Figure
![Best-fit water transport parameters of rat and mouse sperm determined by DSC and fit by FORTRAN (Formula Translation Computer Language) optimization as previously reported in Devireddy et al. [19].](profile/Willem_Wolkers/publication/26300774/figure/tbl1/AS:601583830171662@1520440170997/Best-fit-water-transport-parameters-of-rat-and-mouse-sperm-determined-by-DSC-and-fit-by.png)
Best-fit water transport parameters of rat and mouse sperm determined by DSC and fit by FORTRAN (Formula Translation Computer Language) optimization as previously reported in Devireddy et al. [19].
Source publication
Though cryopreservation of mouse sperm yields good survival and motility after thawing, cryopreservation of rat sperm remains a challenge. This study was designed to evaluate the biophysics (membrane permeability) of rat in comparison to mouse to better understand the cooling rate response that contributes to cryopreservation success or failure in...
Similar publications
Kinematic parameters of thawed ram semen frozen under field conditions were analysed with the use of two commercial (egg yolk vs egg yolk free) semen extenders in different year-seasonal periods. The semen was collected from Suffolk (n = 2) and Charollais (n = 1) rams kept in private breeding farm on 3 test days (pre-mating, at mating, post mating)...
Introduction
In tomcats, epididymal spermatozoa provide an additional source of male gametes available for cryopreservation. While this procedure is feasible, the survival rate and motility of epididymal cat spermatozoa are both low after thawing. Cryopreservation is known to induce oxidative stress in spermatozoa, with mitochondria and the plasma...
Artificial insemination is the main tool for the rapid dissemination of valuable germplasm to improve the genetic quality of farm animals. Freezing of spermatozoa of all the domestic species, is a challenging because of variation in size, shape and lipid composition of sperm from different species. Thus, a cryopreservation protocol for semen of one...
Cryopreservation exposes spermatozoa to stressful conditions, leading to reduced cell viability. Several studies propose that overproduction of reactive oxygen species and decreased antioxidant capacity of semen may increase the damaging effects of the technique. The objective of this work was to evaluate the influence of a skim milk-egg yolk based...
The aim of the present study was to evaluate the effects of holding semen for 4 h at 15 °C in different extenders prior to cryopreservation on sperm characteristics and subpopulation kinematics in INRA180 sheep. Semen samples were collected using an artificial vagina from two animal groups. Group 1: the rams kept next to the lab (G1) considered as...
Citations
... 1. The calorimetric protocol developed to measure fast freezing processes or intracellular ice formation (IIF) is identical to the one detailed in earlier studies to measure water transport in cells and tissues 38,39,[55][56][57][58][59][60][61][109][110][111][112] . The only exception will be that the two heat releases of interest (initial, with live and final, with lysed tissue cells) will be measured at "higher or faster" cooling rates ranging from 10 to 50 ˚C/minsee Fig. 10. ...
The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. Cryopreservation has shown the most promise but is a fairly complex process, necessitating the use of chemicals called cryoprotective agents (CPAs), freezing equipment, and obviously, storage in liquid nitrogen. The purpose of this chapter is to present a general overview of cryopreservation storage techniques and the optimal protocols/results obtained in our laboratory for long-term storage of adult stem cells using freezing storage.
... In slow freezing, cells are cooled in suspension and ice nucleates first in the extracellular space (Hagiwara et al., 2009) leading to the biophysical responses of cellular dehydration. In case a controlled reduction of temperature is maintained, a sufficient osmotic pressure persists that prevents the formation of ice crystals within the cell. ...
Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA's) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed 'kinetic sperm vitrification' has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.
... The DSC has also been used to measure (a) ice nucleation parameters from intracellular ice formation (IIF) heat release readings in yeast and blood cells [27] and in G. Max cells and erythrocytes [28]; (b) heat releases associated with IIF in soybean cells [29], in lymphocytes [30] and in artemia cysts [31]; (c) the volume of freezable water in Drosophila melanogaster embryos [32]; (d) IIF heat release as a function of the cytocrit [33]; and (e) the phase change process in biologically relevant solutions [1,34]. In addition, the DSC has been used to measure the mass transfer (or water transport) across the cell membrane during the freezing of cells in suspension [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52] and cells embedded in tissue [53][54][55][56][57][58][59]. ...
Following an earlier study, we reexamined the latent heat of fusion during freezing at 5 K/min of twelve different pre-nucleated solute-laden aqueous solutions using a Differential Scanning Calorimeter (DSC) and correlated it with the amount of initially dissolved solids or solutes in the solution. In general, a decrease in DSC-measured heat release (in comparison to that of pure water, 335 mJ/mg) was observed with an increasing fraction of dissolved solids or solutes, as observed in the earlier study. In addition, the kinetics of ice crystallization was also obtained in three representative biological media by performing additional experiments at 1, 5 and 20 K/min. A model of ice crystallization based on the phase diagram of a water–NaCl binary solution and a modified Avrami-like model of kinetics was then developed and fit to the experimental data. Concurrently, a heat and mass transfer model of the freezing of a salt solution in a small container is also presented to account for the effect of the cooling rate as well as the solute concentration on the measured latent of freezing. This diffusion-based model of heat and mass transfer was non-dimensionalized, solved using a numerical scheme and compared with experimental results. The simulation results show that the heat and mass transfer model can predict (± 10%) the experimental results.
... These simulations can be used to calculate the amount of ice crystallized or melted in a cryopreservation solution (with or without cells), with the same temperature variation as the one occurring during cryopreservation. These simulations can help optimize cryopreservation, as it allows the quantification of ice crystallization as a function of procedure parameters [4,5,24,36,47] or cell responses [11,12,15,24,33,34,38,43,44]. To precisely reproduce slow-freezing procedures during a DSC experiment, the seeding step must be replicated. ...
... To precisely reproduce slow-freezing procedures during a DSC experiment, the seeding step must be replicated. Many studies have investigated the use of the Snomax® ice-nucleating agent (INA) to trigger nucleation during DSC experiments [9,10,43,44,[11][12][13]15,24,33,34,38]. ...
Controlling the supercooling state during ice crystallization process is important for differential scanning calorimetry (DSC) analysis. In cryopreservation solutions, the control of the nucleation temperature (Tn) is necessary as it allows the simulation of slow-freezing procedures. Herein, the effect of Snomax® on Tn value in aqueous solutions containing dimethyl-sulfoxide (Me2SO) (H2O + 10%(v/v)Me2SO) was studied. The effect of Me2SO, a cryoprotectant, on nucleation control was investigated by evaluating λhet, which describes the influence of a molecule on the nucleation behaviour of an ice-nucleating agent (INA). Note that λhet is specific to the molecule added in solution and to the maximum melting temperature of the solution. The knowledge of λhet helps evaluate the ranges of the supercooling magnitude at which the INA in solution can trigger nucleation. This study shows how Snomax can be used in DSC experiments using the H2O + 10%(v/v)Me2SO solution when ice nucleation must be controlled.
... Since the publication in 2001 of the first successful report concerning the cryopreservation of rat sperm 2 , many papers on the topic have been published [3][4][5][6][7][8][9][10][11][12][13] . However, only one of those papers has reported the successful production of pups derived from embryos that were obtained via in vitro fertilisation using frozen rat sperm 5 . ...
Recently, genome-editing tools have come into common use in the field of rat research, and consequently, many genetically modified rat strains have been preserved and archived as frozen embryos. Although there have been many reports published on the topic of rat sperm cryopreservation, no report has yet provided satisfactory and acceptable protocols for the cryopreservation of rat sperm. In this study, we developed methods for both the cryopreservation of transgenic rat sperm and in vitro fertilisation using frozen sperm, which yielded high fertilisation rates.
... Cryopreservation reduces viability, motility, and mitochondrial activity, as well as imposing the process induces chromatin damage, increases reactive oxygen species, activates apoptosis mechanisms ( Said et al., 2010) and perhaps most importantly, is the premature induction of a capacitation-like process known as cryo-capacitation ( Bailey et al., 2003;Reddy et al., 2010). Cryo-capacitation is characterized by a membrane reorganization with loss of polyunsaturated fatty acids and cholesterol (Maldjian et al., 2005;Chakrabarty et al., 2007) with further development of molecular hallmarks of capacitation such as an intracellular calcium increase ( Hagiwara et al., 2009;Oldenhof et al., 2010) and protein phosphorylation ( Kumar and Atreja, 2012;Singh et al., 2012). All these alterations might reduce longevity of the cryopreserved spermatozoa within the female reproductive tract, decreasing the likelihood of typical oviductal-sperm interactions or affecting the fertilization process. ...
Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 μM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ± 2.95; 40.13 ± 11.82 for 0.3 μM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.
... saate doğru ilerlemesiyle beraber motilite oranında ciddi bir düşme gözlendi. Bu durum, rat spermatozoonunun ebatlarına, non spherik şekillerine bağlı olarak soğutma işlemlerinde zorlukların olmasına ve hücrelerin biyofiziksel cevaplarının farklılık göstermesine bağlanmaktadır (18). Rodent sperm karakterleri memeli spermatozoasından membran lipid içeriği, kompozisyonu ve uzun kuyruk yapısındaki farklılığa bağlı olarak değişiklik göstermektedir (29,30). ...
... The nucleating effect of P. syringae on ice appearance has been previously described [32], and the effect of the Snomax concentration on the T n value was first reported by Maki (1974) [31] and then by other teams [42][43][44][45][46][47], including studies with DSC [27,[48][49][50][51]. Snomax is a particularly efficient INA. Hence, Snomax or P. syringae is widely used to control ice crystal appearance in aqueous solutions [34,44,[52][53][54][55][56], for example, in cryobiology studies [15,[57][58][59][60] with optical experiments [5,6,[61][62][63][64] or in DSC experiments [4,6,[65][66][67][68][69][70][71][72]. However, all these studies were conducted under different experimental conditions, thus preventing comparisons among them. ...
For differential scanning calorimetry (DSC) analysis, controlling the degree of supercooling at which crystallization begins may be required for several studies, experiments, and applications. In this paper, the use of Snomax, an ice nucleating agent (INA), was evaluated to create ice at a desirable temperature range in a DSC aluminum sample pan. The effect of Snomax on the nucleation temperature (Tn) was studied in pure water. Best practices and methods are described in terms of the Tn dependence on three experimental parameters: (i) the Snomax concentration that controls the Tn value for different groups according to the three classes of the Pseudomonas syringae protein aggregates (from which Snomax originates); (ii) the sample volume that affects the presence probabilities of the different INA subpopulations in the solution and that could also favor their deterioration; and (iii) the cooling rate that does not seem to further affect the Tn value. There is no-evidence of time dependence of the nucleation process promoted by Snomax. The presence of artifacts or disturbances introduced by the addition of Snomax into the solution was evaluated. No major disturbances of the thermodynamic characteristics of these solutions were observed with the addition of Snomax below 10³ mg L⁻¹ concentration. This underscores the possible use of Snomax for controlling ice nucleation during DSC experiments.
... The changes are reflected through mitochondrial and membrane dysfunction. This affects the permeability of the sperm surface to water, ions and cryoprotectant (Hagiwara et al. 2009;Oldenhof et al. 2010), reducing the number of viable and progressive motile sperm with uncapacitated and intact acrosomal cap prior to artificial insemination (Cormier and Bailey 2003). The cryocapacitation and acrosomal cap damage are cellular exocyticlike events that influence the fertilizing ability of sperm, a fundamental prerequisite for successful conception after insemination (Lindsay et al. 2005). ...
The present study was conducted to evaluate the effect of egg yolk concentration and washing on sperm quality in cryopreserved Barbari buck semen at higher dilution (100 × 10⁶ cells/ml). Five healthy Barbari bucks of similar age and weight were selected as semen donor. Six ejaculates were collected from each buck during the experiment. Collected semen samples were pooled and diluted with Tris-based semen extender containing 6% glycerol and egg yolk according to the treatments – T-1, that is, 3% egg yolk, T-2, that is, 20% egg yolk and T-3, that is, 20% egg yolk after washing and cryopreserved using the slow freezing technique. Semen was evaluated at three different steps, namely, after dilution, after equilibration and after freeze–thawing. Significantly (p ≤ .01) higher values for motility and viability were observed in T-3 just after dilution while significantly (p ≤ .01) higher values for acrosomal integrity and pattern F (uncapacitated sperm) was observed in T-2. Significantly (p ≤ .01) higher values were observed in T-2 after equilibration and thawing. It may be concluded that incorporation of 20% egg yolk compared to 3% egg yolk or 20% egg yolk after washing in extender confer better cryoprotection to Barbari buck spermatozoa as reflected in semen quality parameters studied.
... With the production of a large number of mutant or transgenic rat strains, there is an increasing demand for preservation and distribution of rat lines. Although rats are relatively easy to house and breed, it is expensive to maintain and transport genetically modified strains [5,6]. For this reason, cryopreservation of gametes and embryos has been developed to maintain genetically valuable materials for an extended period and facilitate transportation of genetic materials at a low cost [7,8]. ...
Successful cryopreservation of rat spermatozoa from various strains still remains a challenge. The objective of this study was to determine if combinations of OptiPrep™ (iodixanol) and adenosine 5′-triphosphate (ATP) can improve rat sperm function during the cryopreservation procedure.
Epididymal rat spermatozoa were frozen under different OptiPrep™ concentrations (0, 1, 2, 3 or 4 %) and were diluted with media supplemented with or without 2 mM ATP after thawing. Post-thaw sperm motility, acrosomal membrane integrity (AMI) and mitochondrial membrane potential (MMP) were then evaluated. In addition, the effect of different OptiPrep™ concentrations on fresh and cooled rat spermatozoa was tested via motility.
There was no effect of OptiPrep™ on motility of fresh and cooled spermatozoa. The supplementation of 1 and 2 % OptiPrep™ increased motility of frozen spermatozoa at 10 min after thawing, while it did not improve motility of spermatozoa at 3 h after thawing in the absence of ATP. During incubation of thawed spermatozoa, the ATP addition protected time-dependent decrease in motility after thawing in OptiPrep™-treated samples. OptiPrep™ had no effect on AMI and MMP in frozen-thawed spermatozoa but combinations of OptiPrep™ and ATP improved MMP in frozen-thawed spermatozoa.
Iodixanol has cryoprotective effects during rat sperm freezing without any toxic effect. Moreover, the combinations of iodixanol and ATP have a beneficial role in maintaining function of frozen-thawed rat spermatozoa for long period of incubation post-thaw.
