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Conservation of sperms: current status and new
trends
Mitsutoshi Yoshida
)Laboratory of Animal Reproduction, Department of Animal Science, Faculty of Agriculture, Kagoshima UniÕersity, Kagoshima 890-0065, Japan
Abstract
Preserving genetic resources for next millennium is of great importance, whereas the major contribution of conserving sperms has the potential in many applications such as agriculture,
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biotechnology, species conservation and clinical medicine. Two major systems liquid and frozen of storage technologies have been developed for sperm conservation until the 20th century. The combinations of storage temperature, the cooling rate, chemical composition of extender, cryopro-tectant concentration and the hygienic control are the key factors that affect the life span of spermatozoa. In the past decades, a slow progress have been made in improvement of storage technology, however, the recent advancement in reproduction technology and well understanding of the reproductive physiology have opened the door to a new era in conservation of sperm. This paper focuses on current sperm conservation systems as well as alternative strategies that would be effective for preserving genetic resources in future.q2000 Elsevier Science B.V. All rights
reserved.
Keywords: Sperm; Conservation; Genetic resource banks
1. Introduction
The sperm is a highly polarized and specialized cell with a tripartite structure of head, midpiece and tail. It has lost the ability of biosynthesis, repair, growth and cell division during the final phase of spermatogenesis. Conservation of sperm generally requires a reduction or arrest of the metabolism of sperm cells, thereby prolonging their life.
)Tel.:q81-99-285-8586; fax:q81-99-285-8586.
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E-mail address: myoshida@bio2.agri.kagoshima-u.ac.jp M. Yoshida .
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.
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Since the first attempt to conserve semen Spallanzani, 1776 there have been many Ž
improvements in conservation technology e.g. the discovery of cryoprotectants, the .
development of semen extender, the progress of reproductive techonology with number Ž
of detailed reviews of conservation of mammalian spermatozoa Hammerstedt et al., 1990; Foote, 1998; Foote and Parks, 1993; Maxwell and Salamon, 1993; Watson, 1995;
. Royere et al., 1996; Holt, 1997; Vishwanath and Shannon, 1997; Woelders, 1997 . The importance of preserving genetic resources for next millennium is widely recognized, whereas the conservation of sperms would have major contribution with great potential applications in agriculture, biotechnology, species conservation and clinical medicine: Ž .1 international exchange importŽ rexport of genetic lines, 2 conservation of manipu-. Ž .
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lated sperm gender selection , 3 conservation of genetic lines having superior genetic
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traits or rare breed or transgenic lines establishment of genetic resource banks , 4 reserve supplies in response to disease-disaster, or in these either semen collected from men before chemotherapy or radiotherapy, or spermatozoa collected from
non-physio-Ž . Ž .
logical situations epididymal or testicular spermatozoa , and 5 conservation of threat-ened or endangered species.
This paper summarizes the current information on sperm conservation systems as well as alternative strategies that would be effective for preserving genetic resources in future.
2. Current status of conservation of sperms
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Two major systems liquid and frozen of storage technologies have been achieved for sperm conservation in the 20th century. The combinations of storage temperature, chemical composition of extender and the hygienic control are the key factors that affect the life span of spermatozoa. The relative advantages and disadvantages of both of these
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storage systems have been well outlined by Vishwanath et al. 1996 .
2.1. Liquid-storage of sperm
The principal advantage of using liquid unfrozen semen is that fertility is maintained with low numbers of sperm in the inseminate: with fewer than 1 million bull sperm per breeding unit, conception rates with liquid semen are similar to those with frozen semen
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with around 15 million sperm Vishwanath et al., 1996; Foote and Kaproth, 1997 . In a liquid environment, at high dilutions, fertility of sperm is maintained for 3–5 days when
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stored at ambient temperatures 10–218C , thereafter, it steadily declines at a rate of 3–6% per day. Bull spermatozoa stored at ambient temperature in a citrate-based
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medium, exhibited a slow decrease in motility over an extended period about 3–4 .
weeks , but the concomitant drop in fertility was significantly higher: fertility, as Ž
measured by the number of females not returning to be inseminated non-return rate,
. Ž .
NRR , was maintained for the first 3–5 days after dilution 69.9% , followed by an
Ž . Ž
.
1997 . This decline in fertility occurs irrespective of whether the sperm are stored at 58C or at 158C, but the rate is greater at storage temperatures higher than 258C. Sperm viability can be maintained for longer periods of time in the presence of antioxidants ŽMaxwell and Stojanov, 1996 , chelating agents Nishimura, 1993; White, 1993 , or. Ž .
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aromatic compounds Bamba and Miyagawa, 1992 . Antibiotics are added to the semen to suppress growth of microorganisms or even reduce their number, and thus, reducing
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the possibility of disease transmission Johnston et al., 1998 . Amikacin sulfate, an aminoglycoside antibiotic, can safely be added to extenders to control
streptomycin-Ž
resistant organisms without detrimental effect on sperm motility Sone et al., 1982; .
Ahmad and Foote, 1985; Foote, 1998 . Since boar spermatozoa are quite sensitive to cold shock, they are usually stored at 15–208C. Recently boar semen diluted with
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Modena based extender Mulberry diluent can be used over a wide temperature range Ž5–158C for up to 12 days without remarkable decrease in litter size 10.7 and. Ž .
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farrowing rate 87.5% Sone et al., 1992; Kuwahara et al., 1996 .
The practical implications for improving the ambient-temperature storage of sperm are to reduce the level of endogenous phosphorylation, or to include a membrane-per-meable scavengers in the diluent thus counteracting the reactive oxygen species
pro-Ž .
duced in situ Vishwanath and Shannon, 1997 .
2.2. Frozen-storage of sperm
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Since the major achievement of using glycerol as a cryoprotectant Polge et al.,
. Ž .
1949 , Polge and Rowson 1952 demonstrated a good conception rate with bull sperm that had been buffered with egg yolk–sodium citrate and equilibrated with glycerol diluter before freezing. Although the survival of cryopreserved spermatozoa does not exceed 50 years, it has been estimated that the biological specimens cryopreserved in liquid nitrogen may remain viable for more than 3000 years, owing to the absence of thermally driven reactions and exceeding background radiation aty1968C. Many works have been done employing variety of methods for the processing and freezing of semen. For different species, including humans and several farm animals, the optimal rate of cooling, freezing and thawing has been described, sometimes in combination with the
Ž
cryoprotectant substances and their concentration Maxwell and Salamon, 1993; Watson, .
1995; Royere et al., 1996; Holt, 1997; Woelders, 1997 . Even though cryopreserved semen is used commercially for breeding of cattle, critical examination revealed that the
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proportion of fully functional bull sperm in a frozen–thawed sample is still low 10% . In addition, artificial insemination in pig and sheep has been hampered by low pregnancy rates or low litter size following cervical insemination with frozen–thawed spermatozoa. It has been suggested that this may be due to the inability of frozen–thawed spermatozoa to survive in the female reproductive tract and subsequently causing
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reduction in sperm transport to the site of fertilization Kemp and Soede, 1997 . In general, the fully functional condition of motile sperm surviving cryopreservation would be similar to their pre-freeze uncapacitatied state. However, recent evidence suggests that spermatozoa surviving freezing and thawing have altered membrane structure or property which may render them functionally similar to capacitated andror
acrosome-Ž .
studies to improve the cryopreservation of spermatozoa given that the ultimate goal of semen preservation is to obtain pregnancies after artificial insemination as effectively as after natural service.
3. New trends for conservation of sperms
The recent advancement in reproductive technology and better understanding of the reproductive physiology provided a new area in conservation of sperm.
3.1. ImproÕement for quality control of preserÕed sperm
Efficient preservation of cells with good fertilizing ability is of great importance to the conservation of sperm. More sensitive criteria of the objective assessment of motility, energy status, damage to the plasma membrane or to subcellular elements, chromatin stability and chromosomal damage have been proposed as complementary
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end-points to define sperm preservation system Royere et al., 1996 . The development of a computer-assisted technology and variety of fluorophores would provide new tools and eritically needed information for assessing the functionality of conserved sperms ŽMaxwell and Johnson, 1997; Tardif et al., 1997; Thomas et al., 1998; Abaigar et al.,
.
1999 . A good quality monitoring system should also be established in order to provide
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maximum reliability. In addition, Rodriguez-Martinez et al. 1997 reviewed the in vitro procedures for the separation and selection of the morphologically normal and viable spermatozoa. Sperm clean-up before andror after preservation may improve the effi-ciency of sperm conservation.
3.2. ConserÕation of gender selected sperm
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According to Johnson 1997 , recent advances of technology on the flow cytometry of sperm with the objective of predetermining gender of offspring have led to a validated method to separate X from Y chromosome-bearing sperm for use in vitro
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fertilization Cran and Johnson, 1996 and embryo transfer, intratubal Johnson, 1997 or
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intra-uterine insemination Seidel et al., 1996 or intracytoplasmic sperm injection ŽHamano et al., 1999 . Conservation of sperm after gender selection would increase the. flexibility with which the separated X and Y sperm could be put in better use for fertilization.
3.3. ConserÕation of sperm in freeze-dried and dehydrated conditions
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been fixed in alcohol or Carnoy’s fluid, a fixative based on alcohol and chloroform, could also develop into pronuclei after direct intracytoplasmic microinjection of oocytes.
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Recently, Wakayama and Yanagimachi 1998 confirmed that freeze-dried mouse sperm nuclei can support normal embryonic development if it was microinjected after three months of storage at room temperature. The only component of the spermatozoa that is crucial for participation in embryological development is likely to be sperm nucleus with
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a stable nuclear matrix Ward et al., 1999 . In personal view, these data may raise the intriguing possibility of scraping spermatozoa from stored smears. For example from archival collections, and using them for recovery of specific genotypes, perhaps rare
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breeds of domestic animals but also extinct species Holt, 1997 .
3.4. ConserÕation of testis cells by harÕesting in culture and transplantation
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Brinster and Nagano 1998 showed that testis cells of a fertile male mouse can be transplanted to the seminiferous tubules of an infertile male, where the donor spermato-gonial stem cells will establish spermatogenesis and produce spermatozoa that transmit the donor haplotype to progeny. In addition, mouse spermatogonial stem cells have been cultured for longer than 3 months and, following transplantation, produced
spermatogen-Ž .
esis Nagano et al., 1998 . This procedure has been taken a step further demonstrating that if rat or hamster spermatogonia were transferred into mouse seminiferous tubules,
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xenogeneic spermatogenesis occur in the tubules Clouthier et al. 1996; Ogawa et al., .
1999 . These techniques provide the system that will permit spermatogonial stem cells to be cultivated and their number increase in vitro to allow genetic modification before
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transplantation to a recipient testis Nagano et al., 1998 . In combination with cryop-reservation of spermatogonia or testicular tissue, this may become a more powerful technology for the storage and regeneration of valuable germplasm. With such approach involving mice that clearly have very limited sperm-producing capacity, but looking into the future one could imagine a scenario in which a pig or sheep testis could be repopulated by spermatogonia from another breed, therefore retaining the capability for
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high sperm output Holt, 1997 . The system could also be applied as therapy for infertile humans. Furthermore, spermatogonia from endangered species could be repopulated into testes from their more common relatives as a way of recovering spermatozoa for
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breeding by assisted reproduction techniques ART , such as artificial insemination, in vitro fertilization or direct intracytoplasmic microinjection of oocytes.
3.5. Establishment of the frozen zoo
In the second half of the 20th century, populations of many wild animal species have Ž
been established in captivity for various purposes e.g. education, conservation, research,
. Ž .
farming and many are kept as companion animals Kirkwood, 1996 . The systematic banking of genome resources using cryopreserved sperm in the frozen zoo offers the opportunity to further conservation strategies of endangered species by assisting in the
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conservation tool that has the potential to play a decisive role in reversing the disturbing trend with many of endangered species. The concept of ex situ genetic management of small captive populations of endangered species with a view to re-introducing them into
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the wild is attracting increasing interest Bainbridge and Jabbour, 1998 . In addition, the exchange of germ from germplasm banks to captive and wild populations would
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increase genetic diversity at reduced risk and expense Ballou, 1992 . The application of ART using conserved germplasm also will play an important role in such programmes. However, many of the reproductive and technical problems with threatened species are
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the same as those to be solved for domestic rare breeds Holt, 1997 . Advances in reproductive technology and better understanding of the reproductive physiology of these animal populations are necessary to permit the application of ART.
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