The anti-oxidant roles of Taurine and Hypotaurine on acrosome integrity, HBA and HSPA2 of the human sperm during vitrification and post warming in two different temperature Publication date: Available online 19 July 2019 Source: Cryobiology Author(s): Mohammad Seify, Mahsa Zarabadipour, Leila Rashki Ghaleno, AliReza Alizadeh, Mojtaba Rezazadeh Valojerdi Abstract Despite advances in vitrification techniques for sperm cryopreservation, cryo-damages of sperm caused by generation of reactive oxygen species (ROS) continue to impede implementation of this technique. This study analyses the effects of taurine and hypotaurine as anti-oxidants during vitrification of human sperms. The study was performed in two steps. In the first step, 20 normospermic semen samples were vitrified in the presence of varying concentrations of taurine and hypotaurine, and their effects as anti-oxidant agents on classical sperm parameters, hyaluronan-binding assay (HBA), lipid peroxidation (LPO) and acrosome reaction (AR) were studied. Statistical analyses showed that the sperm parameters in all vitrified groups decreased significantly (P < 0.05) compared to the fresh group. However, HBA and acrosome integrity in vitrified groups containing taurine and 50 mM of hypotaurine were better than in the control group (P < 0.05). The morphology of the vitrified group was good only in the group that contained 50 mM of hypotaurine (P < 0.05). Based on the results from the first step, 50 mM of hypotaurine was considered the ideal anti-oxidant formulation and further tests were carried out on 10 normospermic semen samples with this protecting agent. In addition to the mentioned parameters, the expression of heat shock proteinA2 (HSPA2) was studied in the vitrified group with 50 mM hypotaurine, warmed under two different warming temperatures 37 and 42 °C. 50 mM Hypotaurine was found to equally improve motility, morphology, HBA, and AR after warming at 37 °C and 42 °C (P < 0.05). However, at both warming temperatures, the expression of HSPA2 was reduced in all vitrified groups comparing to the fresh group (P < 0.05). In conclusion, taurine and hypotaurine antioxidants, especially 50 mM hypotaurine, are able to reduce deleterious cryo-injuries on morphology, acrosome and HBA and improve sperm recovery at both warming temperatures (37 and 42 °C). However, they do not have any protective action on expression of HSPA2.

Pre-conditioning with Xanthine oxidase to improve post thawed quality of bull sperm Publication date: Available online 17 July 2019 Source: Cryobiology Author(s): Mohsen Sharafi, Mahdi Zhandi, Malak Shakeri, Abdolhossein Shahverdi, Sayyed Mohammad Hadi Hussaini Abstract The purpose of this study was to examine the effects of sub-lethal concentration of Xanthine oxidase (XO) on the post-thawed bull sperm quality. Semen samples were collected from four Holstein bulls, twice a week and during three consecutive weeks (n = 24 total ejaculates). After collection in each replicate, semen samples were pooled and then frozen by semen extender containing different concentrations [0 (XO-0), 0.05 (XO-0.05), 0.5 (XO-0.5), 5 (XO-5), 50 (XO-50) and 500 (XO-500) μM] of XO. After thawing, motion parameters (SCA), plasma membrane functionality (HOST), apoptosis status (Phosphatidylserine translocation assay), mitochondrial activity (Rhodamine 123), and acrosome integrity (PSA), were evaluated. The results showed that total motility, VAP, VSL, VCL, STR, and LIN were lower in XO-50 and XO-500 compared to other groups (P < 0.05). Progressive motility were higher in XO-0.05 and XO-0.5 compared to XO-0, XO-50, and XO-500 (P < 0.05). Mitochondrial activity was highest in XO-0.05 and XO-0.5 groups. Sperm plasma membrane functionality was significantly greater in XO-0, XO-0.05, XO-0.5, and XO-5 than that of XO-50 and XO-500. Xanthine oxidase had not significant effects on acrosome integrity and dead spermatozoa. Higher percentage of live spermatozoa was recorded for XO-0, XO-0.05, XO-0.5, and XO-5; however, the lower amount of apoptotic spermatozoa was detected in the aforementioned groups (P < 0.05). In conclusion, it seems that XO at lower doses may have beneficial effects on post-thawed bull sperm quality.

Endovascular hypothermia improves post-resuscitation myocardial dysfunction by increasing mitochondrial biogenesis in a pig model of cardiac arrest Publication date: Available online 6 July 2019 Source: Cryobiology Author(s): Yuanshan Liu, Peng Wang, Cai Wen, Houzhen Zheng, Xinran Tang, Qin Ling, Xuefen Liu, Jiahong Qin, Wanchun Tang, Zhengfei Yang, Zitong Huang Abstract The aim of the study was to investigate the effects of endovascular hypothermia on mitochondrial biogenesis in a pig model of prolonged cardiac arrest (CA). Ventricular fibrillation was electrically induced, and animals were left untreated for 10 min; then after 6min of cardiopulmonary resuscitation (CPR), defibrillation was attempted. 25 animals that were successfully resuscitated were randomized into three groups: Sham group (SG, 5, no CA), normal temperature group (NTG, 5 for 12 h observation and 5 for 24 h observation), and endovascular hypothermia group (EHG, 5 for 12 h observation and 5 for 24 h observation). The core temperatures (Tc) in the EHG were maintained at 34 ± 0.5 °C for 6 h by an endovascular hypothermia device (Coolgard 3000), then actively increased at the speed of 0.5 °C per hour during the next 6 h to achieve a normal body temperature, while Tc were maintained at 37.5 ± 0.5 °C in the NTG. Cardiac and mitochondrial functions, the quantification of myocardial mitochondrial DNA (mtDNA), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF)-1, and NRF-2 were examined. Results showed that myocardial and mitochondrial injury and dysfunction increased significantly at 12 h and 24 h after CA. Endovascular hypothermia offered a method to rapidly achieve the target temperature and provide stable target temperature management (TTM). Cardiac outcomes were improved and myocardial injuries were alleviated with endovascular hypothermia. Compared with NTG, endovascular hypothermia significantly increased mitochondrial activity and biogenesis by amplifying mitochondrial biogenesis factors’ expressions, including PGC-1α, NRF-1, and NRF-2. In conclusions, endovascular hypothermia after CA alleviated myocardial and mitochondrial dysfunction, and was associated with increasing mitochondrial biogenesis.

Changes of pollen viability of ornamental plants after long-term preservation in a cryopreservation pollen bank Publication date: Available online 2 July 2019 Source: Cryobiology Author(s): Ruifen Ren, Zedi Li, Bingling Li, Jin Xu, Xueru Jiang, Yan Liu, Kongying Zhang Abstract This study determined the changes in pollen viability of 102 species/cultivars of ornamental plants (affiliated to 32 genera of 14 families) following long-term liquid nitrogen storage in a cryopreservation pollen bank. The goal was to provide information on the safety and stability of pollen cryopreservation technology. Fresh pollen at the time of storage was used as the control, and the study examined the pollen viability of ornamental plants cryopreserved for 8, 9, or 10 years. The results show that pollen of the 102 species/cultivars in the cryopreservation pollen bank retained viability ranging from 1% to 58%, After long-term storage there were changes in viability: 11.76% (12 species/cultivars) had increased viability, 16.67% (17 species/cultivars) had stable viability, and the viability of 71.57% (73 species/cultivars) showed a decreasing trend.

Animal protein-free OptiXcell and shortened equilibration periods can replace egg yolk-based extender and slow cooling for rhinoceros semen cryopreservation Publication date: Available online 18 June 2019 Source: Cryobiology Author(s): Jessye Wojtusik, Monica A. Stoops, Terri L. Roth Abstract OptiXcell (OP) was tested as an animal protein-free alternative to an egg yolk-based extender for rhinoceros semen cryopreservation and shorter chilling/equilibration periods were evaluated. Semen was collected from three rhinoceros species: black (Diceros bicornis; n = 2), white (Ceratotherium simum; n = 2), and greater one-horned (GOH; Rhinoceros unicornis; n = 3). Controls were diluted with equine extender (EQ) or OP and equilibrated for 1 h. Treatments were diluted with extender and cooled for 15 min (fast: FEQ; FOP) or not cooled (immediate: IEQ; IOP), prior to cryopreservation. Motility decreased post-thaw (EQ: 50.7 ± 5.2%; OP: 52.9 ± 3.4%) from fresh (82.9 ± 2.9%), was higher in OP than IOP (38.6 ± 4.9%; P ≤ 0.05) and decreased over time (P ≤ 0.05). Post-thaw acrosomal integrity was lower in EQ, FEQ, and IEQ (56.9 ± 0.7; 56.6 ± 4.5; 54.9 ± 2.9%) than OP, FOP, IOP (71.8 ± 4.7; 71.9 ± 3.8; 69.9 ± 4.5%) and fresh (72.6 ± 1.4%; P ≤ 0.05). Progression and viability were lower in EQ (2.8 ± 0.2; 61.9 ± 7.4%) and OP (3.1 ± 0.2; 53.4 ± 6.9%) than fresh (3.7 ± 0.2; 87.2 ± 1.3%), decreased over time (P ≤ 0.05) but not different among treatments (P > 0.05). Morphology did not differ between fresh (75.0 ± 4.9% normal) and any treatment group (70.0–77.8%) or over time (P > 0.05). OptiXcell is comparable to egg yolk-based EQ when used for rhinoceros semen cryopreservation. Furthermore, chilling/equilibration can be reduced with little impact on sperm characteristics.

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage, access and transport Publication date: Available online 13 June 2019 Source: Cryobiology Author(s): Asger Kreiner, Frank Stracke, Heiko Zimmermann Abstract When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me2SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me2SO and EG, respectively. Concentration changes in EG/Me2SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.

Cryopreservation of Bangladeshi ram semen using different diluents and manual freezing techniques Publication date: Available online 4 June 2019 Source: Cryobiology Author(s): Pankaj Kumar Jha, M. Golam Shahi Alam, Abdullah AL. Mansur, Nazmun Naher, Taohidul Islam, Musharraf Uddin Bhuiyan, Farida Yeasmin Bari Abstract A study was conducted to establish a sustainable and effective manual freezing technique for cryopreservation of Bangladeshi ram semen. Three diluents and freezing techniques were tested, both as treatment combinations (diluent × freezing technique) and fixed effects (diluent or freezing technique) on post-thaw sperm motility (SM), viability (SV), plasma membrane integrity (SPMI) and acrosome integrity (SAI). Ten rams were selected, based on semen evaluation. Eight ejaculates were used for each treatment combination. Semen samples were diluted using a two-step protocol for home-made Tris-based egg yolk (20%, v/v) diluents: D1 (7% glycerol, v/v) and D2 (5% glycerol, v/v), and one-step for commercial diluent: D3 (Triladyl®, consists of bi-distilled water, glycerol, tris, citric acid, fructose, spectinomycin, lincomycin, tylosin and gentamycin) at 35 °C. Fraction-A (without glycerol) was added at 35 °C, and following cooling of sample to 5 °C (−0.30 °C/min), Fraction-B (with glycerol) was added. The diluted semen samples were aspirated into 0.25 ml French straws, sealed, and equilibrated at 5 °C for 2 h. The straws were frozen in liquid nitrogen (LN) vapour, in a Styrofoam box. The freezing techniques were; One-step (F1): at −15.26 °C/min from +5 °C to −140 °C; Two-step (F2): at −11.33 °C/min from +5 °C to −80 °C, and −30 °C/min from −80 °C-140 °C; and Three-step (F3): at −11.33 °C/min from +5 °C to −80 °C, at −26.66 °C/min from to −80 °C to −120 °C, and at −13.33 °C/min from −120 °C to −140 °C. Two semen straws from each batch were evaluated before and after freezing. The group F3D3 exhibited significantly higher (p < 0.05) post-thaw SM 63.1 ± 2.5%, SV 79.0 ± 2.1% and SPMI 72.9 ± 1.7%, whereas SAI 72.9 ± 1.7% was significantly higher (p < 0.05) in group F3D2. The freezing technique F2 and F3 had significantly higher (p < 0.05) post-thaw sperm values compared to F1. The post-thaw SM and SV were above 50% and 65% with the freezing technique F2 and F3 but differed non-significant. The SPMI 67.6 ± 2.0% and SAI 76.1 ± 1.4% were significantly higher (p < 0.05) with F3. Likewise, the diluent D2 and D3 had significantly higher (p < 0.05) post-thaw sperm values compared to D1. The post-thaw SM, SV and SPMI were above 50%, 65% and 55% with the diluents D2 and D3 but differed non-significant. The SAI 76.1 ± 1.1% was significantly higher (p < 0.05) with D3. We concluded that the use of a simple home-made Tris-based diluent containing 20% (v/v) egg yolk and 5% glycerol (v/v), two-step dilution and a three-step freezing technique is a sustainable and effective method for freezing ram semen. For further validation, the fertility of ewes artificially inseminated with the frozen semen will be observed.

Three-dimensional printing can provide customizable probes for sensing and monitoring in cryobiology applications Publication date: June 2019 Source: Cryobiology, Volume 88 Author(s): Hamed Shamkhalichenar, Jin-Woo Choi, Terrence R. Tiersch Abstract Cryopreservation has been recognized as a powerful tool for long-term preservation of genetic resources. However, the outcomes of cryopreservation by different user groups often vary due to inconsistency in procedures and freezing equipment. Herein, we report on the feasibility of providing customizable sensing probes with three-dimensional (3-D) printing to monitor cryopreservation phenomena. The objectives were to: 1) introduce 3-D printing as a fabrication method for developing customizable probes to be used in cryogenic applications; 2) design and fabricate an example of a 3-D printed sensing probe and multiplexer capable of detecting phase-change phenomena based on quantitative data regarding sample electrical resistance and temperature, and 3) demonstrate the sensing platform in cryopreservation conditions and in combination with a custom-made 3-D printed freezing device. The sensing probe developed was designed to fit within standard 0.5-ml French straws. Phase-transition phenomena were detected by analyzing electrical resistance changes. The quantitative data from this device in conjugation with a 3-D printed freezer rack provided cryopreservation capability with high reproducibility and offered an alternative to expensive programmable freezers. The use of 3-D printing provided flexibility to develop new sensing probes or modify existing designs based on specific needs. After initial prototyping, fabrication, and testing of 3-D printed sensing probes, particularly useful designs can lead to the reduction of variation in performing standardized cryopreservation protocols.

Transcriptome and proteome analyses to investigate the molecular underpinnings of cold response in the Colorado potato beetle, Leptinotarsa decemlineata Publication date: June 2019 Source: Cryobiology, Volume 88 Author(s): Louise Govaere, Mathieu D. Morin, Jacques J. Frigault, Sébastien Boquel, Alejandro Cohen, Simon G. Lamarre, Pier Jr. Morin Abstract The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an insect that can cope with prolonged periods of low temperatures exposure. The molecular changes required to adapt to such conditions have not been thoroughly investigated in this insect. The current work aims at characterizing deregulated transcripts and proteins in adult L. decemlineata exposed to 15 °C and −5 °C using RNA-sequencing-based transcriptomics and liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics approaches, respectively. RNA-sequencing highlighted the differential expression of several transcripts, including ubiquilin-1 and ubiquitin carboxyl-terminal hydrolase 5, in insects submitted to low temperatures when compared with control insects. In addition, proteomics approach detected 2840 proteins in cold-exposed beetles including elevated levels for 409 proteins and reduced levels for 200 proteins. Cuticular proteins CP1, CP4, CP5 and CP7 as well as eukaryotic translation initiation factor 4B were notable proteins with elevated levels in cold insects. Functional analysis of targets modulated at low temperatures using DAVID indicated processes likely affected under cold conditions including select metabolic cascades and RNA-associated processes. Overall, this work presents molecular candidates impacted by low temperatures exposure in L. decemlineata and builds on the current knowledge associated with response to these conditions in this insect. Graphical abstract

Potent humanin analogue (HNG) protects human sperm from freeze-thaw-induced damage Publication date: June 2019 Source: Cryobiology, Volume 88 Author(s): Chao Yang, Ling Xu, Yingdong Cui, Bo Wu, Zhaolin Liao Abstract This study was aimed to investigate the protective effect of potent humanin analogue (HNG) supplementation to freezing media on freezing-thawing induced human sperm damage. We collected semen samples with normal sperm parameters from 15 healthy men. After the swim-up processing, the motile spermatozoa from each of the men were allocated to four equal groups: In the control group, the spermatozoa were frozen in media without HNG supplementation. In the other three groups, the spermatozoa were frozen in media supplemented with different concentrations of HNG (2 μM, 10 μM and 20 μM, respectively). We analyzed the sperm motility, viability, sperm mitochondrial membrane potential, apoptosis, sperm DNA fragmentation index (DFI), reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and caspase-3 activity for the sperm in each group. As a result, supplementation of HNG with 2 μM, 10 μM and 20 μM to the freezing media all significantly improved sperm motility and viability (all p < 0.05) when compared with the control group. Similarly, we found that supplementation of HNG reduced the damage to the mitochondrial membrane and DNA integrity, and inhibited the reaction of oxidative stress and the activity of caspase-3 in sperm. Although these protective effects increased with the elevated concentration of HNG in the freezing media, a final HNG concentration of 20 μM failed to exert significant improvements when compared with the concentration of 10 μM (all p > 0.05). In conclusion, our results suggested that HNG supplementation to the freezing media could protect sperm cells from freezing-thawing induced sperm damage.