Anthropogenic eutrophication of Lake Titicaca (Bolivia) revealed by carbon and nitrogen stable isotopes fingerprinting.

Cultural eutrophication is the leading cause of water quality degradation worldwide. The traditional monitoring of eutrophication is time-consuming and not integrative in space and time. Here, we examined the use of carbon (δ13C) and nitrogen (δ15N) isotopic composition to track the degree of eutrophication in a bay of Lake Titicaca impacted by anthropogenic (urban, industrial and agricultural wastewater) discharges. Our results show increasing δ13C and decreasing δ15N signatures in macrophytes and suspended particulate matter with distance to the wastewater source. In contrast to δ15N and δ13C signatures, in-between aquatic plants distributed along the slope were not only affected by anthropogenic discharges but also by the pathway of carbon uptake, i.e., atmospheric (emerged) vs aquatic (submerged). A binary mixing model elaborated from pristine and anthropogenic isotope end-members allowed the assessment of anthropogenically derived C and N incorporation in macrophytes with distance to the source. Higher anthropogenic contribution was observed during the wet season, attributed to enhanced wastewater discharges and leaching of agricultural areas. For both seasons, eutrophication was however found naturally attenuated within 6 to 8 km from the wastewater source. Here, we confirm that carbon and nitrogen stable isotopes are simple, integrative and time-saving tools to evaluate the degree of eutrophication (seasonally or annually) in anthropogenically impacted aquatic ecosystems.

Diagenetic production, accumulation and sediment-water exchanges of methylmercury in contrasted sediment facies of Lake Titicaca (Bolivia).

Monomethylmercury (MMHg) concentrations in aquatic biota from Lake Titicaca are elevated although the mercury (Hg) contamination level of the lake is low. The contribution of sediments to the lake MMHg pool remained however unclear. In this work, seven cores representative of the contrasted sediments and aquatic ecotopes of Lake Titicaca were sliced and analyzed for Hg and redox-sensitive elements (Mn, Fe, N and S) speciation in pore-water (PW) and sediment to document early diagenetic processes responsible for MMHg production and accumulation in PW during organic matter (OM) oxidation. The highest MMHg concentrations (up to 12.2 ng L-1 and 90% of THg) were found in subsurface PWs of the carbonate-rich sediments which cover 75% of the small basin and 20% of the large one. In other sediment facies, the larger content of OM restricted MMHg production and accumulation in PW by sequestering Hg in the solid phase and potentially also by decreasing its bioavailability in the PW. Diagenetically reduced S and Fe played a dual role either favoring or restricting the availability of Hg for biomethylation. The calculation of theoretical diffusive fluxes suggests that Lake Titicaca bottom sediments are a net source of MMHg, accounting for more than one third of the daily MMHg accumulated in the water column of the Lago Menor. We suggest that in the context of rising anthropogenic pressure, the enhancement of eutrophication in high altitude Altiplano lakes may increase these MMHg effluxes into the water column and favor its accumulation in water and biota.

Cryopreservation of Andalusian donkey (Equus asinus) spermatozoa: Use of alternative energy sources in the freezing extender affects post-thaw sperm motility patterns but not DNA stability.

The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the freezing extender improved total and progressive sperm motility (P < 0.05) and amplitude of lateral head displacement (P < 0.01), but it reduced the values for other sperm motility variables compared with glucose (P < 0.001). The use of fructose resulted in a reduction in values for most CASA variables (P < 0.05), whereas addition of Equex paste did not have any beneficial effect on values for these variables (P > 0.05). Glucose was more effective in maintaining sperm morphology (P < 0.05), while there was no beneficial effect with the addition of Equex paste (P > 0.05). Supplementation of fructose and Equex paste in the freezing extender decreased plasma membrane integrity (P < 0.05) as compared with glucose, but there were no differences between treatments for acrosome and DNA integrity (P > 0.05), even after 24 h of incubation. The use of different sugar sources in the extender could affect the in vitro post-thaw quality of cryopreserved donkey spermatozoa, with sorbitol being an interesting alternative for improving the sperm quality. Results of the present study indicate the use of Equex paste could negatively affect post-thaw outcomes for sperm viability in this species.

Effect of cooling rate on sperm quality of cryopreserved Andalusian donkey spermatozoa.

The aim of this study was to evaluate the effect of different cooling rates on post-thaw quality of cryopreserved donkey spermatozoa. Eighteen ejaculates from six adult Andalusian donkeys (three ejaculates per donkey) were collected using an artificial vagina. Pooled semen samples (two ejaculates per pool) were divided into three aliquots, and frozen in Gent freezing extender using three different cryopreservation protocols (P): P1 (conventional slow freezing, as control): semen pre-cooled in an Equitainer for 2 h and frozen in liquid nitrogen (LN2) vapour; P2 (controlled pre-freeze cooling rate): semen pre-cooled at a controlled rate for 73 min and frozen in LN2 vapour; and P3 (rapid freezing) semen frozen immediately in LN2 vapour. After thawing at 37 °C for 30 s, semen samples were assessed for motility, morphology, acrosome and plasma membrane integrity; spermatozoa were also tested for DNA integrity. Significant (P < 0.01) differences were found between the cryopreservation protocols for all sperm parameters evaluated, except for DNA integrity. Semen samples frozen using P2 showed significantly (P < 0.01) higher values for sperm motility, morphology, sperm membrane integrity, and acrosome integrity. On the contrary, P3 reduced sperm motility (P < 0.01) and increased the percentage of spermatozoa with damaged plasma membrane (P < 0.001). In our study, we demonstrated that the sperm of Andalusian donkey is particularly sensitive to the cooling rate used before freezing. Furthermore, Andalusian donkey semen can be successfully cryopreserved using controlled cooling rates combined with freezing in LN2 vapour.

Cryoprotective effect of glutamine, taurine, and proline on post-thaw semen quality and DNA integrity of donkey spermatozoa.

This study was conducted to evaluate the effect of amino acid addition to semen on post-thaw quality of donkey spermatozoa. Eighteen ejaculates were pooled and divided into aliquots which were cryopreserved in Gent A® containing 1% ethylene glycol (Gent-EG) and supplemented with 0 (as control), 20, 40, or 60 mM of glutamine, proline, or taurine. The greatest concentration (60 mM) of glutamine and taurine resulted in greater (P < 0.001) post-thaw sperm motility. Amino acid supplementation did not improve (P > 0.05) sperm morphology and membrane plasma integrity compared with the control samples. Whereas, improvement (P < 0.05) of acrosome integrity was observed with use of 60 mM glutamine. After thawing, there were no differences (P > 0.05) in the sperm DNA fragmentation index (sDFI) among treatments. The 60 mM glutamine and 40 mM taurine treatments, however, resulted in a reduction (P < 0.05) in sDFI values in the first 6 h of semen incubation, compared with the control samples. At 24 h, the sDFI values were less (P < 0.05) in all supplemented as compared with control samples, except for the 20 mM proline treatment group. In conclusion, supplementation of the Gent-EG extender with glutamine or taurine at 60 mM improved post-thaw donkey sperm quality. The addition of proline to the freezing extender, however, did not provide any significant enhancement in sperm quality, compared with the control group.

Mercury contamination level and speciation inventory in Lakes Titicaca & Uru-Uru (Bolivia): Current status and future trends.

Aquatic ecosystems of the Bolivian Altiplano (∼3800 m a.s.l.) are characterized by extreme hydro-climatic constrains (e.g., high UV-radiations and low oxygen) and are under the pressure of increasing anthropogenic activities, unregulated mining, agricultural and urban development. We report here a complete inventory of mercury (Hg) levels and speciation in the water column, atmosphere, sediment and key sentinel organisms (i.e., plankton, fish and birds) of two endorheic Lakes of the same watershed differing with respect to their size, eutrophication and contamination levels. Total Hg (THg) and monomethylmercury (MMHg) concentrations in filtered water and sediment of Lake Titicaca are in the lowest range of reported levels in other large lakes worldwide. Downstream, Hg levels are 3-10 times higher in the shallow eutrophic Lake Uru-Uru than in Lake Titicaca due to high Hg inputs from the surrounding mining region. High percentages of MMHg were found in the filtered and unfiltered water rising up from <1 to ∼50% THg from the oligo/hetero-trophic Lake Titicaca to the eutrophic Lake Uru-Uru. Such high %MMHg is explained by a high in situ MMHg production in relation to the sulfate rich substrate, the low oxygen levels of the water column, and the stabilization of MMHg due to abundant ligands present in these alkaline waters. Differences in MMHg concentrations in water and sediments compartments between Lake Titicaca and Uru-Uru were found to mirror the offset in MMHg levels that also exist in their respective food webs. This suggests that in situ MMHg baseline production is likely the main factor controlling MMHg levels in fish species consumed by the local population. Finally, the increase of anthropogenic pressure in Lake Titicaca may probably enhance eutrophication processes which favor MMHg production and thus accumulation in water and biota.

Synergistic effects of mining and urban effluents on the level and distribution of methylmercury in a shallow aquatic ecosystem of the Bolivian Altiplano.

Lake Uru Uru (3686 m a.s.l.) located in the Bolivian Altiplano region receives both mining effluents and urban wastewater discharges originating from the surrounding local cities which are under rapid development. We followed the spatiotemporal distribution of different mercury (Hg) compounds and other metal(oid)s (e.g., Fe, Mn, Sb, Ti and W) in both water and sediments during the wet and dry seasons along a north-south transect of this shallow lake system. Along the transect, the highest Hg and metal(oid) concentrations in both water and sediments were found downstream of the confluences with mining effluents. Although a dilution effect was found for major elements during the wet season, mean Hg and metal(oid) concentrations did not significantly differ from the dry season due to the increase in acid mine drainage (AMD) inputs into the lake from upstream mining areas. In particular, high filtered (<0.45 µm) mono-methylmercury (MMHg) concentrations (0.69 ± 0.47 ng L-1) were measured in surface water representing 49 ± 11% of the total filtered Hg concentrations (THgF) for both seasons. Enhanced MMHg lability in relation with the water alkalinity, coupled with abundant organic ligands and colloids (especially for downstream mining effluents), are likely factors favoring Hg methylation and MMHg preservation while inhibiting MMHg photodegradation. Lake sediments were identified as the major source of MMHg for the shallow water column. During the dry season, diffusive fluxes were estimated to be 227 ng m-2 d-1 for MMHg. This contribution was found to be negligible during the wet season due to a probable shift of the redox front downwards in the sediments. During the wet season, the results obtained suggest that various sources such as mining effluents and benthic or macrophytic biofilms significantly contribute to MMHg inputs in the water column. This work demonstrates the seasonally dependent synergistic effect of AMD and urban effluents on the shallow, productive and evaporative high altitude lake ecosystems which promotes the formation of natural organometallic toxins such as MMHg in the water column.

Low total mercury in Caiman yacare (Alligatoridae) as compared to carnivorous, and non-carnivorous fish consumed by Amazonian indigenous communities.

Mercury contamination in the River Beni basin is an important health risk factor, primarily for indigenous communities that live along the river. Among them are the Tacana, living in their original territory with sustainable use of their natural resources, consuming fish, Caiman yacare, and other riverine resources as their main source of protein. To assess mercury exposure to Tacana people, total mercury (THg) was evaluated in the muscle of seven commercial fish, and Caiman yacare (yacare caiman) during 2007 and 2008. THg was extracted by acid digestion and concentrations were determined by atomic absorption spectrometry. Mean mercury concentrations in C. yacare was 0.21 ± 0.22 µg g-1Hg w.w. (wet weight), which is lower than expected given its high trophic level, and its long life-span. It is possible that mercury in C. yacare is accumulated in other organs, not included in this study; but it is also possible that physiological mechanisms are involved that help caimans get rid of ingested mercury, or simply that C. yacare's diverse diet reduces THg accumulation. Carnivorous fishes (Pygocentrus nattereri, Pseudoplatystoma tigrinum, Zungaro zungaro, Plagioscion squamosissimus, and Leiarius marmoratus) had the highest total mercury concentrations, ranging from 0.35 to 1.27 µg g-1Hg w.w. moreover, most were above the limit recommended by WHO (0.5 µg g-1Hg w.w.); except for Leiarius marmuratus, which presented a mean of 0.353 ± 0.322 µg g-1Hg w.w. The two non-carnivorous fish species (Prochilodus nigricans, and Piaractus brachypomus) present mean concentrations of 0.099 ± 0.027, and 0.041 ± 0.019 µg g-1Hg w.w., respectively. Finally, recommendations on the consumption habits of Tacana communities are discussed.

Diurnal variability and biogeochemical reactivity of mercury species in an extreme high-altitude lake ecosystem of the Bolivian Altiplano.

Methylation and demethylation represent major transformation pathways regulating the net production of methylmercury (MMHg). Very few studies have documented Hg reactivity and transformation in extreme high-altitude lake ecosystems. Mercury (Hg) species concentrations (IHg, MMHg, Hg°, and DMHg) and in situ Hg methylation (M) and MMHg demethylation (D) potentials were determined in water, sediment, floating organic aggregates, and periphyton compartments of a shallow productive Lake of the Bolivian Altiplano (Uru Uru Lake, 3686 m). Samples were collected during late dry season (October 2010) and late wet season (May 2011) at a north (NS) and a south (SS) site of the lake, respectively. Mercury species concentrations exhibited significant diurnal variability as influenced by the strong diurnal biogeochemical gradients. Particularly high methylated mercury concentrations (0.2 to 4.5 ng L(-1) for MMHgT) were determined in the water column evidencing important Hg methylation in this ecosystem. Methylation and D potentials range were, respectively, <0.1-16.5 and <0.2-68.3 % day(-1) and were highly variable among compartments of the lake, but always higher during the dry season. Net Hg M indicates that the influence of urban and mining effluent (NS) promotes MMHg production in both water (up to 0.45 ng MMHg L(-1) day(-1)) and sediment compartments (2.0 to 19.7 ng MMHg g(-1) day(-1)). While the sediment compartment appears to represent a major source of MMHg in this shallow ecosystem, floating organic aggregates (dry season, SS) and Totora's periphyton (wet season, NS) were found to act as a significant source (5.8 ng MMHg g(-1) day(-1)) and a sink (-2.1 ng MMHg g(-1) day(-1)) of MMHg, respectively. This work demonstrates that high-altitude productive lake ecosystems can promote MMHg formation in various compartments supporting recent observations of high Hg contents in fish and water birds.

Freezability of Andalusian donkey (Equus asinus) spermatozoa: effect of extenders and permeating cryoprotectants.

The aim of this study was to compare the effect of two semen extenders and four permeating cryoprotectants on post-thaw sperm quality of Andalusian donkeys. First, 32 ejaculates were pooled, split and frozen in either Gent B or INRA 96 with egg yolk and glycerol. Second, 12 pooled semen samples were simultaneously frozen in Gent B (glycerol) or Gent A containing ethylene glycol (EG; 1 or 1.5%) or dimethyl sulfoxide (DMSO; 1.5 or 2%). Finally, nine pooled samples were simultaneously cryopreserved in Gent A containing 1% EG (as control), dimethylformamide (DMFA; 1 or 2.5%) or a combination of 1% EG and 1.5% DMFA. Gent B yielded a higher (P<0.01) post-thaw sperm motility than modified INRA96. EG 1% increased the sperm membrane integrity (P<0.001), whereas DMSO affected sperm motility and membrane integrity (P<0.001). DMFA 2.5% yielded higher (P<0.001) values for sperm motility and membrane integrity. We concluded that Gent B improves in vitro post-thaw sperm quality of donkey spermatozoa, but the replacement of glycerol with 1% EG or 2.5% DMFA increased sperm protection against cryodamage. The use of DMSO for freezing donkey semen was unsuccessful and a toxic effect is suspected. These extenders should be included in the pre-freeze test for each donkey.

Effect of single-layer centrifugation or washing on frozen-thawed donkey semen quality: Do they have the same effect regardless of the quality of the sample?

The aims of this study were to determine the sperm quality of frozen-thawed donkey sperm samples after single-layer centrifugation (SLC) using Androcoll-E in comparison to sperm washing or no centrifugation and to determine if the effect on the sperm quality after SLC or sperm washing depends on the quality of the sample. Frozen-thawed sperm samples from Andalusian donkeys were divided into three aliquots, and they were processed using three different techniques after thawing: uncentrifuged diluted control (UDC), sperm washing (SW), and SLC. Afterward, sperm quality index was estimated by integrating all parameters (total and progressive sperm motility, membrane integrity, and DNA fragmentation) in a single value. The relationship between the sperm quality of thawed UDC samples and the effect on sperm parameters in SW and SLC-selected samples was assessed. Sperm quality index was significantly higher (P < 0.001) in SLC (0.8 ± 0.0) samples than that in UDC (0.6 ± 0.0) and SW (0.6 ± 0.0) samples, regardless of the sperm quality index after thawing of the sperm sample. In conclusion, SLC of frozen-thawed donkey spermatozoa using Androcoll-E-Small can be a suitable procedure for selecting frozen-thawed donkey sperm with better quality, in particular in those samples where an improvement in motility is needed.

Colloid single-layer centrifugation improves post-thaw donkey (Equus asinus) sperm quality and is related to ejaculate freezability.

The aim of this study was to determine whether colloid single-layer centrifugation (SLC) improves post-thaw donkey sperm quality and if this potential enhancement is related to ejaculate freezability. Semen from Andalusian donkeys was frozen following a standard protocol. SLC was performed on frozen-thawed semen and post-thaw sperm parameters were compared with uncentrifuged samples. Sperm quality was estimated by integrating in a single value sperm motility (assessed by computer-assisted sperm analysis), morphology and viability (evaluated under brightfield or fluorescence microscopy). Sperm freezability was calculated as the relationship between sperm quality obtained before freezing and after thawing. Ejaculates were classified into low, medium and high freezability groups using the 25th and 75th percentiles as thresholds. All sperm parameters were significantly (P<0.01) higher in SLC-selected samples in comparison to uncentrifuged frozen-thawed semen and several kinematic parameters were even higher than those obtained in fresh semen. The increment of sperm parameters after SLC selection was correlated with ejaculate freezability, obtaining the highest values after SLC in semen samples with low freezability. We concluded that, based on the sperm-quality parameters evaluated, SLC can be a suitable procedure to improve post-thaw sperm quality of cryopreserved donkey semen, in particular for those ejaculates with low freezability.

Effect of extender and amino acid supplementation on sperm quality of cooled-preserved Andalusian donkey (Equus asinus) spermatozoa.

The main aim of this study was to evaluate the efficacy of two commercially available liquid stallion semen extenders for the preservation of Andalusian donkey semen at 5°C for up to 72h, and to evaluate the effect of amino acid addition on sperm quality of cooled donkey semen. In addition, this study investigated the effect of seasons on semen characteristics of Andalusian jackasses. Throughout a year, 50 ejaculates were collected from ten adult donkeys and a complete semen evaluation was performed immediately after collection. In Experiment 1, semen samples (n=32) were pooled, divided into two aliquots, and cooled in either Gent(®) A or INRA 96(®). In Experiment 2, pooled semen samples (n=9) were cooled in Gent A(®) supplemented with 0 (as control), 20, 40, or 60mM for each glutamine, proline, or taurine. Fresh semen and chilled samples were assessed for sperm motility, morphology, acrosome integrity, and plasma membrane integrity. Sperm motility variables were greater (P<0.05) in Gent(®) A than in INRA 96(®). The presence of glutamine, proline, or taurine in Gent(®) A improved (P<0.001) the motility of Andalusian donkey spermatozoa. Differences (P<0.05) in some sperm variables were observed among seasons. In conclusion, Gent(®) A maintained sperm motility characteristics after 72h of cold storage to a greater extent than INRA 96(®). Moreover, motility was greater when Gent(®) A supplemented at different concentrations of amino acids than Gent(®) A with no supplementation. An effect of seasons on the semen quality of the Andalusian donkey was demonstrated.

Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses.

The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Significant differences (P<0.05) were found between SLC-selected and unselected semen samples for all parameters assessed, obtaining better results after SLC. In the second experiment, semen samples were classified in two groups according to their sperm progressive motility (PM) before SLC. Then, the increments obtained in semen quality parameters after SLC were compared between groups. No significant differences were found between groups, indicating that SLC improved the sperm quality parameters of entire set of semen samples processed with independence to their original PM. In conclusion, SLC with Androcoll-E-Large can be used in donkeys, increasing the sperm quality of cooled-stored donkey semen doses after 24 h of cool storage.

Relationship between conventional semen characteristics, sperm motility patterns and fertility of Andalusian donkeys (Equus asinus).

Sperm quality has an important role in determining fertility. The aims of this study were to compare the conventional sperm parameters, plus the characteristics of the motility patterns of the different sperm subpopulations, of donkey donors with different fertility level, and to determine their relationships to fertility. Thirty ejaculates from 6 Andalusian donkeys were assessed for gel-free volume, pH, sperm concentration, motility and morphology. The fertility of donkeys was classified on the basis of pregnancy rates per cycle, where donkeys with a per cycle pregnancy rate ≥60% were considered to be "fertile" (n=3) and those with a per cycle pregnancy rate <40% were categorized to be "sub-fertile" (n=3). Significant differences (P<0.001) between the "fertile" and the "sub-fertile" group were found for total and progressive motility, and for straight line velocity. Sperm variables associated (P<0.05) with an increase in percent pregnant per cycle included total motility (r=0.37), progressive motility (r=0.53), curvilinear velocity (r=0.44), straightness (r=0.39), beat cross frequency (r=0.44), and gel-free volume (r=0.53). Four sperm subpopulations (sP) were identified in fresh semen: sP1 (slow and non-progressive spermatozoa, 20%), sP2 (moderately slow but progressive spermatozoa, 71.2%), sP3 (highly active but non-progressive spermatozoa, 2.9%), and sP4 (highly active and progressive spermatozoa, 5.9%). The lowest percentage (3.1%; P<0.001) of sP4 spermatozoa was observed in the "sub-fertile" group. Three of the sperm subpopulations were related (P<0.05) to fertility (sP2, r=0.54; sP3, r=0.45; sP4, r=0.56). In conclusion, we were able to relate the fertility of donkeys with in vitro measures of sperm motility using computer-assisted sperm analysis techniques.

Single-layer centrifugation through PureSperm® 80 selects improved quality spermatozoa from frozen-thawed dog semen.

The aim of this study was to investigate whether single-layer centrifugation (SLC) with PureSperm® 80 could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were divided into two aliquots: one of them was used as control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, frozen-thawed control and frozen-thawed SLC treated samples. A multivariate clustering procedure separated 26,051 motile spermatozoa into three subpopulations (sP): sP1 consisting of highly active but non-progressive spermatozoa (40.3%), sP2 consisting of spermatozoa with high velocity and progressive motility (30.0%), and sP3 consisting of poorly active and non-progressive spermatozoa (29.7%). SLC with PureSperm® 80 yielded sperm suspensions with improved motility, morphology, viability and acrosome integrity (P<0.001). The frozen-thawed SLC treated samples were enriched in sP2, reaching a proportion of 44.1% of the present spermatozoa. From these results, we concluded that SLC with PureSperm® 80 may be an alternative and successful method for improving the quality of frozen-thawed dog spermatozoa. Moreover, sP2 (high-speed and progressive spermatozoa) was more frequently observed after SLC. Finally, this study also demonstrated that the general motile sperm structure present in dogs remained constant despite the effect caused by either cryopreservation or separation by SLC through PureSperm® 80.

Sperm motility patterns in Andalusian donkey (Equus asinus) semen: effects of body weight, age, and semen quality.

The aims of this study were to (1) identify sperm subpopulations with specific motion characteristics in fresh Andalusian donkey ejaculates; (2) evaluate the effects of individual donkey and ejaculates within the same donkey on the distribution of the subpopulations found; and (3) explore the relationship between the age and the body weight of donkey donors, the sperm quality parameters, and the sperm subpopulations structure. Sixty ejaculates from 12 Andalusian donkeys (five ejaculates per donkey), ranging in age from 4 to 15 years, were collected. Immediately after collection, sperm characteristics (volume, sperm concentration, objective sperm motility, and sperm morphology) were assessed. Donkeys were evaluated for body weight. Significant (P < 0.05) correlations were established between the body weight of the donkeys and the pH (r = -0.52), sperm motility (percentage of motile spermatozoa: r = -0.31; percentage of progressive motile spermatozoa: r = -0.34), and total sperm abnormalities (r = 0.38). The correlations of the age with the measures of semen quality were low and not significant (P > 0.05). A multivariate clustering procedure separated 65,342 motile spermatozoa into four subpopulations: subpopulation 1, consisting of slow and nonprogressive spermatozoa (15.4%), subpopulation 2, consisting of moderately slow but progressive spermatozoa (35.9%), subpopulation 3, consisting of highly active but nonprogressive spermatozoa (18.5%), and subpopulation 4, consisting of highly active and progressive spermatozoa (30.2%). The distribution of these subpopulations varied significantly (P < 0.05) according to several parameters such as the individual donkey, the ejaculate of the same donkey, the total motility, and the overall sperm concentration. Our results show the existence of four well-defined motile sperm subpopulations in Andalusian donkey ejaculates, and suggest a high heterogeneity in the ejaculate structure in donkey. The relationship between the distribution of the sperm subpopulations and individual donkey, total motility, and sperm concentration shows that the spermatozoa of each have different motility patterns. However, the proportions of sperm subpopulations in the ejaculates did not vary with age and body weight. Finally, the study of discrete subpopulations of motile spermatozoa could lead to a substantial increase in information acquired during donkey semen analysis.

Changes in the structures of motile sperm subpopulations in dog spermatozoa after both cryopreservation and centrifugation on PureSperm(®) gradient.

The aims of the present study were to: (1) determine if discrete motile sperm subpopulations exist and their incidence in fresh dog ejaculates, (2) evaluate the effects of cryopreservation on the distribution of spermatozoa within the different subpopulations, and (3) determine the effect of the discontinuous PureSperm(®) gradient on the sperm subpopulation structure of frozen-thawed dog spermatozoa. Semen from 5 dogs were collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm(®). Sperm motility (assessed by computerized-assisted semen analysis, CASA) was assessed before freezing, just after thawing and after preparation on the PureSperm(®) gradients. Cryopreservation had a significant (P<0.001) effect on CASA-derived parameters. PureSperm(®) centrifugation yielded sperm suspensions with improved motility (P<0.01). A multivariate clustering procedure separated 19414 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (20.97%), Subpopulation 2 consisting of slow and low-linear spermatozoa (18.24%), Subpopulation 3 consisting of highly active but non-progressive spermatozoa (20.75%), and Subpopulation 4 consisting of high speed and progressive spermatozoa (40.03%). Although, cryopreservation had a significant (P<0.001) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after freezing and thawing. The selected sperm samples was enrich in Subpopulation 4, reaching a proportion of 31.9% of the present spermatozoa, in contrast with the unselected sperm samples, where this sperm subpopulation accounted for 24.9% of the total. From these results, we concluded that four well-defined motile sperm subpopulations were present either in fresh semen, in unselected sperm samples or in selected preparations from dogs. The discontinuous PureSperm(®) gradient is a simple method to improve the quality of canine frozen-thawed semen samples, since Subpopulation 4 (high-speed and progressive spermatozoa) was more frequently observed after preparation on the gradient. Finally, this study also demonstrated that the general motile sperm structure present in dog remains constant despite the effect caused by either cryopreservation or separation on PureSperm(®) gradient.

Centrifugation on PureSperm(®) density-gradient improved quality of spermatozoa from frozen-thawed dog semen.

The main objective of this study was to investigate if centrifugation through PureSperm(®) density-gradient can improve the post-thaw semen quality of dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm(®). Assessments of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of Propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, unselected samples and selected preparations. Cryopreservation had a significant (P < 0.001) effect on all studied semen parameters. PureSperm(®) centrifugation yielded sperm suspensions with improved motility and viability (P < 0.001). The washing step significantly reduced (P < 0.001) all of the kinematics parameters evaluated as well as reduced the proportion of viable spermatozoa with intact acrosomes (P < 0.05). We concluded that PureSperm(®) centrifugation is a successful method for improving the quality of frozen-thawed dog spermatozoa. However, washing after density-gradient centrifugation dramatically reduces the post-thaw semen quality, indicating that the inclusion of such a washing step is unnecessary.