Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm

Freeze-drying sperm is an alternative to cryopreservation. Although sperm from various species has been freeze-dried, there are few reports for bovine sperm. The primary objective of this study was to evaluate the protective effect of various freeze-drying media on the structural and functional components of bovine sperm. The media tested were composed of TCM 199 with Hanks salts supplemented with 10% fetal calf serum (FCS) and TCM 199 with Hanks salts supplemented with 10% FCS and 0.2 M trehalose and EGTA solution. The efficiency of each medium on the preservation of freeze-dried sperm structures was evaluated with conventional and electron microscopy, DNA integrity was analyzed by a TUNEL assay, and fertilizing ability of lyophilized sperm was determined with ICSI. Although the plasma membrane was damaged in all media tested, mitochondria were similarly preserved in all freeze-drying treatments. The acrosome was best preserved in the media that contained trehalose (other treatments also conserved this structure). In contrast, media containing EGTA or trehalose most effectively preserved the nuclei in freeze-dried sperm, with only 2 and 5%, respectively, of cells with fragmented DNA. Furthermore, sperm conserved with these media also had higher (P < 0.05) rates of sperm head decondensation (32.5 and 27.5%), pronucleus formation (37.5 and 45.0%) and blastocyst formation (19.4 and 18.3%) than medium supplemented with FCS (15.0, 20.0 and 10.2%, respectively). In conclusion, media with EGTA and trehalose adequately protected bovine sperm during freeze-drying by preserving the viability of their nuclei

Scanning Electron Microscopy of the Mammalian Organ of Corti: Assessment of Preparative Procedures

Different fixation, drying and coating procedures have been applied in preparation of the organ of Corti for scanning electron microscopy (SEM), and structural features of the apical surface of the tissue in unfixed, freeze-fractured preparations used in assessing their effects on morphology. Fixation with glutaraldehyde alone or osmium tetroxide alone causes artefacts that are substantially avoided when tissue is doubly fixed in glutaraldehyde followed by osmium. Significant improvements in preservation are also obtained when tissue is additionally processed through thiocarbohydrazide-osmium (TOTO) processing. In addition to providing a conducting coat, it stabilises the tissue against deformations that might otherwise occur during drying, and reduces the extent of tissue shrinkage. Freeze-drying of TOTO processed tissue produces less tissue distortion than critical point drying (CPD) but is not so easy to apply routinely. The distortions of structure in TOTO-processed CPD tissue are not significant and this may be the preferred procedure for routine use, but air drying from hexamethyldisilazane is a useful alternative, producing results as good as those from CPD samples if TOTO processing is applied beforehand. One particular advantage of freeze-drying, though, is that after freezing, brittle fracture through the tissue can occur making examination of intracellular structure by SEM relatively easy. However, again, TOTO processing prior to freezing is of value as this appears to prevent the formation of large ice-crystal during freezing. Examination of isolated outer hair cells by SEM shows that isolation procedures do not cause significant damage to the stereociliary bundles

Freeze-Drying of Mononuclear Cells Derived from Umbilical Cord Blood Followed by Colony Formation

BACKGROUND: We recently showed that freeze-dried cells stored for 3 years at room temperature can direct embryonic development following cloning. However, viability, as evaluated by membrane integrity of the cells after freeze-drying, was very low; and it was mainly the DNA integrity that was preserved. In the present study, we improved the cells' viability and functionality after freeze-drying. METHODOLOGY/PRINCIPAL FINDINGS: We optimized the conditions of directional freezing, i.e. interface velocity and cell concentration, and we added the antioxidant EGCG to the freezing solution. The study was performed on mononuclear cells (MNCs) derived from human umbilical cord blood. After freeze-drying, we tested the viability, number of CD34(+)-presenting cells and ability of the rehydrated hematopoietic stem cells to differentiate into different blood cells in culture. The viability of the MNCs after freeze-drying and rehydration with pure water was 88%-91%. The total number of CD34(+)-presenting cells and the number of colonies did not change significantly when evaluated before freezing, after freeze-thawing, and after freeze-drying (5.4 x 10(4)+/-4.7, 3.49 x 10(4)+/-6 and 6.31 x 10(4)+/-12.27 cells, respectively, and 31+/-25.15, 47+/-45.8 and 23.44+/-13.3 colonies, respectively). CONCLUSIONS: This is the first report of nucleated cells which have been dried and then rehydrated with double-distilled water remaining viable, and of hematopoietic stem cells retaining their ability to differentiate into different blood cells

The Ability of Bull and Stallion Thawed Spermatozoa Refrozen without Cryoprotectants to Activate Intra- and Interspecies Oocytes

Semen cryopreservation has allowed the establishment of genome banks and the large scale propagation of species. The development of simple techniques to cryopreserve semen or alternatives to efficiently use cryopreserved semen from males of valuable genetics that have become infertile will permit continuous propagation of the genetics from these males and may serve as a model for preservation and propagation of endangered species. Sperm cryopreservation without cryoprotectants is a simple process, and offspring have been produced following intracytoplasmic sperm injection (ICSI); however the ability of frozen-thawed sperm refrozen without the addition of cryoprotectants to activate oocytes following ICSI was unknown. In the series of experiments performed, bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants was used to activate intra- and interspecies oocyte following ICSI. Additionally, equine cumulus-oocyte complexes (COCs) glucose metabolism during in vitro maturation was evaluated. The first experiment demonstrated that bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants had their plasma membrane damaged; however the DNA was unaffected. The second experiment demonstrated that bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants had the ability to activate bovine oocytes following intracytoplasmic sperm injection; although at a lower rate compared to frozen-thawed sperm. The third experiment demonstrated that frozen-thawed stallion sperm refrozen without the addition of cryoprotectants was unable to activate equine oocytes. The exact reason for this failure could not be explained from the experiment; however COC metabolism during in vitro maturation impacts embryo activation/development and required further investigation. The fourth experiment demonstrated that equine COCs consume and metabolize glucose through glycolysis during in vitro maturation; however, results from this experiment were unable to explain the failure of refrozen stallion sperm to activate equine oocytes. To our knowledge, this is the first report of the use of bull or stallion frozen-thawed sperm refrozen without the addition of cryoprotectants to activate oocytes following ICSI. Furthermore, this is also the first report of equine COCs glucose metabolism during in vitro maturation

Intracytoplasmic sperm injection (ICSI) in fishes

Sperm from zebrafish, Danio rerio, and Nile tilapia, Oreochromis niloticus, were microinjected directly into egg cytoplasm to evaluate the potential for developing a novel method of fertilization. In zebrafish, the sperm of two lines (wild-type and gold, long-fin) were injected with or without activation into activated and non-activated eggs. No significant difference (P = 0.997) in fertilization by intracytoplasmic sperm injection (ICSI) was observed between the two lines or when the sperm were activated or not (P = 0.057). There was significance difference in fertilization between activated and non-activated eggs (P = 0.010). The highest fertilization rate was achieved by injection of activated sperm into non-activated eggs (35%). From a total of 188 zebrafish eggs injected, 31 (16%) were fertilized, 10 (5%) developed as abnormal larvae and 3 (2%) developed normally and hatched. Damage of maternal chromosomes by the injection procedure could have caused the developmental abnormalities observed after ICSI. This was investigated by fluorescence microscopy using a DNA-specific stain (Hoechst 33324). Fixed and stained animal poles of zebrafish 30 sec after artificial insemination revealed that female chromosomes were located ~40 mm from the sperm injection site (micropyle). Staining of the animal pole after sperm injection showed no disruption of the formation of the second polar body or its extrusion. Evaluation of two sperm injection sites in zebrafish showed no difference in fertilization rate (P = 0.8264) or reduction of abnormal development. Nile tilapia eggs placed in Hanks\u27 balanced salt solution retained their viability for at least 3 hours after collection. Of a total of 160 Nile tilapia eggs injected with fresh sperm, 16 (10%) were fertilized, 10 (6%) developed abnormally to neurula and 5 (3%) developed normally and hatched, two of which reached adulthood. From 45 eggs injected with cryopreserved sperm, 9 (20%) were fertilized but none developed beyond blastula stage. Injections of sperm fixed in methanol did not yield fertilization. These results demonstrate for the first time that injection of single sperm cells into the cytoplasm of a fish egg allows fertilization and subsequent development of normal larvae to hatching and beyond

X-Ray Microanalysis of Endocrine, Exocrine and Intestinal Cells and Organs in Culture: Technical and Physiological Aspects

In the present study methods for preparation of cultured cells and organ cultures for analytical electron microscopy are investigated. These methods allow qualitative and quantitative analysis of mobile ions in combination with biochemical or morphological studies. Cultured cells can be easily prepared for analytical microscopy and therefore use of in vitro systems for X-ray microanalysis has increased over the last few years. Two major, anhydrous preparation techniques, by which loss or redistribution of ions is minimized, were used: (1) Cells were cryosectioned and analysis carried out on freeze-dried sections obtained from frozen cell monolayers, pelleted cells or organ cultures. (2) Cells cultured on supports compatible with elemental analysis were frozen after removal of experimental media by rinsing, freeze-dried and analyzed. The first technique was applied to the studies of the elemental content of isolated Langerhans islets and thyroid follicles cultured in collagen gel. The second was used in studies of the ionic changes in enterocytes. Data obtained from organotypic cell cultures and cultures of single cells were compared with analytical data obtained from sections of corresponding tissues, where isolation, culturing and steps in processing such as removal of culture or experimental medium were omitted. It was shown that often culture systems fully acceptable to physiologists have an elemental composition different from that of tissue in situ and can not be regarded as fully normal tissue