Benzyladenine and low temperature promote phase transition from juvenile to vegetative adult in bulblets of Lilium?×?formolongi ‘White Aga’ cultured in vitro

Scales excised from lily bulblets were cultured on MS medium supplemented with 0.044 or 4.4 μM BA in the dark for 180 days. The culture period was divided into stage 1 (day 0–30), stage 2 (day 31–90) and stage 3 (day 91–180). The scales were cultured at 25°C in stage 1, 25°C or 8°C in stage 2, and 25°C in stage 3. When the scales were cultured on medium with 4.4 μM BA at 25°C for 180 days, bulblets with and without an elongated stem were produced. The percentage of bulblets with elongated stems greatly increased when the scales had been cultured at 8°C in stage 2. On medium with 0.044 μM BA, only bulblets without elongated stems were produced. The diameter of shoot primodia significantly enlarged in bulblets produced on medium with 4.4 μM BA at 8°C in stage 2 and no such enlargement occurred under the other conditions. Nearly square parenchyma cells were observed in the non-elongated shoot primodia in the former bulblets but not in the latter. These cells changed into longitudinally rectangular ones in the internode of elongated stems. Procambium was arranged almost parallel to the shoot axis in the stem of bulblets in the medium with 4.4 μM BA, but not in the medium with 0.044 μM BA.     

Cryopreservation of transgenic rice suspension cells producing recombinant hCTLA4Ig

Transgenic suspension cells of Oryza sativa L. cv. Dongjin utilized as a host for producing recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) were preserved in liquid nitrogen (−196 °C) after slow prefreezing in a deep freezer (−70 °C). The development of an optimal procedure for long-term storage was investigated by the addition of various concentrations of cryoprotectant mixture and osmoticum in preculture media before cooling. A pre-deep-freezing time of 120 min was the most effective for maintaining cell viability. Compared with mannitol, sorbitol, trehalose, and NaCl under the same osmotic conditions, 0.5 M sucrose was found to be the best osmoticum for preculture media. The cryoprotectant comprising sucrose, glycerol, and dimethylsulfoxide (DMSO) was applied to the precultured cells, and a combination of 1 M sucrose, 1 M glycerol, and 1 M DMSO provided the best result. The viability with this optimized condition was 88% after cryocell-banking for 1 day. The expression of hCTLA4Ig in recovered callus from cryopreservation was also kept stable, and the production level was similar to that observed in noncryopreserved cultures.     

Southern elephant seals: IgM concentration in milk of cows and serum of their pups

Serum and milk Immunoglobulin M (IgM) concentrations in 11 mother-pup pairs were measured in southern elephant seals (Mirounga leonina) throughout lactation during 2 breeding seasons at King George Island. Samples were obtained sequentially throughout the suckling period (approximately 23 days). The IgM concentration was measured by single radial immunodiffusion on agarose plates. Milk IgM concentrations showed significant differences throughout lactation, with the highest concentrations on the 1st day (x=989.7 mg/dL skimmed milk; SD=433.2) followed by a sharp fall during the next 3–6 days of the suckling period. The ratio of milk IgM/serum IgM concentrations from mothers ranged from 0.21 to 21.92, with highest values in the 1st day of lactation (x=8.25, SD=5.4) and a decrease in concentration as lactation progressed. This was due to the fact that, throughout lactation, milk IgM concentrations fell while serum IgM values showed an increasing trend. Pups showed the lowest serum IgM values in the 1st day of the suckling period (x=13.0 mg/dL, SD=4.3) with an increasing trend as lactation progressed. Despite the high IgM concentrations of milk at day 1 of lactation, by 1 week of age pups had serum IgM concentrations only slightly greater than at birth. This suggests that much of this Ig was newly formed and little or no milk IgM was absorbed during the 1st week. Possibly, the function of ingested IgM is to provide local immunity in the pup's gut, during the first few days of postnatal life. Accepted: 26 March 2000     

Inoculum dynamics ofGliocladium virens associated with roots of cotton seedlings

A system was developed to evaluate the effects of root growth of cotton seedlings on the inoculum dynamics ofGliocladium virens in nonsterile soil. In soil infested withG. virens, inoculum densities of the fungus increased when plants remained alive. After 30 days, shoots were excised and the roots allowed to deteriorate. During this portion of the experiment (30–60 days) soil inoculum densities ofG. virens declined. In infested soil without a seedling, inoculum densities remained constant throughout the duration of the experiments. Colonization of roots byG. virens was found to increase throughout the duration of the experiments. At 60 daysG. virens was recovered from approximately 60% of the root pieces (1-cm) sampled. The percentage of primary, secondary, or tertiary roots colonized was different (P = 0.01), but the total colonization of roots at three depths (0–10, 10–20, and 20–30 cm) was not different (P = 0.64). In noninfested soil, colonization of roots by indigenous propagules ofG. virens was never greater than 3%. Offprint requests to: C. M. Kenerley.     

Adventitious rooting is enhanced by methyl jasmonate in tobacco thin cell layers

Adventitious roots (ARs) are induced by auxins. Jasmonic acid (JA) and methyl jasmonate (MeJA) are also plant growth regulators with many effects on development, but their role on ARs needs investigation. To this aim, we analyzed AR formation in tobacco thin cell layers (TCLs) cultured with 0.01–10 μM MeJA, either under root-inductive conditions, i.e., on medium containing 10 μM indole-3-butyric acid (IBA) and 0.1 μM kinetin, or without hormones. The explants were excised from the cultivars Samsun, Xanthii and Petite Havana, and from genotypes with altered AR-forming ability in response to auxin, namely the non-rooting rac mutant and the over-rooting Agrobacterium rhizogenes rolB transgenic line. Results show that NtRNR1 (G1/S) and Ntcyc29 (G2/M) gene activity, cell proliferation and meristemoid formation were stimulated in hormone-cultured TCLs by submicromolar MeJA concentrations. The meristemoids developed either into ARs and xylogenic nodules, or into xylogenic nodules only (rac TCLs). MeJA-induced meristemoid over-production characterized rolB TCLs. No rooting or xylogenesis occurred under hormone-free conditions, independently of MeJA and genotype. Endogenous JA progressively (days 1–4) increased in hormone-cultured TCLs in the absence of MeJA. JA levels were enhanced by 0.1 μM MeJA, on both days 1 and 4. Endogenous IBA was the only auxin detected, both in the free form and as IBA-glucose. Free IBA increased up to day 2, remaining constant thereafter (day 4). Its level was enhanced by 0.1 μM MeJA only on day 1, while IBA conjugation was not affected by MeJA. Taken together, these results show that an interplay between jasmonates and auxins regulates AR formation and xylogenesis in tobacco TCLs.     

Activity of salicylic acid on the growth and biochemism of Chlorella vulgaris Beijerinck

This study was conducted to investigate the influence of salicylic acid (SA) on the growth and changes of nucleic acids, protein, photosynthetic pigments, sugar content and photosynthesis levels in the green alga Chlorella vulgaris Beijerinck (Chlorophyceae). The most significant changes in the content of nucleic acids and proteins was observed at the concentration 10⁻⁴ M SA between 8 and 12 day of cultivation. This concentration of SA increased the number of cells (about 40 %) and content of proteins (about 60 %) and its secretion to the medium. The slight stimulation of protein secretion occurred on the 12th day of cultivation at concentration 10⁻⁴ M, while in the range of 10⁻⁵ M to 10⁻⁶ M the protein secretion was inhibited. SA also stimulated the content of nucleic acids, especially RNA by 20–60 %, compared with the control. The most stimulating influence upon the contents of chlorophylls a and b (50–70 %), total carotenoids (25–57 %), sugar (27–41 %) and intensity of net photosynthesis (18–33 %) was found at 10⁻⁴ M of SA. At the concentration of 10⁻⁶ M SA the slight inhibition of growth and biochemical activity of the algae was recorded at the first days of cultivation.     

Identification and characterization of fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw

A pilot plant for hydrothermal treatment of wheat straw was compared in reactor systems of two steps (first, 80°C; second, 190–205°C) and of three steps (first, 80°C; second, 170–180°C; third, 195°C). Fermentation (SSF) with Sacharomyces cerevisiae of the pretreated fibers and hydrolysate from the two-step system gave higher ethanol yield (64–75%) than that obtained from the three-step system (61–65%), due to higher enzymatic cellulose convertibility. At the optimal conditions (two steps, 195°C for 6 min), 69% of available C6-sugar could be fermented into ethanol with a high hemicellulose recovery (65%). The concentration of furfural obtained during the pretreatment process increased versus temperature from 50 mg/l at 190°C to 1,200 mg/l at 205°C as a result of xylose degradation. S. cerevisiae detoxified the hydrolysates by degradation of several toxic compounds such as 90–99% furfural and 80–100% phenolic aldehydes, which extended the lag phase to 5 h. Acetic acid concentration increased by 0.2–1 g/l during enzymatic hydrolysis and 0–3.4 g/l during fermentation due to hydrolysis of acetyl groups and minor xylose degradation. Formic acid concentration increased by 0.5–1.5 g/l probably due to degradation of furfural. Phenolic aldehydes were oxidized to the corresponding acids during fermentation reducing the inhibition level.     

On the ecology and behavior ofCebus albifrons in eastern Colombia: I. Ecology

A-503 contact-hr study of a 35-member group ofCebus albifrons was conducted in eastern Colombia in 1977 and 1978. The group had a female: male socionomic sex ratio of 2.5:1 and used a home range of 110–120 ha which overlapped the home range of another group ofC. albifrons about 20–30 ha. The animals spent about 80% of their foraging time eating plant material and about 20% of their foraging time eating animal materials. A birth peak at the end of the dry season extending into the wet season was indicated by data available. Attempted predation was recorded by the mustelidEira barbara and the black-and-white hawk-eagleSpizastur melanoleucus. Some association was observed with the red howler monkeyAlouatta seniculus. The group at times spent more than half the day foraging and traveling on the ground, exhibiting a level of terrestriality not reported for other New World primates.     

Activity,movements, and sociality of newborn Mongolian gazelle calves in the Eastern Steppe

We hand-captured, fitted with motion-sensitive VHF transmitters, and monitored 19 newborn (1–2 days old) Mongolian gazelle Procapra gutturosa Pallas, 1777 calves in Dornod, Mongolia during the 2000 and 2003 calving seasons to identify changes in activity, movements, and sociality with age. Overall, activity was highly variable throughout the day, regardless of age or year. Calf activity increased, however, from day 2 (age = 2 days; 18% total activity) to day 7 (29%), and 3-fold by day 25 (54%). By days 5–8, calves had moved an average of 6.6 km (range: 2–21 km) from capture sites but were still only seen alone or with their mother. By day 24–26, however, they were located an average of 41 km (range: 24–63 km) away from their capture site, and most (7 of 9) were in aggregations of >1000 animals. Mongolian gazelles do not display behaviour associated with “hiders” after about 2–3 weeks. This reflects the wide-ranging, nomadic existence of the species.     

Serum chorionic gonadotropin,progesterone, and estradiol-17β levels during pregnancy in the common marmoset,Callithrix jacchus

The serum levels of chorionic gonadotropin (LH/CG), progesterone, and estradiol-17β were measured during pregnancy in the common marmoset. The gestation period in five females was 144±1.5 (141–145) days. The LH/CG level increased from the early stage of pregnancy, reached a maximum of 10–17 ng/ml at 50 to 70 days and decreased to under 40 pg/ml at about 100 days. The progesterone level maintained the same value as that at the luteal phase of 20–40 ng/ml until 90 days of pregnancy, when the serum LH/CG was declining, thereafter increased abruptly, reached a maximum of 140–210 ng/ml at 110–130 days and fell to a low level of under 0.4 ng/ml at 5–10 days before parturition. The estradiol-17β was less than 2 ng/ml until 90 days of pregnancy, thereafter increased abruptly and maintained a high level of 40–135 ng/ml until just before parturition. The 3β-hydroxysteroid dehydrogenase activity in the placenta of the common marmoset was 40 times higher than that in the fetal adrenal, while in the Japanese monkey the former was only about one 40th of the latter. The time course of the serum progesterone and estradiol-17β during pregnancy and the role of the placenta which synthesized and secreted these hormones in the common marmoset showed a similarity to those of humans and anthropoid apes rather than those ofMacaca species. The common marmoset represents a valuable animal model for investigating the feto-placental unit in humans.     

Taurine and Zinc Modulate Outgrowth from Goldfish Retinal Explants

Taurine and zinc, highly concentrated in the retina, possess similar properties in this structure, such as neuro-protection, membrane stabilization, influencing regeneration, and modulating development, maybe by acting in parallel or as interacting agents. We previously demonstrated that there are some correlations between taurine and zinc levels in hippocampus, dentate gyrus and retina of the developing rat. In the present study we evaluate the possible effects of taurine and zinc on outgrowth from goldfish retinal explants. The optic nerve was crushed 10 days before plating and culturing retinal explants in Leibovitz medium with 10% fetal calf serum and gentamicin. Neurites were measured with SigmaScanPro after 5 days in culture. Taurine (HPLC) and zinc (ICP) concentrations were determined in the retina between 1 and 180 days after crushing the optic nerve. Zinc sulfate (0.01–100 μM), N,N, N′,N′-tetrakis (pyridylmethyl) ethylenediamine (TPEN, 0.1–5 nM) and diethylenetriamine penta-acetic acid (DTPA, 10–300 μM), intracellular and extracellular zinc chelators, respectively, were added to the medium. TPEN was also injected intraocular (0.1 nM). Combinations of them were added with taurine (1–16 mM). Taurine concentrations were elevated in the retina 72 h after the crush, but were normalized by 180 days, those of zinc increased at 24 h, preceding the increase of taurine. The axonal transport of [³H]taurine from the optic tectum to the retina was not affected in fish with or without crush of the optic nerve at early periods after the injection, indicating an increase of it post-lesion. Zinc sulfate produced a bell-shaped concentration dependency on in vitro outgrowth, with stimulation at 0.05 μM, and inhibition at higher levels, also increased the effect of 4 mM taurine at 0.02 μM, but diminished it at higher concentrations in the medium. TPEN decreased outgrowth at 1 nM, but not at 0.5 nM, although the simultaneous presence of 4 mM taurine and 0.5 nM TPEN decreased outgrowth respecting the stimulation by taurine alone. The intraocular administration of TPEN decreased outgrowth in vitro, an effect counteracted by the addition of 4 mM taurine to the culture medium. DTPA decreased outgrowth from 10 μM in the medium. The present results indicate that an optimal zinc concentration is necessary for outgrowth of goldfish retinal explants and that, in zinc deficient retina, taurine could stimulate outgrowth. In addition, the observations of variations in tissue concentrations and of the effects of intraocular administration of TPEN indicate that these effects could occur in vivo. Special issue dedicated to Dr. Simo S. Oja     

Impact of Enterococcus faecium on specific activity of disaccharidases in small intestine of gnotobiotic pigs

The aim of the present work was to study the changes in the activity of disaccharidase enzymes (lactase. maltase, saccharase) in the small intestine of gnotobiotic pigs aged 0–35 days and inoculated with Enterococcus faecium. The continual decrease of lactase activity was observed from the 14^th day of age up to the end of the experiment. The most significant decrease of specific lactase activity in the duodenum (2.1 μmol/mg protein/hour) was noted from the 21^st to the 28^th day of age. On the other hand, the specific saccharase activity increased moderately during the post weaning period and maltase activity maintained a constant level. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

An Estimation of the Manganese Requirement for Broilers from 1 to 21 Days of Age

A dose–response experiment was conducted to find the sensitive and consistent biomarker for the estimation of dietary manganese (Mn) requirement and establish the optimal Mn level for broilers fed a practical corn–soybean meal diet from 1 to 21 days of age post-hatching. A total of 480 1-day-old Arbor Acres male chicks were randomly allotted to one of eight treatments with five replicates of 12 birds each and fed diets supplemented with 0, 20, 40, 60, 80, 100, 120, or 140 mg Mn/kg from reagent grade Mn sulfate. Tissue Mn concentrations, manganese-containing superoxide dismutase (MnSOD) activity, and MnSOD mRNA concentration within heart tissue were analyzed at 7, 14, and 21 days of age. Tissue Mn concentrations and heart MnSOD activity showed significant quadratic responses, and heart MnSOD mRNA concentration showed an asymptotic response to dietary supplemental Mn level, respectively. The estimate of dietary Mn for chicks from 1 to 21 days of age was 122–128 for heart Mn concentration, 141–159 for pancreas Mn concentration, 127–138 for liver Mn concentration, and 135–156 mg/kg for heart MnSOD activity, respectively. Heart MnSOD mRNA concentration was a consistent index for the estimation of the Mn requirement of broilers. Based on this index, the estimate of dietary Mn requirement for broilers from 1 to 21 days of age post-hatching was about 130 mg/kg, which was a little more than two times of the current NRC (1994) requirement.     

Body mass dynamic in Eurasian lynxLynx lynx kittens during lactation

Body mass changes of Eurasian lynxLynx lynx Linnaeus, 1758 kittens during the first four months of their life were studied in 1989–1999 in captivity. Four hundred thirteen records of body mass from 63 lynx kittens were analysed. The body mass of lynx kittens up to four months of age increased as a linear function with age. Daily growth rate (in grams) was minimal when the kittens were switching from milk to solid food at the age 41–60 days and was maximal at the age 61–80 days. Body mass dynamic and daily growth rates of kittens depended on the husbandry conditions of adult animals (or probably on their subspecies) and litter size, but did not depend on the sex of the kittens. Specific spontaneous fights during kittens’ ontogenesis showed the trend to affect kittens’ body mass dynamic and daily growth rate on some stages of kittens’ development.     

Relative Bioavailability of Manganese Proteinate for Broilers Fed a Conventional Corn–Soybean Meal Diet

An experiment was conducted to investigate the bioavailability of organic manganese proteinate (Mn) relative to inorganic Mn sulfate for broilers fed a conventional corn–soybean meal basal diet. A total of 448-day-old Arbor Acres commercial male chicks were fed the Mn-unsupplemented basal diet (control) or basal diet supplemented with 60, 120, or 180 mg Mn/kg from each Mn source. At 21 days of age, heart tissue was excised for testing DM, Mn concentration, manganese superoxide dismutase (MnSOD) activity, and MnSOD mRNA level. The Mn concentration, MnSOD activity, and MnSOD mRNA level in heart tissue increased (P < 0.01) linearly as dietary manganese concentration increased. Based on slope ratios from multiple linear regressions of the above three indices on added Mn level, there was no significant difference (P > 0.21) in bioavailability between Mn proteinate and Mn sulfate for broilers in this experiment.     

Obligately and facultatively autotrophic,sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts

A variety of autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria were isolated from thermogenic composts at temperatures of 60–80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non-spore-forming strains were gram-negative rods growing at 60–80°C, with an optimum at 70–75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7–37.6 mol%) and showed a high DNA:DNA homology (70–87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55–75° C, with an optimum at 65–70° C. These bacteria were able to grow heterotrophically, or autotrophically with hydrogen; however, they oxidized thiosulfate under mixotrophic growth conditions (e.g. pyruvate or hydrogen plus thiosulfate). These strains had similar DNA G+C content (60–64 mol%) to and high DNA:DNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermogenic composts as habitats of thermophilic sulfur- and hydrogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This contrasts with the generally held belief that thermogenic composts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Received: 20 July 1995 / Accepted: 25 September 1995     

Verification of daily growth increment formation in saury otoliths by rearing larvae from hatching

Naturally spawned eggs of the Pacific saury,Cololabis saira, were collected in the field and reared in a tank to examine daily periodicity of growth increment formation in the otolith. Larvae were 6.9 mm in knob length at hatching. Their otoliths (sagittae) were 31 μm in radius and had 3–6 faint concentric rings. They started feeding within two days and grew at a rate of 1.1 mm/day on average through larval and juvenile stages feeding on rotifers,Artemia nauplii, and artificial diets. Otolith growth increments showed a concentric pattern with a distance of 3.5–5.0 μm between two adjacent increments. The number of growth increments was almost equal to a known age in days plus 4 or 5. A regression line of number of increments (N) on known age in days (D) between 0–30 days after hatching was N = 4.81 + 1.01D, which shows that one increment was deposited per day.     

Methods of short-term storage of cultures of the blowfly <Emphasis Type="Italic">Calliphora Vicina</Emphasis> R.-D. (Diptera,Calliphoridae)

Three methods of short-term storage of the blowfly Calliphora vicina strains are considered based on the experimental study of 21 strains originating from different parts of the species range. The colony can be preserved as diapausing adults at 6° and darkness for 2–3 months or more, depending on the geographical origin of the population. During the first five days of adult life the flies should be kept at 12° and short day on a sugar diet, after which they should be transferred into a refrigerator. During artificial hibernation the flies also require periodic sugar feeding every 20 days (3–4 h at 20°C) to maintain their vital functions. The combination of temperatures of 20–23°C and a protein diet terminates reproductive diapause, and oviposition starts in 10–17 days. The fly strain may be preserved as reproductive females at 6°C and darkness with sugar feeding. Flies also require periodic sugar feeding at 20°C (3–4 hours). In this case the flies start laying eggs 2–3 days after being transferred to 20–23°C. The preservation of diapausing larvae is a more reliable method of prolonged strain storage. In this case the flies of maternal generation are maintained at 20–23°C on sugar and protein diet. The egg rafts laid during 5–6 hours are then transferred into 12°C and short day until hatchment. The hatched larvae should be immediately placed into a refrigerator (2–3 or 5–6°C), where they feed during 1–1.5 months and enter diapause. For strain restoration, the diapausing larvae should be transferred into 20–25°C, where they pupariate in 3–5 days and the flies emerge in nearly 10 days.     

Some essential elements in maternal and cord blood in relation to birth weight and gestational age of the baby

Maternal and cord blood were collected from 54 Indian women at parturition and analyzed for Zn, Cu, and Fe by flame atomic absorption spectrophotometry to determine the relationship between levels of these elements in mother’s and infant’s blood and maternal age, birth weight, and gestational age of the baby. The blood Zn level of mothers in the age group 24–28 yr was significantly higher than those of mothers in the age group of 18–23 yr (p<0.05). Similarly, mothers in the 24 to 28-yr group also had higher blood Fe level than mothers in the group 29–38 yr (p<0.05). The levels of Zn, Cu, and Fe were higher in the maternal blood and lower, but not significantly, in the cord blood of low-birth-weight babies than in those of normal-birth-weight babies. However, differences in the levels of Zn, Cu, and Fe between maternal and cord blood of the two birth-weight groups was statistically significant. There were no significant differences in the levels of the three elements in maternal or cord blood by the gestational age of the baby. A weak but significant correlation was found between the birth weight of the baby and the Fe level in the cord blood (r=0.26; p<0.05). Also, weak significant correlations were observed between gestational age of the baby and Fe (r=0.23; p<0.05) and Cu (r=0.31; p<0.05) levels in the cord blood. Although, there are many confounders of low birth weight and preterm deliveries, a diminished placental transfer of these essential elements could be one of the several etiological factors for low birth weight of newborns.     

Postnatal development of morphological features and vocalization in the pomona leaf-nosed bat <Emphasis Type="Italic">Hipposideros pomona</Emphasis>

Based on mark-recapture data, we studied the postnatal development of morphological features and vocalization of the pomona leaf-nosed bat (Hipposideros pomona). Morphological changes indicated that body mass and length of forearm followed a linear pattern of growth until 13 days of age at mean growth rates of 0.14 g/day and 1.08 mm/day, respectively, and thereafter, growth rates slowed. The length of the total epiphyseal gap of the fourth metacarpal–phalangeal joint showed a linear increase for up to 10 days, followed by a linear decrease until day 40 at a mean rate of 0.09 mm/day. Together, two equations permitted estimation of the age of H. pomona pups between 1 and 40 days. The logistic equation provided the best fit to the empirical curves for body mass and length of forearm. Studies of vocal development showed that the precursors of echolocation calls were not emitted until day 7 after birth. As the pups grew, the dominant frequency (DF) of isolation calls increased and number of harmonics (NH) decreased, whereas the duration remained relatively stable. The DF and BFM₂ (the bandwidth of the terminal frequency-modulated sweep from the second harmonic) of the early echolocation calls increased; however, the NH and duration decreased.