A method for freezing bovine lymphocytes

A simple two-stage technique for preserving bovine lymphocytes is described. Lymphocytes from animals chosen at random were used. The experiments indicate that the optimum temperature for freezing and the optimum concentration of dimethylsulphoxide (DMSO) as cryoprotectant were in the range -29 degrees C to -31 degrees C and 17.5% to 20% respectively. These concentrations of DMSO are much greater than those reported in most other studies.     

A method for freezing bovine lymphocytes

A simple two-stage technique for preserving bovine lymphocytes is described. Lymphocytes from animals chosen at random were used. The experiments indicate that the optimum temperature for freezing and the optimum concentration of dime-thylsulphoxide (DMSO) as cryoprotectant were in the range -29 °C to -31 °C and 17.5 % to 20 % respectively. These concentrations of DMSO are much greater than those reported in most other studies.     

A simplified method for freezing mouse embryos

Mouse oviducts containing eight-cell embryos were frozen to ?196 °C in 1.45 m DMSO. The cooling rate was 0.3 °C/min and thawing occurred at 3 °C/min. Dilution of DMSO took place either before or after flushing of the thawed oviducts. The yield of intact embryos was higher in the second group.In one particular series involving 21 donor mice (natural ovulation) 88 recovered embryos were transferred to the oviducts of recently mated pseudopregnant mice without prior in vitro culture to the blastocyst stage. Fifty-five live young were born.It is concluded that the freezing of embryos in the oviduct is a reliable method for establishing an embryo bank. Handling and collection of isolated embryos is not required and a large amount of material can be frozen at once. In vitro culturing of embryos is not required immediately after thawing in order to obtain a high yield of live young.     

A high yield method for isolating rat islets of Langerhans using differential sensitivity to freezing

Conventional methods of isolating islets of Langerhans rely upon the differential sensitivity of the pancreatic acinar tissue vs islets to enzymatic dissociation by crude collagenase, however, the yield of intact islets obtainable with this technique is quite low. Higher yields of islets can be obtained with pharmacological or surgical methods which either destroy acinar cells or cause them to release their zymogen granules. However, because of the requirement to pretreat the donor, these methods cannot be scaled-up for potential clinical use. To overcome the limitations of the conventional isolation procedures, we exploited the differential sensitivity of acinar cells and islet cells to freezing damage. Using this approach we are able to isolate greater than 2500 islets from the pancreas of a single rat. Basically, we rapidly mince pancreatic tissue, subject the tissue to a short collagenase digestion, briefly freeze the tissue at -30 degrees C in the presence of glycerol, and immediately thaw it. Subsequent enzyme treatment digests the residual acinar tissue, collagen, DNA, and proteins. Preliminary results indicate that the islets are morphologically indistinguishable from islets isolated using conventional digestion techniques.     

A simple method forin situ freezing of anchorage-dependent cells including rat liver parenchymal cells

We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish. The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at –80°C. After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.     

New preservation method for mouse spermatozoa without freezing

The objective of this study was to investigate the preservation of spermatozoa in a simple medium without freezing and to examine the effects of the preserved sperm on fertilization and development after injection into mature mouse oocytes. Mouse spermatozoa were collected from two caudae epididymides of mature B6D2F1 males and stored under various conditions: 1) in KSOMaa medium (potassium simplex optimized medium with amino acids) supplemented with 0, 1, or 4 mg/ml BSA and held at room temperature (RT, 27 degrees C); 2) in KSOMaa medium containing 4 mg/ml BSA (KSOM-BSA) and held at 4 degrees C, RT, or 37 degrees C (CO2 incubator); 3) in KSOM-BSA with osmolarity ranging from 271 to 2000 mOsmol, adjusted by addition of NaCl and held at 4 degrees C; and 4) a two-step preservation system consisting of storage in 800 mOsmol KSOM-BSA for 1 wk at RT followed by storage at -20 degrees C. Preservation of mouse spermatozoa at 4 degrees C in a medium with high osmolarity (700-1000 mOsmol) resulted in the highest frequency of live births after intracytoplasmic sperm injection (ICSI) into mature oocytes. The optimal conditions for preservation of mouse spermatozoa were 800 mOsmol KSOM containing 4 mg/ml BSA and a holding temperature of 4 degrees C. More than 40% of oocytes injected with sperm heads stored under these conditions for 2 mo developed to the morula/blastocyst stage in vitro and 39% of the embryos developed to term after transfer to recipient mice. Our results also indicate that mouse spermatozoa can be stored in 800 mOsmol KSOM-BSA medium at RT for 1 wk and then at -20 degrees C for up to 3 mo and retain their competence for ICSI. These new preservation methods permit extended conservation of viable spermatozoa that are capable of supporting normal embryonic development and the live birth of healthy offspring after ICSI.     

Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 microg kg(-1) in liver and 5, 15 and 50 microg kg(-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 microg kg(-1), respectively.     

An effective method for freezing White Italian gander semen

Efficiency of freezing method, worked out for the White Italian gander semen was evaluated by comparing motility, morphology and fertilizing ability of spermatozoa in fresh and frozen-thawed semen. A part of pooled semen, collected from 25 White Italian ganders by dorso-abdominal massage was used immediately for artificial insemination of 10 geese (the control group) with a dose of 80 microl. This insemination was performed six times at weekly intervals. The remainder of the semen was diluted 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with 6% (v/v) of dimethylformamide (DMF) and frozen to -140 degrees C at a rate of 60 degrees C/min. Frozen semen was thawed in a 60 degrees C water-bath and inseminated twice weekly in a dose of 100 microl (10 females of the experimental group, 12 inseminations were made). The freezing process affected spermatozoa motility and morphology, but had no effect on their fertilizing ability. Positive movement was observed in 50-60% of the spermatozoa in fresh semen and about 40% of the frozen-thawed cells. The average percentage of total live and live normal spermatozoa decreased due to freezing from 92.2 to 68.4% and from 34.7 to 14.1%, respectively. After the fresh semen insemination with average 12 million of the live normal spermatozoa per week average fertility was 88.24%; hatchability of set eggs was 80.88% and hatchability of fertile eggs was 91.67%. For frozen-thawed semen inseminated with average 9.5 million of the undamaged spermatozoa per week, the average fertility and hatchability rate was 83.78, 73.87, and 88.17%, respectively. Fecundity rates obtained after insemination with the frozen-thawed gander semen allow for the application of the freezing technique into breeding practice, in place of natural mating or to assist natural mating in periods of lowered fertility level.     

Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports

The aim of this study was to improve the freezing protocol of bull sperm, by investigating the influence on sperm viability after freeze/thawing of different freezing medium components, as well as the effect of cooling rates in the different stages of the cooling protocol, in single factor experiments. The experimental variables were: (1) salt-based versus a sugar-based medium (Tris versus sucrose); (2) glycerol concentration; (3) detergent (Equex) concentration; (4) presence of bicarbonate; (5) rate of cooling from 22 degrees C to holding temperature (CR1); (6) holding temperature (HT); (7) rate of cooling from holding temperature to -6 degrees C (CR2); (8) rate of cooling from -10 to -100 degrees C (CR3). All experiments were performed using five bulls per experiment (three ejaculates per bull). Sperm motility after freezing and thawing was assessed by CASA system, and sperm membrane integrity was assessed by flow cytometry. Sucrose-based medium did not offer a clear significant benefit compared to Tris medium. The concentration of Equex that gave the best results in Tris-based media group and sucrose-based media group was in a range between 2-7 and 4-7 g/l, respectively. In both media groups, a glycerol concentration of 800 mM was the best in any post-thaw viability parameters. In the Tris media group, the presence of bicarbonate had a negative effect on sperm viability. CR1 and CR2 had no significant effect on any of the post-thaw sperm viability parameters, but a CR1=0.2 degrees C/min and CR2=4 degrees C/min appeared to give better results in both media. The holding temperature (HT) that gave the best results was found to be in the range of 5-9 degrees C. There was a significant disadvantage of using a low CR3 of 10 degrees C/min, while 150 degrees C/min appeared to be the best cooling rate for either medium.     

Simple, inexpensive method for controlled freezing of cell cultures

We have devised a system for controlled cooling of living cells which eliminates the need for complicated, expensive equipment but allows excellent recovery of viable cells after storage in liquid nitrogen.     

Simple, inexpensive method for controlled freezing of cell cultures

[This corrects the article on p. 616 in vol. 27.].     

A method for freezing synchronous mitotic and G1 cells

A modification of the protocol developed by Kawamoto, J C & Barrett, J N, Brain res (1986), in press for freezing primary neuron cultures in a solution containing low sodium and high lactate and potassium concentrations was used to freeze synchronous mitotic and G1 CHO cells. After thawing, the cells behaved as if they had never been frozen with respect to cell growth, cell division, plating efficiency, and hyperthermic sensitivity.     

A method of parental selection and cross prediction using incomplete partial diallels

Summary Gel electrophoretic investigations were made on the seed albumins of several members of the family Papilionaceae. Relationships were found with taxa of a lower order i.e. between mutants, varieties and subspecies. More distantly related ones, for example species of the same genus or species of different genera, did not show similarities. Thus, it was concluded that the albumin banding pattern is only suitable for studying phylogenetic and taxonomic problems if the material under investigation is not too distantly related.