Cryobiology

Local cryostimulation acutely preserves maximum isometric handgrip strength following fatigue in young women Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): Massimo De Nardi, Sara Silvani, Piero Ruggeri, Livio Luzi, Antonio La Torre, Roberto Codella Abstract Several types of cryostimulation have been recently proposed to rapidly lower skin temperature therefore gaining a possible neuro/muscular recovery after strenuous exercise or, more generally, in sports. Local cryostimulation may be a viable and relatively portable tool to obtain physiological benefits in previously-efforted muscular districts. However, cohesive and standardized cryo-exposure protocols are lacking as well as the righteous procedure to efficaciously combine duration, treatments and temperature in relation to desirable effects on muscular strength. In this randomized-controlled study, fifty young women were tested for maximum isometric handgrip strength, before and after exhausting contractions. Following the fatiguing protocol, the intervention group (cryo, n = 25, 24.7 ± 2.5 years, BMI 21.7 ± 1.8 kg/m2) underwent a 6-min local cryostimulation (−160 °C) on the extensor-flexor muscles of the dominant arm, while control-matched peers sat rested in a thermo-neutral room (22 ± 0.5 °C). Handgrip tests were repeated at baseline (T0), after cryostimulation (T1), and 15 min after T1 (T2). Throughout the protocol, the AUC of the strength performance was significantly higher in the cryo- compared to control group (P = 0.006). In particular, following fatigue and cryostimulation, the cryo group preserved higher strength at T1 with respect to controls (26.8 ± 2.8 vs 23.9 ± 2.8 kg, Bonferroni's post-hoc, P < 0.01). Likewise, ventral and dorsal temperature, recorded with a thermal camera, were lower in cryo- than control group (P < 0.0001). In conclusion, a brief session of local cryostimulation may acutely preserve maximal isometric force in young women following a fatiguing protocol. These findings may have implications in orchestrating strategies of district muscular recovery.

Circulating soluble intercellular adhesion molecule-1 (sICAM-1) after exercise-induced muscular damage: Does the use of whole-body cryostimulation influence its concentration in blood? Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): F. Bieuzen, C. Hausswirth, B. Dugué Abstract As soluble intercellular adhesion molecule-1 (sICAM-1) was recently hypothesized to be a key player in the mechanisms involved in exercise-induced muscular damage (EIMD), we investigated its circulating concentration changes in athletes before and after EIMD with and without the use of whole-body cryostimulation (WBC; 3 min at −110 °C) at the exercise end and repeated once a day during 4 days. We previously characterized plasma specimens from 11 endurance athletes who performed twice (randomized crossover design) strenuous running leading to EIMD, followed by passive recovery or WBC. Muscle soreness and inflammatory response were observed in both cases but the use of WBC induced a significant reduction in these responses (PlosOne 2011; 6:e22748). We now found that sICAM-1 concentration slightly increased in both circumstances and remained elevated for 24 h (p < 0.01). However, no significant WBC effect was observed concerning sICAM-1 changes indicating that this compound is not a major player both in EIMD and WBC physiological impacts.

A preliminary study on the use of jenny colostrum to improve quality in extenders for freezing donkey semen Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): Cristina Álvarez, Victoria Luño, Noelia González, Lydia Gil Abstract Sperm quality in donkeys (Equus asinus) after freezing thawing is still considered lower than that from other animals, including horses. The aim of this study was to test a new freezing extender supplemented with jenny colostrum on donkey sperm. After thawing, we evaluated sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodide (PI), membrane functional integrity by HOS-test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC-PNA) and PI. Ejaculates were collected from five fertile Donkeys. Sperm samples were pooled, diluted and cryopreserved into three experimental extender groups: BotuCrio®, lactose-extender supplemented with egg yolk (20%) and lactose-extender supplemented with jenny colostrum (20%). The results demonstrated that lactose-jenny colostrum samples displayed significantly higher values in almost all parameters evaluated (p < 0.05) compared with the other two extenders after thawing (BotuCrio® and lactose-egg yolk based extender, respectively) –Total Motility, Viability, HOS test, VCL, VSL and VAP. Acrosome status, LIN, STR and WOB despite showing lower values, none of them were statistically significant (p > 0.05). In conclusion, the extender containing jenny colostrum can be successfully used for donkey semen crypreservation and could effectively improve donkey sperm qualities after freezing -thawing. Graphical abstract Schema of the experimental design.

Production of mouse fetuses using spermatozoa exposed temporarily to high temperature or continuously to room temperature after freeze-drying in Na+-free/K+-rich EGTA buffer Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): Hirokazu Kusakabe Abstract Present study aimed to determine to what extent freeze-dried spermatozoa were able to withstand high-temperature conditions: transient increase in storage temperature and long-term exposure to room temperature. Mouse spermatozoa were freeze-dried in EGTA/Tris-HCl buffered solution alkalinized using KOH (K-ETBS, pH 7.7), and then stored for up to 7 months at 4 °C or 25 °C. After 2 months' storage, some of the 4°C-stored spermatozoa were exposed to 40 °C for 1 week or 1 month, then again stored at 4 °C for the remaining storage period. Following storage, rehydrated spermatozoa were injected into mouse oocytes. The resulting zygotes were assessed for chromosome damage, in vitro development up to the blastocyst stage, and post-implantation development to normal fetuses on day 18 of gestation. In storage at 4 °C, one-week exposure to 40 °C had no adverse effect on the chromosome integrity and developmental competence compared to non-exposure to 40 °C (continuous storage at 4 °C). In contrast, one-month exposure to 40 °C caused an increasing level of chromosome damage (36%, P < 0.05) and reduced frequencies of blastocysts (54%, P < 0.05) and normal fetuses (36%, P < 0.05) compared to the frequencies obtained by continuous storage at 4 °C (15%, 82% and 52%, respectively). Storage at 25 °C resulted in accumulation of chromosome damage (27%, P < 0.05), leading to decreased blastocyst formation (63%, P < 0.05). But, the frequency of normal fetus (44%) was not significantly different from that obtained by continuous storage at 4 °C. Consequently, mouse spermatozoa freeze-dried in K-ETBS withstood temporary exposure to 40 °C for 1 week. Chromosome damage accumulated in spermatozoa during storage at 25 °C.

CT-guided percutaneous cryoablation combined with systemic chemotherapy for liver metastases from esophageal carcinoma: Initial experience Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): Yan Wang, Wei-Hao Zhang, Xu Chang, Fei Cao, Hai-Peng Yu, Wen-Ge Xing, Xue-Ling Yang, Zhi Guo Abstract Objective To explore the feasibility, safety and effectiveness of percutaneous cryoablation combined with systemic chemotherapy in the treatment of liver metastases from esophageal carcinoma (ECLM). Materials and methods We retrospectively collected data of 16 patients who received CT-guided percutaneous cryoablation concurrent systemic chemotherapy for liver metastases after primary esophageal carcinoma resection. Functional Assessment of Cancer Therapy-General (FACT-G) was used for the assessment of quality of life (QOL), and overall survival (OS), progression-free survival (PFS) and complications were also evaluated. Results The technical success rate was 96%, and no major complications related to cryoablation procedure were detected. Median OS and PFS after cryoablation were 14.5 months (range, 4–51 months) and 7.5 months (range, 1–31 months), respectively. The 1-year, 2-year, and 3-year survival rates were 56.3%, 31.3%, and 18.8%, respectively. The PFS rate at 6-month, 1-year, and 2-year after procedure were 68.8%, 31.3% and 18.8%, respectively. Furthermore, the QOL of patients was improved after cryoablation therapy compared with preoperative scores (P < 0.05). Conclusions Percutaneous cryoablation combined with systemic chemotherapy is a safe, feasible and effective method to treat liver metastases from esophageal carcinoma. And to a certain extent, this approach is very efficacious in improving the QOL of patients with ECLM.

Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): Zubing Cao, Meiling Zhang, Tengteng Xu, Zhen Chen, Xu Tong, Dandan Zhang, Yiqing Wang, Ling Zhang, Di Gao, Lei Luo, Ibrar Muhammad Khan, Yunhai Zhang Abstract Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.

PD-1 blockade enhances the anti-tumor immune response induced by cryoablation in a murine model of renal cell carcinoma Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): Chenzhuang Zhu, Sihao Lin, Junhao Liang, Yingjian Zhu

Preservation of female genetic resources of common carp through oogonial stem cell manipulation Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): Roman Franěk, Tomáš Tichopád, Christoph Steinbach, Xuan Xie, Jelena Lujić, Zoran Marinović, Ákos Horváth, Vojtěch Kašpar, Martin Pšenička Abstract Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (−1 °C/min) and short-term storage (−80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me2SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at −80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.

Use of trehalose in the semen cryopreservation of Amazonian catfish Leiarius marmoratus Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): Stela Mari M. Gheller, Carine D. Corcini, Camila R.C. de Brito, Izani B. Acosta, Geórgia C. Tavares, Sara Lorandi Soares, Alessandra C. Silva, Diego M. Pires, Antonio Sergio Varela Junior Abstract The current study assessed a semen cryopreservation protocol in the Amazonian catfish Leiarius marmoratus, a freshwater fish, of rheophilic behavior, and of great importance for Brazilian fish farming. Eight males (n = 8) were stripped and the semen was cryopreserved if total motility in fresh semen was higher than 80%. The external cryoprotectant Trehalose was then diluted in Beltsvile Thawing Solution (BTS) extender in the following concentrations: 50, 100, 150, and 200 mM. Semen samples were diluted in the media (1:9 v/v) being tested, then frozen in a container with nitrogen vapor (dryshipper), and stored in liquid nitrogen at −196 °C. Motility parameters assessed post-thawing were performed by CASA-system and sperm cell integrity analyses (membrane integrity, DNA integrity, and mitochondrial function) were performed through fluorescence microscopy. As a result, no significant statistical difference was observed between treatments, independently of Trehalose concentrations tested in the following post-thawing analysis: membrane integrity, DNA integrity, mitochondrial functionality, and sperm motility duration. As of total and progressive motilities, the treatment containing 50 mM trehalose (15.6 and 9.5%, respectively), exhibited inferior results when compared to treatments with 150 mM (22.9 and 17.7%, respectively) and 200 mM (31.4 and 26.3%, respectively) trehalose concentrations (P < 0.05); however, it did not differ from the treatment with 100 mM trehalose (18.6 and 15.3%, respectively). Therefore, treatments with trehalose at higher concentrations exhibited superior results when compared to other treatments in in vitro motility parameters for L. marmoratus.

Effect of insulin on functional parameters of human cryopreserved sperms Publication date: April 2019 Source: Cryobiology, Volume 87 Author(s): Saeed Shokri, Seyyed Meisam Ebrahimi, Sanaz Ziaeipour, Reza Nejatbakhsh Abstract Cryopreservation of sperms is common therapy but with multiple damages to sperms. The aim of this study was to assess the effect of insulin as a prosurvival factor on the most important functional parameters of human spermatozoa during cryopreservation. Semen samples were obtained from 15 normozoospermic men at age 25–40 years of old through masturbation. Cryopreservation of sperms was conducted along with adding 10, 100, 500 and 1000 (ng/ml) insulin and a control group was also considered by adding distilled water. Samples were cryopreserved for 2 weeks in liquid nitrogen. Then, after thawing sperm motility; cytosolic/mitochondrial reactive oxygen species (ROS) levels; and DNA fragmentation were analyzed. Data were analyzed by SPSS software using one-way ANOVA. Results showed that insulin at all doses significantly decreased cytosolic ROS especially in 10 ng/ml group (P˂0.05). Mitochondrial ROS also decreased by adding insulin in comparison to the control group, although unmeaningfully (P˃0.05). Insulin at 1000 (ng/ml) decreased DNA fragmentation, significantly (P˂0.05). Also, the number of motile sperms increased in all insulin groups but it wasn't meaningful (P˃0.05). Based on our findings adding insulin to semen leads to protecting effects against cryopreservation damages and increases sperms motility. Therefore, using insulin for human semen seems to could be suggested for future clinical applications.