Reproduction and larval rearing of amphibians

Reproduction technologies for amphibians are increasingly used for the in vitro treatment of ovulation, spermiation, oocytes, eggs, sperm, and larvae. Recent advances in these reproduction technologies have been driven by (1) difficulties with achieving reliable reproduction of threatened species in captive breeding programs, (2) the need for the efficient reproduction of laboratory model species, and (3) the cost of maintaining increasing numbers of amphibian gene lines for both research and conservation. Many amphibians are particularly well suited to the use of reproduction technologies due to external fertilization and development. However, due to limitations in our knowledge of reproductive mechanisms, it is still necessary to reproduce many species in captivity by the simulation of natural reproductive cues. Recent advances in reproduction technologies for amphibians include improved hormonal induction of oocytes and sperm, storage of sperm and oocytes, artificial fertilization, and high-density rearing of larvae to metamorphosis. The storage of sperm in particular can both increase the security and reduce the cost of maintaining genetic diversity. It is possible to cryopreserve sperm for millennia, or store it unfrozen for weeks in refrigerators. The storage of sperm can enable multiple parentages of individual females' clutches of eggs and reduce the need to transport animals. Cryopreserved sperm can maintain the gene pool indefinitely, reduce the optimum number of males in captive breeding programs, and usher in new generations of Xenopus spp. germ lines for research. Improved in vitro fertilization using genetic diversity from stored sperm means that investigators need the oocytes from only a few females to produce genetically diverse progeny. In both research and captive breeding programs, it is necessary to provide suitable conditions for the rearing of large numbers of a diverse range of species. Compared with traditional systems, the raising of larvae at high densities has the potential to produce these large numbers of larvae in smaller spaces and to reduce costs.     

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca(2+) increase occurs as a Ca(2+) wave at each sperm entry site in the polyspermic egg. Some Ca(2+) waves are preceded by a transient spike-like Ca(2+) increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca(2+) wave was induced by a sperm factor derived from sperm cytoplasm after sperm-egg membrane fusion. The Ca(2+) increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca(2+) store for the Ca(2+) wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Effect of sperm cryopreservation on sperm DNA stability and progeny development in rainbow trout

This study was carried out to test how sperm cryopreservation affected nuclear DNA stability and whether progeny development was modified when eggs were fertilized with cryopreserved spermatozoa. The "comet assay" (alkaline single-cell gel electrophoresis assay) was adapted to trout spermatozoa to estimate DNA stability as measured by alkali-induced DNA strand break formation. Because trout eggs develop in water after fertilization (oviparous species) and that eggshell is easy to clear up after fixative treatment, progeny development was assessed from the blastodisc flattening stage of the embryos to the first feeding stage of the hatched fries by direct observation. All parameters under study were analyzed on each sperm and comparisons between parameters were made using paired data. Freeze-thawing of sperm slightly but significantly increased the percentage of nuclei showing altered DNA after comet assay. This increase was correlated to the decrease in fertilization rates of sperm, but the absolute percentage of altered nuclei was not predictive of the absolute fertilization ability of sperm. Assessment of progeny development showed that survival rate and abnormality rate obtained after fertilization with cryopreserved sperm were not different from those obtained with fresh sperm. It is concluded that trout sperm cryopreservation only slightly affected sperm DNA stability and that the use of cryopreserved spermatozoa did not impair offspring survival and quality.     

Function of multiple sperm storage organs in female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae)

Sperm storage organs allow females to temporally separate insemination from fertilization, manipulate ejaculates and control fertilization. In the reproductive tract of female fruit flies (Diptera: Tephritidae), sperm are found in two different organs--a pair or triplet of spermathecae, and a "fertilization chamber". In order to understand the specific function of each of these organs, we tested the following hypotheses: (1) Sperm are distributed equally amongst the various sperm storage organs; (2) Both organ types maintain sperm viability; and (3) Sperm used in fertilization come from the fertilization chamber. We counted sperm in spermathecae and fertilization chamber of Mediterranean fruit flies (Ceratitis capitata) every 3 days for 18 days following insemination, and used a live/dead staining technique to determine the viability of sperm in these organs. Finally, by extirpating spermathecae from inseminated females and allowing them to oviposit, we were able to identify the fertilization chamber as the source of fertilizing sperm. Numbers of sperm in the spermathecae declined from an average of 3575 on the day of copulation to 649, 18 days later. Conversely, the fertilization chamber maintained a fairly constant level of sperms, ranging between an average of 207 cells on day 3 to 115 sperms on day 18. Throughout the period we monitored, we found high levels of sperm viability in both organs (> 80%). Sperm viability was similarly high in the fertilization chambers of females without spermathecae. However, fertility of eggs laid by these females declined rapidly, as did the number of sperm in the fertilization chamber. We conclude that both the spermathecae and the fertilization chamber are active sperm storage organs, with separate functions: the spermathecae for long-term storage and the fertilization chamber, periodically filled by the spermathecae, a staging point for fertilizing sperm. We suggest that the use of both organs by females results in sperm economy, which adaptively prolongs the intervals between copulations.     

Insemination factors related to timed AI in cattle

Six-day-old bovine ova/embryos were recovered non-surgically and used as biomonitors to evaluate time of artificial insemination. These embryos/ova provided information regarding fertilization status and embryo quality, as well as quantitative and qualitative data regarding associated accessory sperm. Both sperm access to the ovum (addressed by accessory sperm) and fertilization status/embryo quality were important in addressing pregnancy rate for specific intervals from the onset of estrus to insemination. Based on these biomonitors, early insemination failed to achieve optimum pregnancy rate due to inadequate access of sperm to the ovum (i.e., low fertilization rate, manifested by low accessory sperm numbers). However, embryo quality was high in early inseminations, which favors pregnancy. Late insemination failed to achieve optimum pregnancy rate (due to reduced embryo quality), however, sperm access to the ovum was highest. Thus, the selection of an insemination time to achieve optimum pregnancy rate appeared to be a compromise between the two extreme intervals. For timed-AI programs, consideration of the time of ovulation (and its variability) becomes important, in addition to conventional considerations, such as semen handling, site of insemination, and bull selection.     

Current trends in evaluation of sperm function: in vitro selection and manipulation of male gametes for assisted conception

With increasing medical utilization of assisted reproductive technology (ART), scientists and clinicians have been able to study extensively multiple cell functions operating synchronously and flawlessly during the events preceding, before and after fertilization. Critical evaluation of the functional status of spermatozoa for in vitro techniques such as sperm-mucus interaction, acrosome reaction status, sperm-zona pellucida binding and penetration tests, hyaluronic acid binding assay, and computer assisted semen analysis etc. can direct a male partner of an infertile couple to more aggressive forms of treatments. In vitro selection of functionally competent sperm cells is a pre-requisite for successful outcome in in vitro fertilization or in intracytoplasmic sperm injection (ICSI). Direct injections of acrosome-intact spermatozoa into oocyte during ICSI bypassing the normal events of sperm oocyte interaction and fusion events have raised concerns with regard to fertilization abnormalities and genetic issues. The present communication briefly reviews the sperm function tests with emphasis on its correlation with fertility outcome, and the currently employed sperm selection and manipulation procedures which may have implications in assisted conception programs.     

Advances in the cryopreservation of salmonid semen and suitability for a production-scale artificial fertilization program

During the past decade, semen cryopreservation techniques in salmonid fish have progressed to where fertilization rates obtained with preserved sperm approach those produced with fresh sperm. Recent advances in the collection, dilution and storage of salmonid spermatozoa are reviewed. The problems encountered during the development and examination of artificial insemination programs in warm-blooded animals are contrasted with those encountered in fish. This paper discusses the advantages of artificial fertilization, and the manner in which advances in cryopreservation techniques could aid in the development and expansion of concentrated, production-scale artificial fertilization programs in salmonid fish.     

Function of multiple sperm-storage organs in female damselflies (Ischnura senegalensis): difference in amount of ejaculate stored, sperm loss, and priority in fertilization

We studied changes in the number of sperm within two kinds of female sperm-storage organ in the damselfly Ischnura senegalensis (Odonata: Coenagrionidae): the bursa copulatrix and the spermatheca. We counted the number of sperm within each storage organ and tested their viability after a single copulation in female damselflies kept for seven days with and without oviposition. We also counted sperm and tested their viability in females that underwent an interrupted second copulation after the sperm-removal stage, and after subsequent oviposition. Our results showed that the bursa copulatrix and spermatheca have different sperm storage roles. Immediately after copulation, most eggs appear to have been fertilized with bursal sperm, which were positioned near the fertilization point. By seven days after copulation, a greater proportion of spermathecal sperm were used for fertilization, as the number of bursal sperm had decreased. We hypothesize that female damselflies use the spermatheca for long-term storage and the bursa copulatrix for short-term storage: bursal sperm are more likely to be used for fertilization but may have a higher risk of mortality due to sperm removal by a competing male and/or sperm expelling by the female, whereas spermathecal sperm are safer but will be used for fertilization only after their release from the spermatheca.     

The Ca increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Egg-jelly structure promotes efficiency of internal fertilization in the newt, Cynops pyrrhogaster

Egg-jelly is composed of a network of fibrous components and contains substances regulating the sperm-egg interaction. Many studies on the latter have been conducted, whereas the role of the egg-jelly structure in fertilization has not yet been fully assessed. In this study, we examined the fertilization efficiency in the presence and absence of the structure around the egg of the newt, Cynops pyrrhogaster, using a gelatin gel system. Although de-jellied eggs of C. pyrrhogaster can be fertilized with an adequate number of sperm, the fertilization rate was dramatically increased through the use of the gelatin gel. Sperm showed forward motility with straight morphology in the gel, whereas they swam in circles in solution. This result indicates that the gel structure is significant for sperm guidance to the egg surface, and its presence raises the fertilization efficiency in C. pyrrhogaster. When sperm were entangled in the gel structure, they were immediately folded and never showed any forward motility. Sperm with zigzag morphology were observed in the gelatin gel as well as in the egg-jelly, indicating the elimination of sperm by the gel structure. The effect of sperm elimination on successful fertilization was estimated using gelatin gels of different thickness. Though the variation did not affect the fertilization rate, the rate of normal development gradually increased in the thicker gels. This result indicates that sperm elimination in egg-jelly can function in the fertilization system. The roles of sperm guidance and sperm elimination under the physiological condition of internal fertilization of the newt are discussed.     

Evidence that the voltage-dependent component in the fertilization process is contributed by the sperm

To investigate the mechanisms that account for the voltage dependence of fertilization and provide an electrical block to polyspermy, we studied cross-fertilizations between three species of amphibians having different degrees of voltage dependence. Anurans, such as the toad Bufo japonicus, as well as the primitive urodele Hynobius nebulosus, have voltage-dependent fertilization; other urodeles, such as Cynops pyrrhogaster, have voltage-independent fertilization (Y. Iwao, 1989, Dev. Biol. 134, 438-445). Entry of Hynobius sperm into Cynops eggs was blocked by clamping the egg's membrane potential at +40 mV, as is the case for fertilization of Hynobius eggs with Hynobius sperm, but not for fertilization of Cynops eggs with Cynops sperm. Therefore, fertilization was voltage dependent in an experimental condition where only the sperm could be contributing this characteristic. The voltage-dependent properties of fertilization between Bufo eggs and Hynobius sperm were also characteristic of the sperm species; fertilization was blocked at +50 mV as in Hynobius fertilization, but not at +20 mV as in Bufo fertilization. These results support the conclusion that the voltage dependence of fertilization results from a component contributed by the sperm.     

Effect of steroids and oviductal cells, from the different parts of the oviduct, on the incidence of monospermy in porcine in vitro fertilization

A high incidence of polyspermy occurs in porcine in vitro fertilization. It is also known that in vivo, the oviductal cells and their secretions play an important role in fertilization and early development. Vesicles from oviductal cells from different parts of the oviduct (isthmus or ampulla) pretreated with estradiol or progesterone or ethanol were used to assess their role in the fertilization process. Oviductal cells were co-cultured with 0.5 million motile sperm/ml for 30 min. A 10-microl sample (spermatozoa bound with the cells) was added to 40-microl droplets of fertilization medium containing 5 oocytes. After 15 to 18 h, oocytes were examined for penetration and monospermy. The results show a lower penetration rate with oviductal cells than that of the control. The use of oviductal cells from the isthmus treated with estradiol significantly decreased the percentage of polyspermy compared with that of ampulla treated with the estradiol or with the control. When the isthmus cells were treated with progesterone, an increase in the incidence of polyspermy was observed. Therefore, it is possible to use oviductal cells to increase the incidence of monospermy in porcine in vitro fertilization; moreover, estradiol increases the proportion of monospermy when added to isthmus-derived oviductal cells.     

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.     

Copulation duration and fertilization success in a damselfly: an example of cryptic female choice?

Copulation duration is highly variable (0.5-3 h) in the damselfly, Ceriagrion tenellum (Coenagrionidae). Using laboratory experiments, we tested four adaptive hypotheses to explain this variation: the effect of time constraints, in-copula mate guarding, sperm displacement and cryptic female choice. Copulation duration was negatively correlated with time of day, as predicted by the first two hypotheses, and positively correlated with male density, as predicted by the mate-guarding hypothesis. Males prolonged copulation in response to the volume of sperm stored by females, suggesting they were able to detect and quantify the amount of sperm stored. This behaviour is not explained by mate guarding or time constraint effects. Males removed all the sperm from the bursa copulatrix in just 10 min. Our results also suggest that, because the duct is too narrow to allow male genitalia to enter, males do not remove spermathecal sperm. Therefore, direct sperm removal could not explain long copulations. Prolonged copulations could also have evolved as a result of cryptic female choice if they increase male fertilization success by female-mediated processes. Our results support this idea: male fertilization success was greater after long copulations. Apparently, male copulatory behaviour elicits female responses that increase male fertilization success. Copyright 2000 The Association for the Study of Animal Behaviour.     

In vitro fertilization in inbred BALB/c mice I: isotonic osmolarity and increased calcium-enhanced sperm penetration through the zona pellucida and male pronuclear formation

To optimize IVF conditions for BALB/c mice, which are known to have poor in vitro fertilizability, the requirements for sperm-ova interaction were studied by use of modified simplex optimization medium (mKSOM) as a basic medium. Modified human tubal fluid (mHTF) was used for sperm preincubation and acted as a positive control. When the two media were compared, neither capacitation nor fertilization was supported in mKSOM. Increasing the calcium concentration in mKSOM to 5 mM or more during sperm: ova coincubation improved zona penetration but not male pronuclear (MPN) formation to the same level as those cells incubated in mHTF. When medium osmolarity was varied from 230-305 mOsmol by NaCl at 5 mM CaCl2, MPN formation improved at 280 mOsmol or higher osmolarity to the same level as that found when using mHTF. When NaCl equivalent to 25-75 mOsmol was substituted with trehalose, no significant reduction in fertilization was observed. Substitution of NaCl equivalent to 75 mOsmol with other osmotic reagents (sucrose, choline chloride and sorbitol) resulted in similar levels of fertilization as found with mHTF, except for sorbitol, which reduced fertilization significantly caused by its detrimental effect on sperm viability. At isotonic osmolarity (305 mOsmol), maximum fertilization was observed at 5 mM CaCl2; lower or higher concentrations of CaCl2 resulted in reduced fertilization. Calcium and osmolarity, therefore, are important for sperm : ova interaction in BALB/c mice and the increases in calcium to 5 mM and osmolarity to 305 mOsmol are optimal for BALB/c sperm to penetrate through the zona and to form MPN.     

Sperm use economy of honeybee (Apis mellifera) queens

The queens of eusocial ants, bees, and wasps only mate during a very brief period early in life to acquire and store a lifetime supply of sperm. As sperm cannot be replenished, queens have to be highly economic when using stored sperm to fertilize eggs, especially in species with large and long‐lived colonies. However, queen fertility has not been studied in detail, so that we have little understanding of how economic sperm use is in different species, and whether queens are able to influence their sperm use. This is surprising given that sperm use is a key factor of eusocial life, as it determines the fecundity and longevity of queens and therefore colony fitness. We quantified the number of sperm that honeybee (Apis mellifera) queens use to fertilize eggs. We examined sperm use in naturally mated queens of different ages and in queens artificially inseminated with different volumes of semen. We found that queens are remarkably efficient and only use a median of 2 sperm per egg fertilization, with decreasing sperm use in older queens. The number of sperm in storage was always a significant predictor for the number of sperm used per fertilization, indicating that queens use a constant ratio of spermathecal fluid relative to total spermathecal volume of 2.364 × 10^?6 to fertilize eggs. This allowed us to calculate a lifetime fecundity for honeybee queens of around 1,500,000 fertilized eggs. Our data provide the first empirical evidence that honeybee queens do not manipulate sperm use, and fertilization failures in worker‐destined eggs are therefore honest signals that workers can use to time queen replacement, which is crucial for colony performance and fitness.     

Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs

In the fertilization of sea urchin eggs, intracellular [Ca2+] (Cai) increases transiently and intracellular pH (pHi) elevates accordingly. Unlinking these two activating factors experimentally, the requirement of the increase in pHi for sperm aster formation in the sea urchin, Clypeaster japonicus, was investigated. When the eggs were injected with an EGTA or BAPTA solution, they incorporated sperm but did not organize the sperm aster. Using these sperm-incorporated eggs under the condition that an increase in Cai was blocked, pHi was regulated by two methods: (i) perfusing ammonium acetate-containing seawater; and (ii) injecting pH buffer solutions of various pH values. By either of the two methods, the sperm aster formed at pHi 7.0 or more and functioned in female pronuclear migration when the sperm aster reached the female pronucleus. Hence, the step of the transient increase in Cai at fertilization can be bypassed. In contrast, a pHi increase is indispensably required for sperm aster formation in sea urchin eggs. Moreover, under the condition that there was the transient increase in Cai, the threshold pHi value for sperm aster formation was pHi 7.0 or more. Consequently, whether a Cai increase on fertilization occurs or not, the threshold pHi value for sperm aster formation is constant in sea urchin eggs.     

Production of F1 interspecies hybrid offspring with cryopreserved sperm from a live-bearing fish, the swordtail Xiphophorus helleri

Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.     

Liposome-mediated DNA uptake by sperm cells.

To investigate the potential use of sperm cells as vectors to transfer exogenous DNA via the fertilization of oocytes into the germ line of mice, we have used liposomes to transfect DNA into the sperm head. Although the DNA transfer into sperm mediated by liposomes was very efficient and no obvious reduction in the fertilization frequency of oocytes could be detected, we were unable to generate transgenic mice by this method.     

Apoptosis and the processes of DNA fragmentation in native and cryopreserved human sperm cells with normo- and pathosperma

Cryopreserved human sperm is widely applied in the treatment of infertility using methods of assisted reproductive technologies. Comprehensive studies of the sperm condition after cryopreservation and subsequent cultivation will update the efficiency of the programs based on assisted reproductive technologies. The aim of this study was to investigate the effect of cryopreservation factors on the state of DNA and apoptotic processes in isolated human spermatozoa fractions in normo- and pathospermia. The analysis of DNA fragmentation and the assessment of apoptotic processes (human spermatozoa necrosis) were performed using the sperm chromatin dispersion test and the flow cytometry method. According to our study, sperm cryopreservation has not affected the DNA fragmentation index in normozoospermia and significantly increased it in oligoastenoteratozoospermia. The postcultivation increase of apoptotic and necrotic processes in oligoastenoteratozoospermia gives reason for choosing an alternative technique for oocyte fertilization— intracytoplasmic sperm injection.