Methylene blue-mediated antimicrobial photodynamic therapy on chicken semen.

BACKGROUND: Artificial insemination is widely employed in poultry, but high degrees of bacterial contamination are often observed in semen because of its passage through the cloaca. Consequently, most semen extenders for birds have antibiotics that could aggravate bacterial resistance. METHODS: We evaluated the potential of antimicrobial photodynamic therapy (PDT) as an alternative to the use of antibiotics, and assessed whether changes in concentration and incubation time with methylene blue (MB), radiant exposure, and irradiance of light affect spermatozoa activity and bacteria in chicken semen. RESULTS: Incubation with MB (< 25 µM) did not alter sperm motility, regardless of the pre-irradiation time (PIT, 1 or 5 min). Following 1 min of PIT with MB at 10 µM, samples were irradiated for 30, 60, 120, and 180 s at irradiances of 44, 29, and 17 mW/ cm² (660 nm LedBox). MB and light alone did not interfere with the analyzed parameters. However, when both factors were associated, increases in light dose led to greater reductions in sperm parameters, regardless of the irradiance used. Besides, PDT conditions that were less harmful to spermatozoa were not able to significantly reduce bacterial colonies in chicken semen. CONCLUSIONS: A failure in MB selectivity could explain unsuccessful bacterial reduction following PDT. Further research involving other photosensitizers or conjugating molecules to MB to target microbial cells is needed for PDT application in poultry breeders.

Insights on sperm assays and cryopreservation in six Neotropical pit vipers.

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane - EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.

Insights on sperm assays and cryopreservation in six Neotropical pit vipers

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane – EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.