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.

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.

Beneficial Impact of Hypercapnic Conditions During Early Incubation on Broiler Hatchability. Embryo Mortality and Postnatal Performance

We assessed the extent to which CO2 levels altered different hatching and chick parameters. In Experiments 1 and 2, a total of 16,184 eggs from Cobb 500 breeders were incubated in single stage incubators under three different conditions: (a) standard ventilated incubator (CON, Exp.1 and 2); (b) increasing CO2 levels during the first 10 days of incubation until 0.7% (V7000, Exp. 1) and (c) until 0.8% (V8000, Exp. 2). High levels of CO2 improved hatchability, possibly due to lower embryo mortality from ED18 to ED21. Internal and external pipping in experiment V8000 started later than in CON; nevertheless, the hatch still occurred before in V8000 as a result of the shorter durations of external pipping and hatch. In Experiment 3, a total of 12,138 eggs from Cobb 500 were incubated in single stage incubators under three different conditions: (a) standard ventilated incubator (CON); (b) increasing CO2 levels until 1.0% with ventilation (V10000); and (c) increasing CO2 levels until 1.0% without ventilation (NV10000). Hypercapnic conditions led to better hatchability and lower embryo mortality from ED18 to ED21. Internal pipping started earlier in NV10000, but only V10000 differed from CON in terms of the average time for hatch. Hypercapnic groups also showed shorter durations of external pipping and hatch when compared to CON. Post-hatch analysis revealed no differences among incubation conditions in terms of body weight gain, feed conversion ratio, mortality by sudden death syndrome, and production factor. Nevertheless, V10000 showed a lower mortality by ascites and a better viability when compared to CON, while NV10000 presented a higher mortality by other causes. Altogether, our findings indicate that in addition to not being detrimental to embryo survival, high CO2 levels reduce embryonic mortality at 18-21 days of incubation and increase hatchability.(AU)

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.