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The freeze-thawing process induces osmotic changes that may affect the membrane domain location of aquaporins' (AQP) in spermatozoa. Recent studies suggest that changes in AQP3 localization allows better sperm osmo-adaptation, improving the cryoresistance. Ultra-rapid freezing is an alternative cryopreservation technique that requires less equipment than conventional freezing, and it is faster, simpler and can be used in the field. This study aimed to determine the influence of freezing-thawing rates (slow (control) vs. ultra-rapid) on AQP3 expression and location in the spermatozoa from small ruminants (sheep and goats) and its relationship with sperm cryo-damage. Spermatozoa were collected from 10 Merino rams and 10 Murciano-Granadina bucks. The presence and distribution of AQP3 were assessed by Western blotting and immunocytochemistry (ICC), employing a commercial rabbit polyclonal antibody. Sperm motility was CASA system-analyzed, and membrane and acrosome integrity assessed by fluorescence (PI/PNA-FITC). Western blotting did not detect a significant effect of freezing-thawing rate on the amount of AQP3 while ICC found freezing-thawing rate affecting AQP3 location (P < 0.05). In both species, the percentages of spermatozoa showing AQP3 in the post-acrosome region, mid-piece, and principal piece of the tail were greater in samples cryopreserved by slow freezing-thawing (control) than ultra-rapid freezing-thawing rates (P < 0.05). Spermatozoa cryopreserved using ultra-rapid freezing-thawing showed decrease motility, plasma membrane, and acrosome integrity (P < 0.05), which might be related, at least in part, to a lower expression of AQP3. In conclusion, the cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat, which might be associated with sperm cryosurvival. • This is the first study identifying AQP3 in spermatozoa of goat. • Cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat. • Changes in the location of AQP3 appears to be related to sperm cryosurvival. [ABSTRACT FROM AUTHOR]

Inhalation exposure to fine particulate matter (PM 2.5) represents a global concern due to the adverse effects in human health. In the last years, scientific community has been adopted the assessment of the PM 2.5 -bound pollutant fraction that could be released (bioaccessible fraction) in simulated lung fluids (SLFs) to achieve a better understanding of PM risk assessment and toxicological studies. Thus, bioaccessibility of 49 organic pollutants, including 18 polycyclic aromatic hydrocarbons (PAHs), 12 phthalate esters (PAEs), 11 organophosphorus flame retardants (OPFRs), 6 synthetic musk compounds (SMCs) and 2 bisphenols in PM 2.5 samples was evaluated. The proposed method consists of a physiologically based extraction test (PBET) by using artificial lysosomal fluid (ALF) to obtain bioaccessible fractions, followed by a vortex-assisted liquid-liquid microextraction (VALLME) and a final analysis by programmed temperature vaporization-gas chromatography-tandem mass spectrometry (PTV-GC-MS/MS). The highest inhalation bioaccessibility ratio was found for bisphenol A (BPA) with an average of 83%, followed by OPFRs, PAEs and PAHs (with average bioaccessibilities of 68%, 41% and 34%, respectively). Correlations between PM 2.5 composition (major ions, trace metals, equivalent black carbon (eBC) and UV-absorbing particulate matter (UVPM)) and bioaccessibility ratios were also assessed. Principal Component Analysis (PCA) suggested that PAHs, PAES and OPFRs bioaccessibility ratios could be positively correlated with PM 2.5 carbonaceous content. Furthermore, both inverse and positive correlations on PAHs, PAEs and OPFRs bioaccessibilites could be accounted for some major ions and metal (oid)s associated to PM 2.5 , whereas no correlations comprising considered PM 2.5 major ions and metal (oid)s contents and BPA bioaccessibility was observed. In addition, health risk assessment of target PM 2.5 -associated PAHs via inhalation was assessed in the study area considering both total and bioaccessible concentrations, being averaged human health risks within the safe carcinogenic and non-carcinogenic levels. [Display omitted] • Bisphenol A showed the highest inhalation bioaccessible ratio, accounting for 83%. • A correlation between logK ow and bioaccessibility was found for some compounds. • Bioaccessibilities seemed to be positively correlated with PM 2.5 carbon content. • Human health risks for PM 2.5 -bound PAHs via inhalation were within the safe range. [ABSTRACT FROM AUTHOR]

• In the conditions of the study, bucks were more seasonal than rams. • Plasma testosterone and thyroxine were negatively related to buck sperm freezability. • Plasma testosterone and thyroxine were unrelated to ram sperm cryoresistance. • For cryopreservation, it is recommended to collect buck sperm in spring and winter. This study examines the relationship between seasonal changes in plasma testosterone and thyroxine concentrations and the relationship between these changes and the sperm resistance to the freezing-thawing process in Merino x Merino Precoz crossbreed rams and Florida breed bucks. Plasma testosterone and thyroxine concentration, sperm head area, sperm variables and their cryoresistance ratios were analyzed throughout a year. Both species showed a rise in thyroxine preceding testosterone fall. Plasma thyroxine concentrations in rams were higher during autumn than in spring and summer (P <0.05), whereas testosterone concentrations were not influenced by season. Conversely, bucks showed seasonal differences in plasma testosterone levels, with higher concentrations during summer and autumn than winter and spring (P <0.05), whereas thyroxine concentrations were not influenced by season. In bucks, a negative correlation was detected between plasma testosterone concentrations and the cryoresistance ratio for membrane integrity (R=-0.88; P <0.05) from April to September and between plasma thyroxine concentrations and the cryoresistance ratios for total motility (R=-0.66; P <0.05), curvilinear velocity (R=-0.61; P <0.05), the amplitude of lateral head displacement (R=-0.77; P <0.05) and the beat-cross frequency (R=-0.74; P <0.05), throughout the year. The results suggest that the annual variation in sperm cryoresistance may be related to seasonal endocrine changes, at least in goat. Winter and spring are the most appropriate seasons to collect and freeze sperm from Florida goat. [ABSTRACT FROM AUTHOR]

The expression of aquaglyceroporin 3 (AQP-3) has been demonstrated in the spermatozoa of several mammalian species and its role has been associated with cryotolerance. Post-thaw sperm quality from individual dromedary males with different response to freezing-thawing process was evaluated through sperm head morphometry. In order to understand the cellular mechanisms affected by cryoinjury we have explored the presence and distribution of sperm AQP-3 using western blotting and immunocytochemistry. WB showed different intensity of the specific signal bands at 28 kDa. Immunofluorescence assessments allowed us to identify five different and clear AQP-3 distribution patterns of labelling in the sperm plasma membrane; acrosome, post-acrosome, mid-piece, and principal and final tail. Although expression of AQP-3 varied among male ejaculates, the individual sperm response to freeze-thawing was not associated with AQP-3 expression. Thus, AQP3 expressions do not seem like a reliable predictor of sperm response to freeze-thawing process in this species. This work is the first to describe the morphometric characteristics of the heads of dromedary spermatozoa. No correlation was found between sperm head dimensions and sperm quality variables after freeze-thawing suggesting that dromedary camel sperm head morphometry is also not a reliable predictor of cryosurvival. • The dromedary sperm response to freeze-thawing was not associated with aquaporin 3 expression. • The sperm head morphometry is not predictive of cryosurvival in dromedary camel. • Aquaporin 3 was identified in different regions of dromedary sperm. [ABSTRACT FROM AUTHOR]

The objective was to characterize the stress response and the seminal parameters obtained with electroejaculation (EE) or transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) in two captive but nondomestic ruminants, the mouflons and the Iberian ibex under general anesthesia. In mouflons, the physiological responses (heart and respiratory rate, rectal temperature, cortisol, creatine kinase, potassium and glucose concentrations) changed similarly with both procedures. The TUMASG procedure was faster than EE in mouflons (21.7 ± 1.4 vs. 12.4 ± 1.2 minutes, P < 0.01). In ibexes, respiratory rate, cortisol and creatine kinase concentration changes were greater with EE than with TUMASG (final respiratory rate: 62.7 ± 5.5 vs. 38.1 ± 5.6 breaths/min [P < 0.05]; final cortisol: 51.4 ± 5.1 vs. 25.3 ± 5.6 ng/mL [P < 0.001]; and final creatine kinase: 300.9 ± 99.9 vs. 87.1 ± 16.9 U/L [P < 0.001]). Electroejaculation provided better results in some sperm parameters (mouflons: sperm score: 3.4 ± 0.3 vs. 2.6 ± 0.2 [P < 0.01]; total number of sperm ejaculated: 982.4 ± 299 vs. 710.0 ± 542.2 [P < 0.05]; ibexes: sperm with progressive motility: 47.7 ± 6.2 vs. 20.5 ± 8.3 [P < 0.05]). The transrectal ultrasound-guided massage of the accessory sex glands appears to be an alternative technique to collect sperm from wild ruminants, reducing the need for electrical stimuli and thus decreasing the undesired responses of EE in the more sensitive species. On the other hand, better fresh sperm may be collected with EE. However, TUMASG provides practical advantages in animal welfare, firstly in these wild species more sensible to stress management and capture myopathy. [ABSTRACT FROM AUTHOR]