Long-term Semen Storage Research Articles

Fowl (Gallus domesticus) sperm motility depends upon mitochondrial calcium cycling driven by extracellular sodium.

A relationship between extracellular Ca(+2), fowl sperm phospholipase A2 activity, long-chain acylcarnitine content, and motility was demonstrated in previous work. Sperm motility appeared to depend upon Na+-dependent Ca(+2) cycling when sperm were incubated at body temperature without glucose. In the present work, motility decreased as a function of time when sperm were incubated in 2 mM Ca(+2) prepared with either buffered isotonic sucrose or LiCl. However, this effect was less pronounced in the case of LiCl. The sparing effect of Li+ was attributed to the mitochondrial Na+/Ca(+2) exchanger. Motile concentration decreased exponentially in response to micromolar concentrations of CGP 37157, a specific inhibitor of the mitochondrial Na+/Ca(+2) exchanger. KB-R7943 mesylate, an inhibitor of the reverse mode of the Na+/Ca(+2) exchanger, prevented re-initiation of motility when exogenous Ca(+2) was added to sperm rendered immotile by incubation with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a high-affinity Ca(+2) chelator. The presence of voltage-gated Ca(+2) channels was confirmed by the effect of nifedipine on motile concentration. Neither motile concentration nor straight line velocity was affected by either ouabain or orthovanadate, which inhibit Na+-K+ ATPase and Ca(+2)-ATPase, respectively. In summary, we infer that 1) fowl sperm motility is dependent upon extracellular Ca(+2) cycling through mitochondria; 2) such cycling is dependent upon extracellular Na+; and 3) fowl sperm conserve ATP by moving neither Na+ nor Ca(+2) by active transport. Understanding the relationship between mitochondrial Ca(+2) cycling and ATP production may be applicable to long-term semen storage.

Parameters affecting sperm survival after thawing in long-term frozen storage.

Objective: Long-term sperm storage in semen banks for future use is a common practice in those patients undergoing contraception or cancer treatment. Nowadays it is impossible to predict the parameters that potentially could influence the rates of sperm survival and motility. This is an important topic to counsel and manage those patients about the time that the samples can be maintained in the banks and the number of them that should be frozen to ensure good post-thawing expectatives. Moreover, other factors that can influence on, and that must be improved in the laboratory, are containers and freezing methods. To this end, our aim in this study was to determine the influence of the parameters involved in long-term semen storage on sperm post-thawing quality.

Semen collection, characterization, and cryopreservation in a Magellanic penguin ( Spheniscus magellanicus )

A cooperative method was developed for collecting semen from a Magellanic penguin. Ejaculate parameters and semen production during a breeding season were characterized. Experiments were performed to study the effect on penguin spermatozoa of two temperatures (4°C and 21°C) for short-term storage, and two cryoprotectants (dimethylsulfoxide [DMSO] and ethylene glycol [EG]) for long-term storage (cryopreservation). All dilutions were made using modified Beltsville Poultry Semen Extender. Sperm quality was assessed by evaluating motility and forward progression (sperm motility index [SMI]), viability, and morphology. A total of 39 ejaculates was collected over the 40-day study period. Thirty-eight ejaculates contained spermatozoa, but semen quality decreased toward the end of the study period. Varying levels of urate contamination were present in all ejaculates. Sperm quality parameters were similar for diluted samples held at 4°C and 21°C, and samples maintained high numbers of viable (77.8 ± 5.4%) and morphologically normal (67.9 ± 2.5%) spermatozoa at 3 hr. SMI and percentage of viable sperm decreased (P 0.05). Both SMI and viability of frozen-thawed spermatozoa were higher (P < 0.05) for clean than for contaminated ejaculates. This is the first report on penguin ejaculate parameters, semen production, and preliminary methods for short- and long-term semen storage. Zoo Biol 18:199–214, 1999. © 1999 Wiley-Liss, Inc.

Physiological characteristics of epididymal spermatozoa from postmortem rams

A specific problem in goat semen preservation is the detrimental effect of seminal plasma on sperm viability in extenders containing yolk or milk. Thus, the use of chemically defined extenders will have obvious advantages. Although previous studies indicate that the initial pH of an extender is crucial to sustain high sperm motility, changes in extender pH during long-term semen storage have not been observed. Monitoring extender pH at different times of semen storage and modeling its variation according to nonlinear models is thus important for protocol optimization for long-term liquid semen preservation. The present results showed that during long-term liquid storage of goat semen, both sperm motility and semen pH decreased gradually, and a strong correlation was observed between the two. Whereas increasing the initial extender pH from 6.04 to 6.25 or storage with stabilized pH improved, storage with artificially lowered pH impaired sperm motility. Extender renewal improved sperm motility by maintaining a stable pH. Sperm coating with chicken (Gallus gallus) egg yolk improved motility by increasing tolerance to pH decline. A new extender (n-mZAP) with a higher buffering capacity was formulated, and n-mZAP maintained higher sperm motility, membrane integrity and acrosome intactness than the currently used mZAP extender did. Goat semen liquid-stored for 12 d in n-mZAP produced pregnancy and kidding rates similar to those obtained with freshly collected semen following artificial insemination. In conclusion, maintenance of a stable pH during liquid semen storage dramatically improved sperm viability and fertilizing potential.

Lipid peroxides in spermatozoa; formation, rôle of plasmalogen, and physiological significance.

In view of the high content of unsaturated fatty acids in the phospholipids of mammalian spermatozoa, the possibility was investigated that during aerobic incubation of spermatozoa these fatty acids, in particular arachidonic (20:4) and docosahexanoic (22:6) acid, undergo peroxidation, which in turn is responsible for the decline in viability of spermatozoa. The formation of lipid peroxides was followed by the thiobarbituric acid (TBA) reaction, iodometry and fluorimetry; it was found that during aerobic incubation of washed ram spermatozoa at 37 °C in the presence of ascorbic acid (0.1 mg/ml) and ferrous iron (2.8 μg Fe/ml), peroxides became detectable after 30 min incubation and their level reached a maximum after 60 min. Coincident with the formation of peroxides motility declined and the spermatozoa became agglutinated, but their oxygen uptake was not grossly different from that of motile spermatozoa in mixtures free of ascorbic acid or Fe. Peroxide formation could be prevented by the addition of either chelating agents, such as EDTA and sodium citrate, or antioxidants, such as tocopherol and butylated hydroxyanisole, to the incubation medium. Conclusive evidence that the substrate for the peroxidation reaction is a lipid, was obtained by extracting lipids from spermatozoa with chloroform: methanol and incubating aerobically in the presence of catalytic amounts of ascorbate and Fe, two fractions: the extracted lipid and the ‘defatted’ sperm residue; very high peroxide values were recorded in the former, but not the latter fraction. Examination by thin-layer and gas-liquid chromatography of the phospholipid and neutral lipid fractions obtained from spermatozoa, revealed that peroxidation caused a loss of approximately 50% in the content of plasmalogens, 20:4 and 22:6 fatty acids, and palmitaldehyde. Changes in the other phospholipids and their bound fatty acids were relatively small, but there was some increase in the amount of a substance which behaved chromatographically as lysolecithin. The content of cholesterol and glycerides in the neutral lipid fraction was not appreciably different in non-peroxidized and peroxidized spermatozoa, but the latter contained a higher proportion of free fatty acids. Our results indicate that under aerobic conditions the lipids in spermatozoa, in particular the plasmalogens, are susceptible to peroxidation. The peroxidation reaction apparently takes place within the lipoprotein, and is associated with a decline in the viability of spermatozoa. This decline could be due either to an increase in cellular permeability resulting from changes in membrane-bound lipids, or to a direct toxic effect of lipid peroxides on spermatozoa. Development of means for the prevention of adverse effects of the peroxidation reaction, may be of practical importance in relation to the problem of long-term storage of semen for artificial insemination.