Cryopreservation and the age of the allotransplant

For centuries, reconstructive surgeons have restored form and function with autografts. These techniques are highly effective, but they are associated invariably with donor site morbidity. To avoid this, surgeons have long dreamed of using cadaveric sources for reconstructive material. However, allografts have two major limitations: rejection and limited donor tissue. In order to limit rejection, the allograft must be rendered more tolerable to the host or the host must be rendered more tolerant of the allograft. Both strategies have been used with considerable success in recent years. As understanding of the human immune response increases, clinical immunosuppressive regimens will undoubtedly become less morbid, and the indications for allotransplantation will broaden. This will place an even greater burden on the already small donor pool. One way to relieve this burden would be through the development of strategies for the long-term preservation of donated tissues and organs. Cryopreservation has been used clinically for decades, and recent advances in the field have allowed the preservation of an ever widening array of tissues and organs. As cold storage has been shown to reduce the antigenicity of parts, cryopreservation may actually serve to improve the survival rate of transplanted parts, as well as increase their availability. As the era of autotransplantation gives way to the age of allotransplantation, cryopreservation will play an increasingly important role.     

Immobilization of Cryopreserved Primary Rat Hepatocytes for the Development of a Bioartificial Liver System

Primary rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing 40% (v/v) fetal bovine serum (FBS) and 10% (v/v) dimethyl sulfoxide (DMSO) in liquid N₂ for 6 months. After thawing, the cells were immobilized using 2% (w/v) alginate and 0.5% (w/v) chitosan solutions. The capacities of ammonia removal and urea synthesis of the immobilized-thawed hepatocytes were similar to those of immobilized hepatocytes without cryopreservation. This result shows that immobilized hepatocytes after cryopreservation are useful for the development of a bioartificial liver system.     

Ultrastructural characterization of spermatozoa in euglossine bees (Hymenoptera, Apidae, Apinae)

Summary. Euglossine spermatozoa are the longest described to date for the Hymenoptera. This cell includes a head and a flagellar region. In transverse sections, the acrosome is circular at the tip but has an oval contour along most of its length. The perforatorium penetrates into a deep cavity in the nuclear tip. The flagellum consists in an axoneme, a pair of mitochondrial derivatives, a centriolar adjunct and a pair of accessory bodies. The axoneme has a 9+9+2 microtubule pattern which becomes gradually disorganized in the final portion, with the central microtubules and the nine doublets terminating simultaneously, followed by the accessory microtubules. The mitochondrial derivatives are asymmetric both in length and diameter. Sectioned transversally, the derivatives are ellipsoidal or have a pear shape. The larger one has a more obvious paracrystalline region. The centriolar adjunct begins at the nuclear base and extends parallel to the axoneme until it encounters the smaller mitochondrial derivative, on which it fits, making a concave groove. In addition to these consistent euglossine features, species-specific differences that might be useful in phylogenetic work on the group are also noted.Received 18 October 2003; revised 4 September 2004; accepted 4 October 2004.     

Spermiogenesis and sperm morphology in the marine gastropod Nucella crassilabrum with an account of morphometric patterns of spermatozoa variation in the family Muricidae

Abstract

Spermatid differentiation and the morphology of mature spermatozoa in Nucella crassilabrum, a muricid snail, was investigated. Five phases of spermiogenesis considering the polarization of organelles, nuclear elongation and chromatin condensation are described. Characteristics observed are compared to those of other muricaceans gastropods. The comparison of the proportional size of the different sperm segments (head, middle- and principal piece) between these species, is emphasized.

Two morphometric patterns of sperm structure are distinguished and their functional significance discussed. The relative size attained by the different sperm segments within each of these patterns, could be the result of the combined effect of at least two types of factors: first, those determining the proportional size (length) of the head and, secondly, those factors conditioning the energy requirements of the gamete thus influencing the relative development of the middle- and principal piece.     

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm® gradient between freeze–thaw cycles

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze–thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing–thawing cycles on sperm quality and to analyze three different elapsed times between freezing–thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing–thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen–thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.     

Identification of a rabbit sperm autoantigen as a Ricinus communis I receptor

The isolated rabbit sperm plasma membrane autoantigen RSA-1 has been identified as a receptor for the lectin, Ricinus communis I (RCA). Using purified RSA-1 labeled with¹²⁵ I, the autoantigen was shown to bind to RCA affinity columns and the eluted fraction bound to specific anti-RSA-1 alloantiserum immunoadsorbent columns.     

Asian low‐androstenone haplotype on pig chromosome 6 does not unfavorably affect production and reproduction traits

European pigs that carry Asian haplotypes of a 1.94‐Mbp region on pig chromosome 6 have lower levels of androstenone, one of the two main compounds causing boar taint. The objective of our study was to examine potential pleiotropic effects of the Asian low‐androstenone haplotypes. A single nucleotide polymorphism marker, rs81308021, distinguishes the Asian from European haplotypes and was used to investigate possible associations of androstenone with production and reproduction traits. Eight traits were available from three European commercial breeds. For the two sow lines studied, a favorable effect on number of teats was detected for the low‐androstenone haplotype. In one of these sow lines, a favorable effect on number of spermatozoa per ejaculation was detected for the low‐androstenone haplotype. No unfavorable pleiotropic effects were found, which suggests that selection for low‐androstenone haplotypes within the 1.94 Mbp would not unfavorably affect the other eight relevant traits.     

High-throughput cryopreservation of spermatozoa of blue catfish (Ictalurus furcatus): Establishment of an approach for commercial-scale processing

Hybrid catfish created by crossing of female channel catfish (Ictalurus punctatus) and male blue catfish (Ictalurus furcatus) are being used increasingly in foodfish aquaculture because of their fast growth and efficient food conversion. However, the availability of blue catfish males is limited, and their peak spawning is at a different time than that of the channel catfish. As such, cryopreservation of sperm of blue catfish could improve production of hybrid catfish, and has been studied in the laboratory and tested for feasibility in a commercial dairy bull cryopreservation facility. However, an approach for commercially relevant production of cryopreserved blue catfish sperm is still needed. The goal of this study was to develop practical approaches for commercial-scale sperm cryopreservation of blue catfish by use of an automated high-throughput system (MAPI, CryoBioSystem Co.). The objectives were to: (1) refine cooling rate and cryoprotectant concentration, and evaluate their interactions; (2) evaluate the effect of sperm concentration on cryopreservation; (3) refine cryoprotectant concentration based on the highest effective sperm concentration; (4) compare the effect of thawing samples at 20 or 40 °C; (5) evaluate the fertility of thawed sperm at a research scale by fertilizing with channel catfish eggs; (6) test the post-thaw motility and fertility of sperm from individual males in a commercial setting, and (7) test for correlation of cryopreservation results with biological indices used for male evaluation. The optimal cooling rate was 5 °C/min (Micro Digitcool, IMV) for high-throughput cryopreservation using CBS high-biosecurity 0.5-ml straws with 10% methanol, and a concentration of 1 × 10⁹ sperm/ml. There was no difference in post-thaw motility when samples were thawed at 20 °C for 40 s or 40 °C for 20 s. After fertilization, the percentage of neurulation (Stage V embryos) was 80 ± 21%, and percentage of embryonic mobility (Stage VI embryo) was 51 ± 22%. There was a significant difference among the neurulation values produced by thawed blue catfish sperm, fresh blue catfish sperm (P = 0.010) and channel catfish sperm (P = 0.023), but not for Stage VI embryos (P ? 0.585). Cryopreserved sperm from ten males did not show significant variation in post-thaw motility or fertility at the neurulation stage. This study demonstrates that the protocol established for high-throughput cryopreservation of blue catfish sperm can provide commercially relevant quantities and quality of sperm with stable fertility for hybrid catfish production and provides a model for establishment of commercial-scale approaches for other aquatic species.     

Effective surface-based cryopreservation of human embryonic stem cells by vitrification

Human embryonic stem cells (hESCs) are candidates for many applications in the areas of regenerative medicine, tissue engineering, basic scientific research as well as pharmacology and toxicology. However, use of hESCs is limited by their sensitivity to freezing and thawing procedures. Hence, this emerging science needs new, reliable preservation methods for the long-term storage of large quantities of functional hESCs remaining pluripotent after post-thawing and culturing.Here, we present a highly efficient, surface based vitrification method for the cryopreservation of large numbers of adherent hESC colonies, using modified cell culture substrates. This technique results in much better post-thaw survival rate compared to cryopreservation in suspension and allows a quick and precise handling and storage of the cells, indicating low differentiation rates.