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Semen freezability is positive correlated with the cholesterol content in the sperm cell. Freeze-thawing mainly cause temperature chock and change on media osmolarity, which can modify plasma membrane lipids content and sperm conformation, resulting in decreased fertility. Therefore, the aim of this study is to investigate the effect of adding cholesterol-loaded cyclodextrin (CLC) to the cryopreservation process of ram semen with low freezability. For that, two experiments were performed using 5 ejaculates of 6 rams, totalizing 30 samples. For experiment 1 the following treatments were tested: in natura (IN), Tris solution (CON), CLC + Tris solution (CLC), and pure methyl-β-cyclodextrin + Tris solution (MCD). For experiment 2 treatments CON and CLC were tested in samples subdivided into three freezability classes: high (n = 10), intermediate (n = 10) and low (n = 10). Freezability classes were based on the variation of sperm motility between IN and CON groups from the first experiment. Sample analyzes included sperm motility, sperm morphology, plasma and acrosome membrane integrity, mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and fluidity of plasma membrane. Results showed that CLC treatment was more efficient in maintaining sperm motility, integrity of plasma membrane, integrity of acrosome, and mitochondria membrane potential. In addition, CLC treatment in the groups with low and intermediate freezability showed improvement on progressive motility and percentage of rapid cells. In contrast, no difference was noted between CLC and CON treatments in the high freezability group. Therefore, the addition of CLC to semen extender improved sperm cryopreservation, especially in rams with low freezability., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Reestablishment of testicular normal temperature after testicular heat stress is unknown and its effect varies widely. The aim of this study was to investigate the impact of scrotal insulation (IN) on testicular temperature and its relation to semen quality and testosterone blood serum concentration. For this, 33 rams were used; 17 submitted to IN for 72 hours (using bags involving the testes) and 16 not submitted to IN (control group). The experiment was performed between August and December 2013 in Pirassununga, Brazil (21°56″13″ South/47°28'24″ West). Seminal characteristics, testosterone blood serum concentration, rectal temperature (RT), respiratory frequency, scrotal superficies mean temperature (SSMT), and eye area mean temperature (EAMT) were analyzed 7 days before IN and 21, 35, 49, 63, and 90 days afterward. Scrotal superficies mean temperature and EAMT were measured by thermography camera FLIR T620. Testosterone was evaluated by radioimmunoassay. Analysis of variance was used to determine the main effects of treatment, time, and treatment-by-time interaction using PROC MIXED of SAS software adding command REPEAT. Pearson correlation test was used to verify correlation between SSMT, EAMT, RT, and respiratory frequency. Significant difference was considered when P ≤ 0.05. At the end of IN, SSMT was higher (P < 0.05) in insulated group (32.26 ± 0.19(o)C) than in control group (30.58 ± 0.18(o)C), and the difference between rectal and testicular (deduced from SSMT) temperatures was 1.12 °C; in the other times of the evaluation this difference was between 2.91 and 4.25 °C in IN group. Scrotal superficies mean temperature was reestablished 24 hours after IN. Rectal temperature and EAMT presented correlation (r = 0.59; P < 0.0001). There was time-by-treatment interaction for total sperm (P = 0.0038) and progressive motility (P = 0.01), abnormal spermatozoa (P < 0.0001), membranes integrity (P < 0.0001), induced thiobarbituric acid reactive substances (TBARSs; P = 0.05), and DNA integrity (P = 0.0004). These semen characteristics were negatively affected 21 days after IN, and excluding induced TBARSs and abnormalities, recovered 35 days afterward; induced TBARSs just were affected after 49 days of IN; sperm abnormalities just recovered after 63 days. Testosterone blood serum concentration was lesser in insulated rams (P = 0.03). Thus, the difference of 1.12 °C between RT and testicular temperature impacts semen quality and testosterone blood serum concentration. Moreover, this study shows that rams can recover testes temperature efficiently toward IN and that infrared thermography is an efficient tool to identify differences on SSMT., (Copyright © 2016 Elsevier Inc. All rights reserved.)