ± 0.59 vs 5.40 ± 1.90; ETd: 65.66 ± 6.27 vs 86.93 ± 10.65 and ETD: 92.35 ± 11.24 vs 132.10 ± 16.35, p ≤ 0.001). Results suggest acquired TD stallions were suffering testicular degeneration with loss of architecture and function as all Ecotext parameters were altered in relation to controls. Induced TD horses only showed a reduction in ETD (116.2 ± 8.59 vs 132.10 ± 16.35, p ≤ 0.001), despite all sperm parameters being worse. These findings suggested immunized stallions probably only experience an acute loss of testicular functionality and parenchyma architecture is likely not affected since differences in Ecotext parameters with control stallions were not detected. ETD was the best parameter to identify animals with TD (AUC: 0.84, optimal cut-off value of 124.3 seminiferous tubules/cm2). Correlations were found between ETD and Doppler indices (PI: 0.60; RI: 0.47 p ≤ 0.001), total testicular volume (r: 0.48; p ≤ 0.05) and sperm motility (TM:0.51; and PM:0.54; p ≤ 0.001) and production (r:0.51; p ≤ 0.001). In summary, Ecotext could identify changes in testicular echotexture of stallions with TD. Results open the possibility for new research focused on establishing the relationship between Ecotext parameters and histomorphometry features in stallion testes.

The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae.

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 µM Sulfasalazine (SS) and 0.5 mM CysS + 500 µM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 105 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation.

Power Doppler can detect the presence of 7-8 day conceptuses prior to flushing in an equine embryo transfer program.

In order to determine whether differences in uterine blood flow between pregnant and non-pregnant mares can be used to predict the presence of the equine embryo prior to flushing in an embryo transfer program, power Doppler ultrasonography was used on a total of 52 mares on days 7 or 8 post-ovulation. Computer analysis of Doppler images was subsequently performed using ImageJ v1.48 software. Vascular perfusion of the endometrium was analyzed using spot meter techniques, measuring mean pixel intensity and area of blood flow. Mares with positive flushings presented a higher uterine blood flow area (one embryo: 54.01 ± 2.27 mm2 or two embryos: 61.01 ± 6.73 mm2) prior to embryo recovery compared to barren mares (21.77 ± 2.22 mm2) (p≤0.05). However, significant differences in vascular perfusion were not detected between single or twin pregnancies. Blood flow area appears to be a good predictor for differentiation between pregnant and non-pregnant mares with an AUC: 0.869; p≤0.001 and an optimal cut-off value of 37.21 mm2. Both the mare's age and day of embryo recovery caused effects on uterine vascular perfusion. According to Youden's J statistics the uterine blood flow area of young pregnant mares was greater than 25.4 mm2 on day 7 (with a sensitivity of 75% and a specificity of 87.5%) and greater than 21.02 mm2 on day 8 post-ovulation (with a sensitivity of 93.8% and a specificity of 100%). The uterine blood flow area in adult pregnant mares was greater than 41.4 mm2 on day 7 (with a sensitivity of 80% and a specificity of 85.5%) and greater than 35.55 mm2 on day 8 after ovulation (with a sensitivity of 97.2% and a specificity of 85.7%). Evaluation on day 8 is therefore considered to be more reliable. Older and middle aged pregnant mares (5-18 years old) had increased uterine vascularization compared to young pregnant mares (2-5 years old) (p≤0.001). Conversely, older barren mares showed higher endometrial vascularity (35.06 ± 2.56 mm2) than young (17.21 ± 1.26 mm2) and middle aged non-pregnant mares (23.84 ± 1.50 mm2) (p≤0.05). We hypothesized that the higher blood flow area seen in older barren mares may be a consequence of a subclinical endometritis due to repeated flushing for embryo recovery. The results of the present study indicate that power Doppler ultrasound combined with computer assisted analysis of images are reliable techniques to detect early pregnancy prior to embryo recovery.

How does the microbial load affect the quality of equine cool-stored semen?

Contaminating bacteria present in stallion ejaculates may compromise sperm quality during storage. Different procedures have been used to reduce the load of microorganisms in semen and avoid bacterial growth during storage. The aims of this study were: 1) to evaluate different techniques to eliminate bacteria in semen 2) to study the relationship between total microflora load (TML) and ROS production; and 3) to determine if TML affects the functionality of cool-stored sperm. Ejaculates from 11 stallions were split and processed in 3 ways: A. extended semen; B. conventional centrifuged semen, and C. Single layer centrifugation through Androcoll-E (SLC). All samples were preserved in INRA 96 at 5 °C for 72 h. Aliquots from native semen and from different treatments were taken for bacteriological analysis at T0, T24, T48 and T72h of storage and Total microbial load (TML: CFU (colony-forming units/ml) was calculated. The ROS production (dichlorodihydrofluorescein diacetate for H2O2, dihydroethidium for superoxide anion and CellROX deep red for total ROS), viability (YO-PRO-1-Ethidium) and lipid peroxidation (BODIPY-C11) were assessed by flow cytometry, and motility by CASA. The bacteria isolated were Corynebacterium spp, Arcanobacterium spp, Bacillus spp, Dermobacter, Staphylococcus spp, Streptococcus spp, Penicilium spp. TML of semen showed correlations with live sperm (r: -0.771), dead sperm (r: 0.580), H2O2 production (r: 0.740), and total ROS production (CellROX (+)) (r: -0.607), Total motility (r: 0.587), Progressive motility (r: -0.566), VCL (r: -0.664), VSL (r: -0,569), VAP (r: -0.534) (p ≤ 0.05). SLC removed 99.34% of the microbial load, which was assicated with a significanlty reduced H2O2 production (p ≤ 0.05). However, only samples treated with Androcoll-E had a higher total ROS production (CellROX +) (p ≤ 0.05). These results suggest that CellROX stain probably identifies superoxide production rather than H2O2 and this higher superoxide production may reflect an intense sperm functionality. The bacterial load increased the production of H2O2 in cool-stored semen which was associated with lower tolerance to refrigeration. SLC was the sperm processing technique that was most efficient at removing bacteria, reducing H2O2 production and selecting the most functional sperm.

Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH•) only induces spermptosis.

Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO4 exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8-iso-PGF2α and 4-hydroxynonenal (4-HNE). While both treatments induced changes indicative of spermptosis (caspase-3 activation and decreased mitochondrial membrane potential) (p < 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.

A simple flow cytometry protocol to determine simultaneously live, dead and apoptotic stallion spermatozoa in fresh and frozen thawed samples.

Spermatozoa undergo apoptotic changes during the cryopreservation process. These changes, recently termed spermptosis, resemble the cryopreservation induced delayed onset of cell death observed after thawing of somatic cells. Due to its importance in cryobiology, methods to easily identify spermptotic cells are warranted. In this study, a well-validated method for identification of spermatozoa with caspase 3 activity was compared with use of the combination of Hoechst 33342 (H-42) and ethidium homodimer (Eth-1). Live, dead and apoptotic spermatozoa assessed with each method were compared using descriptive statistics and method agreement analysis. No differences were observed in the percentages of spermatozoa in each of the categories investigated with each method. Moreover the method agreement analysis indicated there were consistent findings using both methods The combination H-42/Eth-1 can be successfully used to determine apoptosis in addition to dead and live spermatozoa. Moreover the intensity of H-42 fluorescence (bright and dim populations) allows for distinguishing of live and dead sperm cells.

Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na.

In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na+ content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na+ -K+ ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (p < 0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (p < 0.05) in intracellular Na+ . These changes occurred in relation to activation of caspase 3 (p < 0.001). Cryopreservation reduced the activity of the Na-K+ pump and inhibition of the Na+ -K+ ATPase pump with ouabain-induced caspase 3 activation. It is concluded that inactivation of Na+ -K+ ATPase occurs during cryopreservation, an inhibition that could play a role explaining the accelerated senescence of the surviving population of spermatozoa.

Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes.

The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes. However, there is a lack of studies investigating both phenomena simultaneously. In order to improve our knowledge in this particular point, we studied in raw and frozen-thawed samples apoptotic and capacitative markers using a wide battery of test based in flow cytometry. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. Conventional and computational flow cytometry using nonlinear dimensionally reduction techniques (t-distributed stochastic neighbor embedding (t-SNE)) and automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) were used. Most of the changes induced by cryopreservation were apoptotic, with increase in caspase 3 activation (P < 0.01), PS translocation to the outer membrane (P < 0.001), loss of mitochondrial membrane potential (P < 0.05), and increase in intracellular Na+ (P < 0.01). Average values of markers of capacitative changes were not affected by cryopreservation; however, the analysis of the phenotype of individual spermatozoa using computational flow cytometry revealed the presence of subpopulations of spermatozoa experiencing capacitative changes. For the first time advanced computational techniques were applied to the analysis of spermatozoa, and these techniques were able to disclose relevant information of the ejaculate that remained hidden using conventional flow cytometry.

NSDUAZ unfolding package for neutron spectrometry and dosimetry with Bonner spheres.

NSDUAZ (Neutron Spectrometry and Dosimetry from the Universidad Autónoma de Zacatecas) is a user friendly neutron unfolding package for Bonner sphere spectrometer with (6)LiI(Eu) developed under LabView(®) environment. Unfolding is carried out using a recursive iterative procedure with the SPUNIT algorithm, where the starting spectrum is obtained from a library initial guess spectra to start the iterations. The NSDUAZ performance was evaluated using (252)Cf, (252)Cf/D(2)O, (241)AmBe neutron sources and the neutrons outside the radial beam port of a TRIGA Mark III nuclear reactor running to 10 W.