The cryobiology of spermatozoa Benson, J.D; Woods, E.J; Walters, E.M ...
Theriogenology,
11/2012, Letnik:
78, Številka:
8
Journal Article
Recenzirano
The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective ...and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm cryobiology, provide a synopsis of the development of sperm cryobiology as a discipline, and present the current state and directions for future research in sperm cryobiology in the three major areas outlined above—agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.
The fertility of mice after autologous transplantation of ovaries, before or after cryopreservation, was investigated in this study. Female mice were randomly assigned to either sham-operated (n = ...14), ovariectomized (n = 11), fresh (n = 12) or cryopreserved (n = 11) ovarian transplant groups. Ovaries were cryopreserved in 1.4 M dimethyl sulphoxide (DMSO) by cooling to -55 degrees C at 0.5 degree C/min (ice nucleation at -7 degrees C), plunged in liquid nitrogen and then thawed at room temperature. Oestrous cyclicity was observed 7 days after sham operation or 15 days after fresh or cryopreserved ovarian transplant. Ovariectomized animals did not demonstrate oestrous cyclicity but were mated, and no pregnancies resulted. Live births were recorded from all sham-operated, all fresh transplant, and 8/11 (73%) cryopreserved transplant animals. Overall mean +/- SEM litter sizes from fresh (4.32 +/- 0.44) and cryopreserved (4.71 +/- 0.57) transplant groups were smaller (P < 0.05) than those of sham-operated animals (12.54 +/- 0.44), although the sizes were not significantly different (P > 0.05) from each other. Animals were mated at least four times, with four litters of live pups from 4/4 sham-operated, 1/10 fresh and 1/9 cryopreserved ovarian transplant animals. Litter sizes from pups of sham-operated and transplant animals were not significantly different from each other. Following autologous transplantation of mouse ovaries, before or after cryopreservation, offspring appeared normal, with high rates of fertility.
A novel cryogenic heat pipe, oscillating heat pipe (OHP), which consists of an 4
×
18.5
cm evaporator, a 6
×
18.5
cm condenser, and 10
cm length of adiabatic section, has been developed and ...experimental characterization conducted. Experimental results show that the maximum heat transport capability of the OHP reached 380
W with average temperature difference of 49
°C between the evaporator and condenser when the cryogenic OHP was charged with liquid nitrogen at 48% (v/v) and operated in a horizontal direction. The thermal resistance decreased from 0.256 to 0.112 while the heat load increased from 22.5 to 321.8
W. When the OHP was operated at a steady state and an incremental heat load was added to it, the OHP operation changed from a steady state to an unsteady state until a new steady state was reached. This process can be divided into three regions: (I) unsteady state; (II) transient state; and (III) new steady state. In the steady state, the amplitude of temperature change in the evaporator is smaller than that of the condenser while the temperature response keeps the same frequency both in the evaporator and the condenser. The experimental results also showed that the amplitude of temperature difference between the evaporator and the condenser decreased when the heat load increased.
A detailed mathematical model predicting the effect of contact angle on the meniscus radius, thin film profile and heat flux distribution occurring in the micro-trapezoidal grooves of a heat pipe has ...been presented. The model can be used to determine the maximum evaporating heat transfer rate in the evaporator including the effects of disjoining pressure and surface tension. The equation of meniscus radii calculation in the evaporator at given heat load based on the liquid wicks configuration has been put forward. The numerical results show that while the capillary limitation governs the maximum heat transport capability in a grooved heat pipe, the thin film evaporation determines the effective thermal conductivity in a grooved heat pipe. The ratio of the heat transfer through the thin film region to the total heat transfer through the wall to the vapor phase decreases when the contact angle increases. The superheat effects on the heat flux distribution in the thin film region also have been conducted and the results show that the disjoining pressure plays an important role in this region. The current investigation will result in a better understanding of thin film evaporation and its effect on the effective thermal conductivity in a grooved heat pipe.
This study was conducted to determine the osmotic properties of bull spermatozoa, including the effects of osmotic stress
and cryoprotectant agent (CPA) addition and removal, on sperm motility. Semen ...from beef bulls was collected by electroejaculation
and extended 1:3 in TL-Hepes containing 100 μg/ml pyruvate and 6 mg/ml BSA. In solutions of 150â1200 mOsmolal (mOsm), bull
spermatozoa behaved as linear osmometers ( r 2 = 0.97) with an osmotically inactive cell volume of 61%. The isosmotic cell volume was 23.5 μm 3 . Motility was determined after exposure to anisosmotic solutions ranging from 35 to 2400 mOsm and after return to isosmotic
conditions. Retention of at least 90% of isosmotic motility could be maintained only between 270â360 mOsm. Bull spermatozoa
were calculated to retain 90% of their isosmotic motility at 92â103% of their isosmotic cell volume. Motility following a
one-step addition and removal of 1 M glycerol, dimethyl sulfoxide, and ethylene glycol was reduced by 31%, 90%, and 6%, respectively,
compared with CPA addition only. These data indicate that, during bull spermatozoa cryopreservation, osmotically driven cell
volume excursions must be limited by exposure to a very narrow range that may be facilitated by the use of ethylene glycol
as a CPA.
BACKGROUND: Knowing osmotic tolerance limits is important in the design of optimal cryopreservation procedures for cells. METHODS: Mature human oocytes were exposed to anisosmotic sucrose solutions ...at concentrations of 35, 75, 150, 600, 1200, or 2400 (±5) milliosmolal (mOsm) at 37°C. A control treatment at 290 mOsm was also utilized. Oocytes were randomly allocated to each experimental treatment. After the treatment, the oocytes were cultured for 1 h, then fixed in cold methanol. Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II (MII) spindle. Logistic regression was used to determine if media osmolality had a significant effect on spindle structure. RESULTS: Osmolality was a significant predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150 mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles, respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted in 44, 44, and 100% of the spindles with abnormal structure, respectively. CONCLUSIONS: Anisosmotic conditions lead to disruption of the MII spindle in human oocytes. Applying this fundamental knowledge to human oocyte cryopreservation should result in increased numbers of cells maintaining viability.
The rat is an important system for modeling human disease. Four years ago, the rich 150-year history of rat research was transformed by the sequencing of the rat genome, ushering in an era of ...exceptional opportunity for identifying genes and pathways underlying disease phenotypes. Genome-wide association studies in human populations have recently provided a direct approach for finding robust genetic associations in common diseases, but identifying the precise genes and their mechanisms of action remains problematic. In the context of significant progress in rat genomic resources over the past decade, we outline achievements in rat gene discovery to date, show how these findings have been translated to human disease, and document an increasing pace of discovery of new disease genes, pathways and mechanisms. Finally, we present a set of principles that justify continuing and strengthening genetic studies in the rat model, and further development of genomic infrastructure for rat research.
As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. ...However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN₂). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a “closed” system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease.
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