Establishment of methods to analyze the structural and functional integrity of the quail (Coturnix coturnix japonica) sperm plasma membrane.

1. The objectives of this study were to establish the use of the fluorophores Hoechst 33342 and propidium iodide for the evaluation of sperm plasma membrane integrity and to identify an adequate hypoosmotic solution for the evaluation of sperm membrane functionality in quails.2. Sperm samples were collected from the vas deferens of nine quails. After initial evaluation, the samples were subjected to a flash-frozen assay. Three treatments with the following proportions of fresh sperm and sperm subjected to flash freezing were prepared as follows: 100:0 (T100), 50:50 (T50), and 0:100 (T0). The hypoosmotic swelling test used distilled water (0 mOsm/l) and fructose solutions (50, 100, and 200 mOsm/l).3. Immediately after recovery, the samples showed 75.6 ± 5.0% motility with vigour of 3.7 ± 0.3 and 96.1 ± 0.5% of the sperm appeared normal. The membrane integrity test showed 62.2 ± 5.2% intact sperm at T100, 29.0 ± 4.1% at T50 and 0.1 ± 0.1% at T0. Moreover, a greater number of reactive sperm (74.7 ± 6.7%) were observed when incubated in distilled water (0 mOsm/l) in comparison to other solutions (P < 0.05).4. The association of fluorescent probes composed of Hoechst 33342 and propidium iodide provided an efficient assessment of the integrity of the plasmatic membrane of quail spermatozoa. However, the study identified that the hypoosmotic swelling test has little predictive value regarding sperm membrane functionality in this species.

Characterization of the ovarian preantral follicle populations and its correlation with age and nutritional status in Brazilian Northeastern donkeys (Equus assinus).

This study aimed to characterize the ovarian follicular population and to determine its correlations with the age and nutritional status of donkeys of the Northeastern Brazilian breed. A total of 10 females with a mean age of 5.1±3.2years were submitted to ovariectomy by videolaparoscopy to obtain the ovaries. In the laboratory, the ovaries were sectioned into 6-12 fragments of approximately 7mm in diameter, which were fixed in Carnoy, dehydrated in increasing concentrations of alcohol (85%, 95% and absolute), clarified using xylol and embedded in blocks of histological paraffin. The blocks were cut in sections of 7µm and each 120th section was mounted on a slide for observation using optical microscopy. The follicle counting and identification allowed the characterization of the population of the preantral follicles. A total of 21.135±10.821 preantral follicles was counted, of which, 91.3% were primordial, 8.3% were primary follicles and 0.4% were secondary follicles. There were no differences between the two ovaries of each animal regarding the follicular population (P>0.05). There was a rate of 9.77% degenerated follicles. Values of 0.99% follicles containing two oocytes were also identified and classified as multi-oocyte follicles, always of the primordial category. The thickness of the granulosa cell layer was 1.85µm±1.39, 3.56µm±2.08 and 21.85µm±17.27, for primordial, primary and secondary follicles, respectively. There was a significant inverse correlation (r=-0.66; P<0.001) between the age of the animals and the population of ovarian follicles. A negative influence of the weight and body score on the ovarian follicular population was also observed, when donkeys had very little or a great amount of body condition. This is the first study to describe the morphometric characteristics and estimation of the population of preantral ovarian follicles in Northeastern Brazilian donkey, showing that number of preantral follicles decreased with increasing age of the animals and this finding may be affected by nutritional status of the animals.

Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina).

We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes-epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide - DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing-thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.