Fertilization potential of follicular oocytes classified by stage of cycle and size of follicle

Bovine follicular oocytes, collected from two sizes of vesicular follicles and from donor animals from three stages of the estrous cycle, were matured and fertilized in vitro . Frequency of fertilization and ability to form male pronuclei after in vitro maturation were found to be independent of both estrual stage and follicular size.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

Glycerol permeability of human spermatozoa and its activation energy

Glycerol has commonly been employed as a cryoprotectant in cryopreservation of human spermatozoa. However, the addition of glycerol into the sperm before freezing and the removal of glycerol from the sperm after freezing and thawing result in anisotonic environments to the cells, which can cause cell injury. To define optimal procedures for the addition/removal of glycerol and to minimize the cell injury, one needs to know the kinetics of glycerol permeation across the sperm plasma membrane at different temperatures. For this, one has to determine the permeability coefficient of glycerol (Pg) and its activation energy (Ea). Values of Pg at different temperatures and at different glycerol concentrations were determined by measuring the time required for 50% spermolysis in hyperosmotic glycerol solutions which were hypotonic with respect to electrolytes. Value of the Ea was determined assuming an Arrhenius type temperature dependence of Pg. A dual fluorescent staining technique (propidium iodide and 6-carboxyfluoroscein diacetate) and flow cytometry were used to measure the spermolysis. The values of Pg in 0.5, 1.0, 1.5, and 2.0 M glycerol at 22 degrees C are 1.62, 1.88, 1.68, and 1.54 x 10(-3) cm/min, respectively. The values of Pg in 1 M glycerol at 0, 8, 22, and 30 degrees C are 0.33, 0.54, 1.88, and 2.60 x 10(-3) cm/min, respectively. The value of Ea is 11.76 kcal/mol.     

Improved islet survival and in vitro function using solubilized small intestinal submucosa

In vitro proliferation of isolated pancreaticislets has become an area of great interest given the scarcity of clinicalisletdonors and the islet mass requirements for clinical islet transplantation.Smallintestinal submucosa (SIS), a naturally occurring extracellular matrix, hasbeeninvestigated to promote wound healing, tissue remodeling and cell growth. Thisstudy evaluated recovery and function of isolated canine pancreatic isletsfollowing in vitro tissue culture. Pancreatic islets wereisolated from mongrel dogs using standard surgical procurement followed byintraductal collagenase distension, mechanical dissociation and EuroFicollpurification. Groups of purified islets were cultured in a humidifiedatmosphereof 95% air and 5% CO₂ for 48 hours in standard islet cultureconditions of CMRL 1066 tissue culture media (Gibco) which had beensupplementedwith 25M HEPES, penicillin/streptomycin and either 10% heat inactivatedfetal calf serum (FCS, Gibco) or solubilized SIS solution (Cook Biotech, Inc.,West Lafayette, IN). The mean recovery of islets following the culture periodwas determined by sizing duplicate counts of a known volume and viability wasassessed by static incubation with low glucose (2.8 mM), highglucose (20 mM) and high glucose solution supplemented with 50m IBMX solution. Remaining islets were embeddedhistologically.From a consecutive series of six culture experiments, a significantly higher (p< 0.05) recovery of islets co-cultured with SIS was observed when comparedtocontrols. Mean islet recovery was 84.5 ± 2.9% (mean ± SEM) fromthe SIS cultured group compared with 64.7 ± 4.5% from the control groupcultured in FCS (p < 0.05, n=6). Islets from the SIS treated group exhibiteda significantly higher (p <, 0.05) insulin response to the high glucosestimulus than islets cultured in the standard FCS cultured solution. Thecalculated stimulation index was 12.3 ± 3.4 for the SIS-treated groupcompared with 5.6 ± 1.8 for the standard cultured group (p < 0.05).The overall mean numbers of islets recovered following invitro culture was also higher in the SIS-treated group. Theproportion of islets with a mean diameter >150 m increasedfrom 24% to 31% in the SIS-treated group, whereas the same proportion decreasedto 18% from 22% in the control (FCS-treated) group. Histological evaluation offixed tissue samples collected following the culture period identified insulinand glucagon-secreting cells in the SIS and FCS treated groups, however ahigherfrequency of insulin positive cells were detected consistently in the SIStreated group. A proliferation marker (PCNA) identified positive cells withinboth groups as well. This study suggests that co-culture of freshly isolatedcanine islets in medium supplemented with solubilized SIS can improve thepost-culture recovery and in vitro islet function. Futureinvestigations will focus on the cellular interactions of SIS, bothinvitro and in vivo.     

Canine RBC osmotic tolerance and membrane permeability

The objective of this study was to determine the cryobiological characteristics of canine red blood cells (RBC). These included the hydraulic conductivity (L(p)), the permeability coefficients (P(s)) of common cryoprotectant agents (CPAs), the associated reflection coefficient (sigma), the activation energies (E(a)) of L(p) and P(s) and the osmotic tolerance limits. By using a stopped-flow apparatus, the changes of fluorescence intensity emitted by intracellularly entrapped 5-carboxyfluorescein diacetate (CFDA) were recorded when cells were experiencing osmotic volume changes. After the determination of the relationship between fluorescence intensity and cell volume, cell volume changes were calculated. These volume changes were used in three-parameter fitting calculations to determine the values of L(p), P(s), and sigma for common CPAs. These volume measurements and data analyses were repeated at three different temperatures (22, 14, 7 degrees C). Using the Arrhenius equation, the activation energies of L(p) and P(s) in the presence of CPAs were determined. The osmotic tolerance limits for canine RBC were determined by measuring the percentage of free hemoglobin in NaCl solutions with various osmolalities compared to that released by RBC incubated in double distilled water. The upper and lower osmotic tolerance limits were found to be 150mOsm (1.67V(iso)) and 1200mOsm (0.45V(iso)), respectively. These parameters were then used to calculate the amount of non-permeating solute needed to keep cell volume excursions within the osmotic tolerance limits during CPA addition and removal.     

Osmotic characteristics of mouse spermatozoa in the presence of extenders and sugars

Successful cryopreservation requires cells to tolerate volume excursions experienced during permeating cryoprotectant equilibration and during cooling and warming. However, prior studies have demonstrated that mouse spermatozoa are extremely sensitive to osmotically induced volume changes. A series of three experiments were conducted 1) to test the efficacy of two commonly used extender media components, egg yolk (EY) and skim milk (SM), in broadening the osmotic tolerance limits (OTL) of ICR and B6C3F1 murine spermatozoa; 2) to determine if the extender components affected sperm plasma membrane permeability coefficients for water and cryoprotective agent (CPA) characteristics; and 3) to test the effects of permeating and nonpermeating CPA on mouse sperm morphology. In experiment 1, sperm samples were added to 150, 225, 300, 450, or 600 mOsm NaCl, EY, SM, sucrose, or choline chloride at 22 degrees C and then returned to isosmotic conditions. In experiment 2, epididymal sperm were preequilibrated in 1 M glycerol (Gly) or 2 M ethylene glycol (EG) prepared in SM extender, abruptly exposed to isosmotic conditions at 22, 15, or 2 degrees C, and the corresponding volume excursions were measured and analyzed. In experiment 3, the effects of permeating CPA (0.3 M EG or dimethyl sulfoxide) or nonpermeating CPA (12% sucrose or 18% raffinose) on sperm morphology (i.e., principle midpiece folding and putative membrane fusion) were evaluated. Experiment 1 showed that spermatozoa from ICR and B6C3F1 mice have effectively broader OTL when exposed to EY or SM extenders. The results of experiment 2 indicated that, for ICR sperm, the activation energy (E(a)) for the hydraulic conductivity (L(p)) was unchanged in SM extender. However, for B6C3F1 sperm, there were significant differences in E(a) of L(p) in the presence of Gly and EG. The result of experiment 3 indicated that permeating CPAs damage sperm membrane integrity, causing a high frequency of head-to-tail or tail-to-tail membrane fusion, whereas this occurrence in the presence of nonpermeating CPA was less than 3%. Finally, the results of experiments 1 and 2 were combined in a mathematical model to predict Gly and EG addition and removal in the presence of SM extender, which would prevent mouse sperm membrane damage. These predictions indicated that, for ICR sperm, both Gly and EG may be added and removed in a single step. However, for B6C3F1 spermatozoa, Gly required a two-step addition while EG only required a single step. For removal from B6C3F1 sperm, Gly required a three-step removal process while EG required a two-step removal.     

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I—Comparative fundamental cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols

The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols.     

Variants in the interleukin-1 alpha and beta genes,and the risk for periodontal disease in dogs

Elevated levels of interleukin-1 (IL-1) have been shown to amplify the inflammatory response against periodontopathogenic bacteria. In humans, polymorphisms in the IL1A and IL1B genes are the most well-studied genetic polymorphisms associated with periodontal disease (PD). In contrast to human, there is a lack of knowledge on the genetic basis of canine PD. A case–control study was conducted in which a molecular analysis of dog IL1A and IL1B genes was performed. Of the eight genetic variants identified, seven in IL1A gene and one in IL1B gene, IL1A/1_g.388A >C and IL1A/1_g.521T >A showed statistically significant differences between groups (adjusted OR (95% CI): 0.15 (0.03–0.76), P= 0.022; 5.76 (1.03–32.1), P= 0.046, respectively). It suggests that in the studied population the IL1A/1_g.388C allele is associated with a decreased PD risk, whereas the IL1A/1_g.521A allele can confer an increased risk. Additionally, the IL1A/2_g.515G >T variation resulted in a change of amino acid, i.e. glycine to valine. In silico analysis suggests that this change can alter protein structure and function, predicting it to be deleterious or damaging. This work suggests that IL1 genetic variants may be important in PD susceptibility in canines.