RESUMEN

The objectives of the study were to (1) describe the kinematic parameters of spermatozoa (2) compare methods of evaluating sperm viability (3) validate assays of functionality and integrity of the sperm membrane and (4) evaluate possible changes between spermatozoa from the epididymis and the vas deferens of the greater rhea. Semen samples were recovered from 7 adult individuals. Sperm motility was characterized by adjusting the set-up for Computer-assisted semen analysis (CASA) to that new species. For sperm viability evaluation, smears of bromophenol blue and eosin-nigrosine dyes were used. Five solutions of different osmolarities were then tested for the hypoosmotic swelling test (HOST). The combination of fluorescent probes (propidium iodide - IP and Hoechst 33342) was also used to assess plasma membrane integrity. Data were presented as mean ± SEM. Rhea spermatozoa from the vas deferens had an overall motility of 14.6 ± 2.5%. The bromophenol blue staining technique revealed that 64.6 ± 5.2% sperm were viable, while that proportion was 72.1 ± 2.5% using eosin-nigrosine. An average of 77.6 ± 4.8% of spermatozoa reacted to the HOST with distilled water at 0 mOsm/l. Fluorescent probes indicated that 65.3 ± 2.6% of spermatozoa had intact membranes. Interestingly, no statistical differences were observed between the parameters analyzed in the epididymal spermatozoa and the vas deferens. These new assays set reference values that can now be used to further exploration of sperm handling conditions and freezing protocols in rheas.

RESUMEN

It is unquestinable that artificial insemination (AI) offers many benefits to avian conservation programs, but a serious impediment towards implementing AI for wild species is the development of effective techniques to consistently collect good quality ejaculates. Thus, we aimed to examine the success rate of electro-stimulation (ES) in collecting semen from 49 unconditioned males from orders Piciformes, Strigiformes, Accipitriformes, Cathartiformes, Galiformes, Anseriformes and Psittaciformes at different times of the year. Sixty out of 299 ES attempts provided ejaculates with sperm, but collection success rates varied widely (0-50%) depending on the species. Except for swans whose greater results were registered during spring-summer, males from most orders responded better to ES during winter-spring, suggesting seasonal variations on semen collection success rates. Overall, ES enabled successful semen collection from males of unproven and proven fertility under mixed pairing conditions. However, the highest success rate occurred in paired males with fertile clutches (40.6%) followed by unpaired males (22.1%), paired males without clutches (13.9%), and paired males with infertile clutches (6.8%). Behavioral responses of male birds to electrical impulses were also recorded to assess any discomfort during semen collection. Furthermore, macroscopic and microscopic analysis provided ejaculate parameters from several species, even from orders that hitherto have never been assessed for semen collection, which may serve as a starting point in the future. Altogether, these findings demonstrate the feasibility of ES in collecting semen from unpaired, unconditioned and non-imprinted males from a variety of bird orders. In the medium to long term, the use of this technique in both captive and free-ranging populations offers new perspectives to ensure genetic diversity in avian conservation programs.

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RESUMEN

Several methods have been developed to evaluate spermatozoa function in birds but many of these are sometimes complicated, costly and not applicable to field studies (i.e., performed within poultry breeding facilities). The objective was, therefore, to validate efficient, practical and inexpensive procedures to determine DNA fragmentation, acrosomal integrity, and mitochondrial activity in poultry spermatozoa. Initially, ejaculates were individually diluted and divided into control (4°C, 4h) and UV-irradiated aliquots (room temperature, 4h), and then samples containing different percentages of DNA-damaged spermatozoa (0%, 25%, 50%, 75% and 100%) were subjected to Toluidine Blue (TB) and Sperm Chromatin Dispersion assessments (SCD). Fast Green-Rose Bengal (FG-RB) and FITC-PSA staining protocols were subsequently used to assess acrosome status in aliquots comprising assorted amounts of acrosome-reacted spermatozoa. Furthermore, to validate 3,3'-diaminobenzidine (DAB) assay, ejaculates containing different gradients of spermatozoa with great amounts of mitochondrial activity were concurrently evaluated using DAB and JC-1 stains. The proportion of spermatozoa with abnormal DNA integrity when evaluated using the TB assessment correlated significantly with the expected percentages of UV-irradiated spermatozoa and with SCD results. A significant linear regression coefficient was also observed between expected amounts of acrosome-intact spermatozoa and FG-RB readings, and there was a significant correlation of the data when FG-RB and FITC-PSA were used. Likewise, the use of the DAB assay enabled for accurately ascertaining percentages of rooster spermatozoa with greater and lesser mitochondrial function, and results were highly correlated to results with staining with JC-1. Altogether, findings of the present study indicate acrosomal status, DNA integrity and mitochondrial activity in rooster spermatozoa can be easily and reliably determined using FG-RB, TB and DAB stains.

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