The effects of different cryoprotectants and the temperature of addition on the survival of red deer epididymal spermatozoa

With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa conservation, we evaluated the effects of four most commonly used cryoprotectants (CPAs), Glycerol (G), Ethylene glycol (EG), Propylene glycol (PG), and Dimethyl sulfoxide (DMSO), on the sperm survival. Besides, the effects of two temperatures of CPA addition--22 degrees C (ambient temperature) and 5 degrees C--on sperm quality were also tested. For each temperature tested, sperm samples were evaluated after 0, 15, 30 and 60 min of spermatozoa exposition to CPAs. Thus, sperm quality was in vitro judged by microscopic assessments of individual sperm motility (SMI), and of plasma membrane (Viability) and acrosome (NAR) integrities. Overall, DMSO showed the highest toxicity for red deer epididymal spermatozoa, and glycerol the lowest. Thus, at 60 min of incubation SMI results showed that the toxicity to red deer epididymal spermatozoa of the four CPAs are in the following sequence: G approximately = EG approximately = PG < DMSO ('less than' symbol means P < 0.05, and approximate symbol means P = 0.08). Furthermore, our results also showed a differential response of acrosome membrane to temperature of CPAs addition. Regardless of the CPA used, statistically significant variations (P < 0.05) were found between the two temperatures of addition of CPAs for acrosome integrity, the best being 22 degrees C (NAR = 83.8% vs. 69.8%). These data indicate that sperm quality of red deer epididymal spermatozoa, in addition to be affected by the cryoprotectant, can also be influenced by the temperature at which CPAs are added prior to freezing.     

Characteristics of fresh gander semen and its susceptibility to cryopreservation in six generations derived from geese inseminated with frozen-thawed semen

The paper summarises seven years experiments designed to determine the effect of continuous insemination with frozen-thawed semen on fresh semen quality and sperm susceptibility to freezing stress in succeeding generations. During course of experiments, semen was collected from 10-12 White Koluda ganders at the age of 8-9 months, then subjected to freezing and used after thawing for insemination of 10 geese in order to obtain the subsequent generation of males. Semen was diluted 1 to 0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with 6 percent (v/v) of dimethyl-formamide (DMF), frozen to temp. -140 degrees C at a rate 60 degree C per min and then transferred into liquid nitrogen container. Semen samples were thawed prior to insemination in a 60 degree C water-bath. It is difficult to conclude whether freezing stress affected the fresh semen quality, since average volume of SQF (index comprising ejaculate volume, sperm concentration and percentage of live normal cells) varied between generations from 19.3 to 56.2. Continuous goose reproduction by insemination with frozen-thawed semen resulted in significant increase (P less than 0.01) in spermatozoa resistance to cryoinjury in every subsequent generation. In the relation to adequate fresh semen the percentage of live morphologically intact spermatozoa which withstood freezing procedure increased from 27.2 in first generation to 74.4 in sixth generation.     

A simple osmotic stress test to predict boar sperm cryosurvival

This work was carried out to test whether viability of pig spermatozoa subjected to an osmotic test is correlated to sperm cryosurvival. Spermatozoa were cooled from 22 degrees C to -5 degrees C, aliquots were exposed to a series of hyperosmotic solutions (300-2100 mOsm/kg) for 15 min, immediately spermatozoa were re-warmed to 37 degrees C and isosmolarity was restored. Spermatozoa were cooled from 22 degrees C to -5 degrees C and one aliquot was exposed to the osmotic test while diluted spermatozoa were frozen-thawed. Plasma membrane-intact spermatozoa decreased as osmolarity increased (P < 0.0001), a further decreased (P < 0.0001) was observed when isotonicity was restored. Proportions of plasma membrane-intact and acrosome-intact cells from the osmotic test were no different from those after freeze-thawing: 36% vs. 35%, 80% vs. 80%, respectively. A significant correlation was found between the proportion of acrosome-intact cells after freeze-thawing and that from the osmotic test (r = 0.81, P <0.01). This test provides a useful and economical mean to predict in vitro boar sperm cryosurvival.     

Evaluation of damage in Pacific oyster (Crassostrea gigas) spermatozoa before and after cryopreservation using comet assay

We examined the applicability of the comet assay (single cell gel electrophoresis assay) to estimate the quality of frozen-thawed Pacific oyster (Crassostrea gigas) spermatozoa. Comet assay was performed on semen before and after cryopreservation followed by fluorescent staining with propidium iodide to assess DNA integrity. After cryopreservation, the percentage of spermatozoa with damaged DNA significantly increased, while only about half of the cells displayed intact DNA, even when protected with 10 percent DMSO. All the considered parameters (head length, head area, head intensity, total length, total area, total intensity, tail length percent, tail area percent, and tail intensity percent) were higher than the oyster sperm protected with 10 percent DMSO-artificial sea water after freezing and thawing. Only tail length percent, tail area percent, and tail intensity percent were increased significantly after cryopreservation. The tail length percent was found to be the most sensitive indicator of the cryopreservation-induced DNA damage. Our freeze-thawing procedure significantly affected oyster sperm DNA, as indicated by the reduced fertilization rate when frozen-thawed oyster sperm are used. Irreversible alteration of the genome may prevent fertilization or alter normal embryonic development. This study is the first to demonstrate that the comet assay is an inexpensive, rapid and sensitive method for determining DNA damage in Pacific oyster sperm quality assessments.     

Effect of rapid freezing–thawing techniques on the sperm parameters and ultrastructure of Chinese Taihang black goat spermatozoa

Supercooling sperm in liquid nitrogen vapour is a feasible and economic technique for the practical production. The study aimed to reveal the negative effects of this rapid freezing and thawing processes on Taihang black goat spermatozoa and to find out the changing of spermatozoa motility and ultrastructure by using CASA and TEM. Qualified semen samples, which collected from twenty Chinese Taihang black goats using artificial vagina were pooled and investigated the kinematics parameters and ultrastructural morphology. The results showed that freezing–thawing caused a significant reduction in the spermatozoon total motility (P < 0.001), in rapid and medium cell numbers (P < 0.001) and motility parameters (VAP, VSL, VCL, ALH and BCF) (P < 0.01). Immotile spermatozoa number was increased significantly after freezing–thawing (P < 0.001). In the ultrastructural analysis, the shape with a sperm nucleus characterized by ruptures, bend and deformity was observed. The plasma membranes were broken, and nucleoplasm erupted. The mitochondria in the middle piece were disturbed by partial absence or additional accumulations. Swelling, coiling, vacuolization and structural disorganization of mitochondria were also observed. In conclusion, Freezing–thawing procedure has a detrimental effect on motility, membrane integrity and mitochondria of goat spermatozoa. Transmission electron microscopy provides an intuitive observation to investigate deformity spermatozoa.     

Production of normal offspring from partially zona-incised vitrified mouse oocytes fertilized with cryopreserved spermatozoa using an optimized protocol

The present study was designed to investigate the optimized conditions for cryopreservation of Kunming (KM) mice spermatozoa (Experiment 1) and to compare the developmental potential of IVF embryos produced from fresh oocytes (Group 1), vitrified-warmed oocytes without (Group 2) or with partial zona pellucida incised by a piezo manipulator (ZIP) (Group 3) fertilized with frozen-thawed spermatozoa (Experiment 2). In experiment 1, spermatozoa were cryopreserved with the medium containing raffinose and egg yolk with different concentrations (0 to 60 percent) and then followed by fertilization with fresh oocytes after thawing. The highest cleavage (76.2 percent) and blastocysts formation rates (63.6 percent) were obtained when the egg yolk concentration was adjusted to 30 percent. To optimize the equilibration time, the spermatozoa were equilibrated in the optimized medium for 0, 10, 30, 50, 70, 90 min at 40 degree C before plunging into liquid nitrogen. After thawing, the highest cleavage rate (87.4 percent) of IVF embryos was observed when equilibrated for 30 min. In experiment 2, the cleavage and blastocyst rates in Group 1 (81.2 percent, 65.4 percent) and Group 3 (72.5 percent, 45.0 percent) were higher (P less then 0.05) than those in Group 2 (22.2 percent and 13.9 percent), respectively. When 2-cell embryos obtained in Group 1 and 3 were transferred, 32.1 percent and 22.7 percent of embryos in the pregnant receipts developed to term, respectively. In conclusion, the optimized protocol is highly efficient for the cryopreservation of KM mice spermatozoa; the ZIP technique is very useful for improvement of the fertilization efficiency using the cryopreserved gametes and normal offspring can be produced efficiently.     

Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei: Istiophoridae)

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation, mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).     

Comparative sperm ultrastructure of three species in Siniperca (Teleostei: Perciformes: Sinipercidae)

Siniperca chuatsi, Siniperca kneri, and Siniperca scherzeri are three of the most economically important sinipercid species. The ultrastructure and morphology of the mature spermatozoa of them are examined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The sperm consists of an acrosome-less head, a short midpiece and a long flagellum. Ultrastructurally, it has a homogeneously electron-dense nucleus in a granular pattern with nuclear lucent and a nuclear fossa excluding the centriolar complex. One to four mitochondria with lamellar cristae encircle the basal body of the flagellum in the midpiece. The cytoplasm surrounding the centrioles and the cylindric cytoplasmic channel contains glycogen granules and vesicles. Comprising the conventional 9 + 2 axoneme, vesicles and lateral fins, the sperm flagellum is inserted laterally on the nucleus, therefore the spermatozoon is asymmetrical. All of the spermatozoa of the three species are of the primitive or ect-aquasperm form and conform to the teleostean type II spermatozoa instead of the previously supposed type I. Variations in the shape of the heads, angles between the two centrioles, location of the cytoplasmic vesicles, mitochondrial number and structural characteristics of the lateral fins are notable among the three species. S. chuatsi is a sister-group of the other two species and is the most differentiated. The present study provides fresh insights to the comparative spermatology of Siniperca fishes and will be useful to the existing knowledge of the sinipercid fishes in systematic characters, biodiversity conservation and reproduction.     

Cryopreservation of human sperm using rapid cooling rates

The effect of cryopreservation on human spermatozoa has been investigated during the past few decades. The majority of current cryopreservation protocols are carried-out using low cooling rates. However, theoretical calculations have shown that for human spermatozoa an optimal cooling rate is about 7,000 C/min. In our work we have studied the effect of cryopreservation with high cooling rates, variation of osmolarity of the cryoprotectant medium and the glycerol content. The results of experiments have demonstrated that within the range of high cooling rates, after thawing the dependency of sperm survival on the cooling rate has a maximum recovery at 2,500-3,300 C/min in moderately hyperosmolar medium, containing 4-5% glycerol. When decreasing the cooling rate down to 1,750-2,500 C/min there was a statistically significant reduction in sperm motility. Using the cooling rate of 8,000-11,000 C /min only a small percentage of spermatozoa retained their motility.     

Actin localisation and the effect of cytochalasin D on the osmotic tolerance of cauda epididymidal kangaroo spermatozoa

This study examined the hypothesis that filamentous actin associated with the complex cytoskeleton of the kangaroo sperm head and tail may be contributing to lack of plasma membrane plasticity and a consequent loss of membrane integrity during cryopreservation. In the first study, the distribution of G and F actin within Eastern Grey Kangaroo (EGK, Macropus giganteus) cauda epididymidal spermatozoa was successfully detected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam), respectively. G-actin staining was most intense in the acrosome but was also observed with less intensity over the nucleus and mid-piece. F-actin was located in the sperm nucleus but was not discernable in the acrosome or sperm tail. To investigate whether cytochalasin D (a known F-actin depolymerising agent) was capable of improving the osmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypo-osmotic media (61 and 104 mOsm) containing a range of cytochalasin D concentrations (0-200 microM). Cytochalasin D had no beneficial effect on plasma membrane integrity of sperm incubated in hypo-osmotic media. However, when EGK cauda epididymidal sperm were incubated in isosmotic media, there was a progressive loss of sperm motility with increasing cytochalasin D concentration. The results of this study indicated that the F-actin distribution in cauda epididymidal spermatozoa of the EGK was surprisingly different from that of the Tammar Wallaby (M. eugenii) and that cytochalasin-D does not appear to improve the tolerance of EGK cauda epididymidal sperm to osmotically induced injury.     

Effect of the extender supplement Equex-STM on cryopreserved semen in the Assaf sheep

This study was designed to evaluate the effect of adding the detergent Equex-STM to the extender used to dilute semen for cryopreservation on several indicators of sperm preservation. Two consecutive ejaculates per day were obtained from 5 Assaf sheep on two days out of every week over three alternate months. The freezing protocol involved diluting the semen in Fiser's extender, to which 0.7 percent Equex-STM was added or omitted before cryopreserving the semen in straws by exposure to nitrogen vapor. Equex-STM supplementation gave rise to significantly (p=0.05) improved sperm quality variables after different periods of freezing (0 hours, 1 week and 1 month). The variables examined were: individual motility, viability, acrosome integrity, plasma membrane integrity (HOS test) and morphological anomalies. This improvement was independent of the ram and month of testing. In a second experiment in which we incubated the semen (0 and 6 hours) at 37 degree C after thawing, Equex-STM also showed a beneficial effect on sperm quality.     

Cryopreservation of gametophytes of Laminaria japonica (phaeophyta) with two-step cooling: interactions between variables related to post-thaw survival

Little attention has been given to the effect of interactions between different cryogenic parameters on the viability of cryopreserved algae. Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen by two-step cooling, and interactions were tested for optimizing the cooling protocol and improving freeze-tolerance. UNOVA of a general linear model suggested that interactions between both cooling rate and holding time and between holding temperature and holding time significantly affected the survival of thawed gametophytes. Based on the magnitude of effect, the importance order of factors was found to be: holding temperature, holding time, cooling rate, cooling rate x holding temperature, holding temperature x holding time. UNOVA also suggested significant main effects of pre-culture conditions and sex on survival of thawed gametophytes. Under the optimal procedure, post-thaw survivals obtained were 84 percent for male and 69 percent for female gametophytes. Male gametophytes were observed to be more tolerant to the whole procedure of cryopreservation than females. Following thawing viable gametophytes could grow asexually or give rise to morphologically normal sporophytes.     

Thawing of frozen vegetables observed by a small dedicated MRI for food research

The thawing process for boiled and frozen edible vegetables was traced by a dedicated MRI for food research. The MRI system is small, with a 1.0-T static magnetic field, and can be placed in an ordinary research room with a light air conditioner. Images of green soybeans, broad beans, okra, asparagus and taro were measured by the spin-echo method (echo time=7 ms) with 0.1 or 0.2 s and 1 s repetition times. The images appeared along with the thawing time, and signals uniformly covered the sliced plane of the samples in the thawed condition. Information about the thawing process and tissue structures of the materials was obtained during transit thawing conditions. The thawing kinetics were examined with increased signal intensity, which were divided into two types. The signal increased linearly and saturated for okra and asparagus but exhibited convex curves for soybeans, broad beans and taro. The small MRI was stable, its handling was simple, and the internal structures of food materials could be accurately identified, although the grey-scale of the images was insufficient for determining precise textural fluctuations of tissue organization. We conclude that the devised MRI is useful for examining the quality of frozen foods and for developmental research into frozen foods.     

Acrosome reaction in Octopus tankahkeei induced by calcium ionophore A23187 and a possible role of the acrosomal screw

The octopod sperm is unique especially in two aspects: the screw-shaped acrosome and its inner layered substructure (striation). The present study aims to investigate morphological changes of Octopus tankahkeei spermatozoa during the acrosome reaction (AR) and to pursue functions of the internal substructures revealed by inducing AR with the calcium ionophore A23187. Gradual changes of the spermatozoa were traced using fluorescence and electron microscopy. The AR process included the bulging, vesiculation, and dehiscence of the plasma membrane around the acrosome and the nucleus, as well as the vesiculation of the mitochondrial sheath. Membrane vesiculation outside the nucleus has never been reported in the order Octopoda. The rigid screw and the inner striation of the acrosome remained intact surmounting the nucleus, suggesting that these two structures have potential functions during fertilization. In addition, the detachment of the sperm head and the tail was commonly observed in this study, both in intact and acrosome-reacted sperm. Fluorescence microscopy revealed that the detached mitochondrial sheath usually gave weaker and more dispersive signals than the joint ones. This phenomenon implied that the intense energy release might promote the detachment of the mitochondrial sheath.     

Measuring body length of male sperm whales from their clicks: the relationship between inter-pulse intervals and photogrammetrically measured lengths

Sperm whales (Physeter macrocephalus) emit short, broadband clicks which often include multiple pulses. The time interval between these pulses [inter-pulse interval (IPI)] represents the two-way time for a pulse to travel between the air sacs located at either end of the sperm whale's head. The IPI therefore, is a proxy of head length which, using an allometric relationship, can be used to estimate total body length. Previous studies relating IPI to an independent measure of length have relied on very small sample sizes and manual techniques for measuring IPI. Sound recordings and digital stereo photogrammetric measurements of 21 individuals were made off Kaikoura, New Zealand, and, in addition, archived recordings of whales measured with a previous photogrammetric system were reanalyzed to obtain a total sample size of 33 individuals. IPIs were measured automatically via cepstral analysis implemented via a software plug-in for pamguard, an open-source software package for passive acoustic monitoring. IPI measurements were highly consistent within individuals (mean CV=0.63%). The new regression relationship relating IPI (I) and total length (T) was found to be T=1.258I+5.736 (r(2)=0.77, p<0.001). This new regression provides a better fit than previous studies of large (> 11 m) sperm whales.     

Fine structure of the spermatozoon of Perinereis macropus (Claparède, 1870) (polychaeta,Nereidae)

The mature spermatozoa of Perinereis macropus were investigated by transmission electron microscopy. The spermatozoon is composed with a large anterior part (head), a short middle piece and a long flagellum. The head contains a large acrosomal complex with a convex acrosomal vesicle, a subacrosomal space, a fibrillar crown and an acrosomal rod which penetrates into the nucleus invagination. The later is U shaped (in longitudinal section). The short middle piece contains about nine to eleven mitochondria and a centriole associated with the flagellum. This centriole, slightly eccentric to the sperm axis, anchors to the plasma membrane by nine satellite rays of the pericentriolar complex. The axoneme has a “9 + 2″ arrangement of microtubules. In cross section, the flagellar membrane extends in two lateral protuberances, aligned with the axis formed by the two central microtubules of the axoneme. The spermatozoon of P. macropus conforms to the primitive type with an acrosomal extension. Nevertheless, the acrosome complex ultrastructure shows noticeable modifications from the basic form. This finding agrees with the previously observed reproductive pattern (broadcast spawning – free-swimming larvae), and may be helpful to classify the sperm type of P. macropus.     

Cryopreservation of human skeletal muscle impairs mitochondrial function

Previous studies have investigated if cryopreservation is a viable approach for functional mitochondrial analysis. Different tissues have been studied, and conflicting results have been published. The aim of the present study was to investigate if mitochondria in human skeletal muscle maintain functionality after long term cryopreservation (1 year). Skeletal muscle samples were preserved in dimethyl sulfoxide (DMSO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry. The capacity of oxidative phosphorylation was significantly (P < 0.05) reduced in cryopreserved human skeletal muscle samples. Cryopreservation impaired respiration with substrates linked to Complex I more than for Complex II (P < 0.05). Addition of cytochrome c revealed an increase in respiration indicating cytochrome c loss from the mitochondria. The results from this study demonstrate that normal mitochondrial functionality is not maintained in cryopreserved human skeletal muscle samples.     

High-pressure freezing and freeze-substitution fixation reveal the ultrastructure of immature and mature spermatozoa of the plant-parasitic nematode Trichodorus similis (Nematoda; Triplonchida; Trichodoridae)

The spermatozoa from testis and spermatheca of the plant-parasitic nematode Trichodorus similis Seinhorst, 1963 (Nematoda; Triplonchida; Trichodoridae) were studied with transmission electron microscopy (TEM), being the first study on spermatogenesis of a representative of the order Triplonchida and important to unravel nematode sperm evolution. Comprehensive results could only be obtained using high-pressure freezing (HPF) and freeze-substitution instead of chemical fixation, demonstrating the importance of cryo-fixation for nematode ultrastructural research. The spermatozoa from the testis (immature spermatozoa) are unpolarized cells covered by numerous filopodia. They contain a centrally-located nucleus without a nuclear envelope, surrounded by mitochondria. Specific fibrous bodies (FB) as long parallel bundles of filaments occupy the peripheral cytoplasm. No structures resembling membranous organelles (MO), as found in the sperm of many other nematodes, were observed in immature spermatozoa of T. similis. The spermatozoa from the uterus (mature or activated spermatozoa) are bipolar cells with an anterior pseudopod and posterior main cell body (MCB), which include a nucleus, mitochondria and MO appearing as large vesicles with finger-like invaginations of the outer cell membrane, or as large vesicles connected to the inner cell membrane. The peripheral MO open to the exterior via pores. In the mature sperm, neither FBs nor filopodia were observed. An important feature of T. similis spermatozoa is the late formation of MO; they first appear in mature spermatozoa. This pattern of MO formation is known for several other orders of the nematode class Enoplea: Enoplida, Mermithida, Dioctophymatida, Trichinellida but has never been observed in the class Chromadorea.     

Ferroelectric properties of ultrasonochemically prepared SbSI ethanogel

This article presents for the first time the electrical properties of sonochemically synthesised, high-surface-area SbSI ethanogel made up of large quantity nanowires with lateral dimensions of about 10-50 nm and lengths reaching up to several micrometers. The composition, morphology, dimensions, microstructures, and optical energy gap of the new form of SbSI were characterized. This material is a semiconducting ferroelectric as in the case of bulk SbSI crystals. The maximum of dielectric constant epsilon=1.6x10(4) is observed at Tc=292(1) K. The activation energies in temperature dependences of electric conductivity of SbSI ethanogel are different for ferroelectric and paraelectric phases during heating and cooling of the sample.     

Effect of combined source (F0) and filter (formant) variation on red deer hind responses to male roars

Studying female response to variation in single acoustic components has provided important insights into how sexual selection operates on male acoustic signals. However, since vocal signals are typically composed of independent components, it is important to account for possible interactions between the studied parameter and other relevant acoustic features of vocal signals. Here, two key components of the male red deer roar, the fundamental frequency and the formant frequencies (an acoustic cue to body size), are independently manipulated in order to examine female response to calls characterized by different combinations of these acoustic components. The results revealed that red deer hinds showed greater overall attention and had lower response latencies to playbacks of roars where lower formants simulated larger males. Furthermore, female response to male roars simulating different size callers was unaffected by the fundamental frequency of the male roar when it was varied within the natural range. Finally, the fundamental frequency of the male roar had no significant separate effect on any of the female behavioral response categories. Taken together these findings indicate that directional intersexual selection pressures have contributed to the evolution of the highly mobile and descended larynx of red deer stags and suggest that the fundamental frequency of the male roar does not affect female perception of size-related formant information.