Viability assessment of spermatozoa in large falcons (Falco spp.) using various staining protocols.

Viability assessment is an important part of semen analysis, and various live/dead staining protocols have been used in semen of avian species. Results of live/dead count differed between dyes, staining protocols and bird species, impeding comparability between studies and requiring species-specific comparisons of viability stains. In raptor semen, similar comparisons are absent. Thus, the aim of the present study was to compare eight conventional viability stains. Eosin blue 2% [EB], eosin blue 2% with the addition of 3% sodium citrate [EB2], eosin blue-nigrosin 5% [EBN5], eosin yellow-nigrosin 5% [EYN5], eosin yellow-nigrosin 10% [EYN10], eosin blue-aniline blue [EBA], eosin yellow-aniline blue [EYA] and bromophenol blue-nigrosin [BBN] were evaluated in comparison with the fluorescence stain SYBR® Green-propidium iodide [SYBR-PI] in spermatozoa of falcons. The comparison was performed using conventional light microscopy which is applicable in breeding centres, veterinary practices and field studies. Additionally, live/dead stains were correlated to motility values of the same samples to validate sperm viability. Light microscopy using EB and using SYBR-PI enabled an effective and clear differentiation between alive and dead spermatozoa of falcons. Motility values correlated significantly and strongly with EB only (r = .629; p < .001), but not with any other stain used in the study. Therefore, our results suggest EB as the most suitable stain for viability assessment in the semen of large falcons.

Computer-assisted semen analysis in large falcons (Falco spp.) and comparison to results of conventional semen analysis

OBJECTIVE: Numerous raptor species including some falcon species are facing continuous population decline in the wild and some are threatened by extinction. To support these species, captive breeding and reintroduction programs are attempted. Besides conservation, some large falcon species are commonly used in falconry and therefore bred commercially. Assisted reproduction is established in falcon breeding since the 1970s and semen analysis is an integral part to enable assessment of breeding males, inclusion or exclusion of semen donors and quality control of semen prior to artificial insemination. Conventional methods for semen analysis are widely used, but are time consuming and depend on the investigator's experience and ability. Computer-assisted semen analysis (CASA) would offer an objective, fast and reproducible alternative, but as they have not been established in large falcon species, this was the aim of this study. MATERIAL AND METHODS: To this end, we examined in 3 breeding seasons 109 semen samples of gyr-saker hybrid falcons (n=2) and peregrine falcons (n=4) in 940 fields of view using the Minitube CASA SpermVision and compared these results to results of conventional methods of semen analysis. We used a preprogrammed setting and adapted two settings of CASA according to specific semen characteristics of falcons. RESULTS: Sperm velocity, motility and viability parameters were recorded successfully using CASA. Correlation of conventional and computer-assisted motility analysis improved during the process of adaptation of CASA settings, but both methods differed significantly due to misinterpretation of round bodies and semen impurities by CASA. Viability values of conventional and computer assisted viability analysis using SYBR-PI correlated significantly while sperm concentration did not at all. CONCLUSION: CASA failed to replace conventional semen analysis regarding sperm motility and sperm concentration using 3 different settings, as a reliable differentiation between spermatozoa, spermatids and round bodies was not achieved. CLINICAL RELEVANCE: Using CASA, sperm velocity parameters were measured in spermatozoa of captive-bred large falcons for the first time and may be used as orientation values.

Modification and Clinical Application of the Inner Perivitelline Membrane Test in Different Avian Species.

The aim of this study was to adapt an inner perivitelline membrane (IPVM) test as an interspecies penetration assay for avian spermatozoa. The IPVM of different bird species was evaluated to test the penetrating ability of avian spermatozoa in an intra- and interspecies design. Isolation of the IPVM via acid hydrolysis was tested in pre-incubated chicken eggs and in six other avian species. The separation protocol was modified (time, acid concentration) to facilitate practicability. Separated membranes were evaluated with dark field microscopy for the presence of holes produced by penetrating spermatozoa. In chicken eggs, the influence of different membrane storage conditions was tested. In the penetration assay, the IPVM of chicken eggs was used as a model for fresh and frozen-thawed rooster sperm and for fresh spermatozoa of cockatiels and falcons. Results demonstrated that the time of egg-incubation had a significantly negative influence on the isolation ability of the IPVM (p < 0.0001). IPVM-separation was successful for a maximum of two days after preincubation. In the experiments with eggs from other avian species, results were heterogenous: there was no isolation in geese and cockatiels, 20% in the European kestrel, and 40% in pheasant, quail, and duck. In the penetration assay, holes were found in 100% of the IPVM of chicken eggs after incubation with native and frozen-thawed rooster semen and in 10% with fresh cockatiel semen. Falcon spermatozoa failed to produce visible holes. In conclusion, the IPVM of chicken eggs seems to be unsuitable to establish a functional sperm assay in other species tested but is suitable for quality evaluation of cryopreserved rooster sperm.

Potassic zeolites from Brazilian coal ash for use as a fertilizer in agriculture.

Brazilian coal has an ash content ranging from 30 to 50% by weight. Consequently, its use in coal-fired thermoelectric for power production generates a lot of waste. The construction sector is the largest consumer of coal ash, but it cannot absorb the entire amount generated. Thus, other applications for coal ash should be studied in aim to optimize the use of this industrial waste. This research had as focus to synthesize potassic zeolite from of the coal ash into on potassium fertilizer for the grown wheat plant. In this work, it was used a subbituminous coal from Mina do Leão (RS, Brazil) presenting 48.7% ash content on a dry basis. Concerning the synthesis of potassic zeolite, it was adopted the conventional method of hydrothermal treatment with potassium hydroxide. A schedule of experiments was conducted in order to define the optimum condition of zeolite synthesis that was then used an alkaline solution of 5M KOH with a reaction time of 24h at 150°C. According to this procedure, it was obtained a zeolite with a single crystalline phase, identified through X-ray diffraction as Merlinoite. Subsequently, it was performed a set of tests using potassic zeolite asa fertilizer for plants in a greenhouse. The synthesized potassic zeolite showed a good potential for its use as fertilizer in agriculture.

Impact of protective agents and drying methods on desiccation tolerance of Salix nigra L. seeds.

Willow seeds are classified as orthodox, but they show some recalcitrant characteristics, as they lose viability in a few weeks at room temperature. The aim of this work was to improve the desiccation tolerance of willow seeds (Salix nigra L.), as a model of sensitive materials to dehydration, through imbibition in solutions and later vacuum (VD) or freeze-drying (FD). Imbibition was conducted with 45% w/v trehalose or polyethylene glycol 400 -PEG- or water prior to dehydration treatments. Water- and especially trehalose-imbibed seeds subjected to VD showed better germination capability with respect to the freeze-dried ones. Water crystallization was mainly responsible for the great loss of capability germination observed in water- or trehalose-imbibed seeds subjected to FD. PEG behavior was better when seeds were FD instead of VD. DSC thermograms of seeds allowed to identify two thermal transitions corresponding to lipids melting and to proteins denaturation. This last transition reveals information about proteins state/functionality. Dehydration of control and PEG- or water-imbibed seeds affected proteins functionality leading to lower germinability. In the case of trehalose-imbibed seeds subjected to VD, proteins maintained their native state along dehydration, and the seeds showed a great germination capacity for all the water content range. Germinated seeds showed higher luminosity (L*), greenness (a*) and yellowness (b*) values than not-germinated seeds independently of the employed agent. Present work reveals that the presence of adequate protective agents as well the dehydration method were the main critical factors involved in willow seed desiccation tolerance.