7 Tesla (T) human cardiovascular magnetic resonance imaging using FLASH and SSFP to assess cardiac function: validation against 1.5 T and 3 T.

We report the first comparison of cardiovascular magnetic resonance imaging (CMR) at 1.5 T, 3 T and 7 T field strengths using steady state free precession (SSFP) and fast low angle shot (FLASH) cine sequences. Cardiac volumes and mass measurements were assessed for feasibility, reproducibility and validity at each given field strength using FLASH and SSFP sequences. Ten healthy volunteers underwent retrospectively electrocardiogram (ECG) gated CMR at 1.5 T, 3 T and 7 T using FLASH and SSFP sequences. B1 and B0 shimming and frequency scouts were used to optimise image quality. Cardiac volume and mass measurements were not significantly affected by field strength when using the same imaging sequence (P > 0.05 for all parameters at 1.5 T, 3 T and 7 T). SSFP imaging returned larger end diastolic and end systolic volumes and smaller left ventricular masses than FLASH imaging at 7 T, and at the lower field strengths (P < 0.05 for each parameter). However, univariate general linear model analysis with fixed effects for sequence and field strengths found an interaction between imaging sequence and field strength (P = 0.03), with a smaller difference in volumes and mass measurements between SSFP and FLASH imaging at 7 T than 1.5 T and 3 T. SSFP and FLASH cine imaging at 7 T is technically feasible and provides valid assessment of cardiac volumes and mass compared with CMR imaging at 1.5 T and 3 T field strengths.

Nutritional scientist or biochemist?

When invited by the editors to provide a prefatory article for the Annual Review of Nutrition, I attempted to decide what might be unique about my experiences as a nutritional biochemist. Although a large proportion of contemporary nutritional scientists were trained as biochemists, the impact of the historical research efforts related to nutrition within the Biochemistry Department at the University of Wisconsin 50 to 60 years ago was, I think, unique, and I have tried to summarize that historical focus. My scientific training was rather standard, but I have tried to review the two major, but greatly different, areas of research that I have been involved in over my career: inorganic fluorides as an industrial pollutant and the metabolic role of vitamin K. I have also had the opportunity to become involved with the activities of the societies representing the nutritional sciences (American Society for Nutrition), biochemistry (American Society for Biochemistry and Molecular Biology), Federation of American Societies for Experimental Biology, the Food and Nutrition Board, the Board on Agriculture and Natural Resources, and the U.S. Department of Agriculture National Agricultural Research, Extension, Education, and Economics. These interactions can be productive or frustrating but are always time-consuming.

Effects of testosterone on pedicle formation and its transformation to antler in castrated male, freemartin and normal female red deer (Cervus elaphus).

Pedicles and antlers are male deer secondary sexual characters. As such, development of these structures is under the control of androgen hormones. Pedicle growth is caused by increasing and elevated plasma testosterone (T) levels, whereas first antler transformation from a fully formed pedicle occurs when the T levels are decreasing. Castration prior to pedicle initiation abrogates future pedicle and antler formation. Female deer also have the potential to develop pedicles and antlers, but they do not normally express this phenotype due to lack of sufficient androgen stimulation. Previous studies have shown that female white-tailed deer could be readily induced to grow pedicles as well as antlers by singular administration of exogenous androgens (EA), but in red deer (Cervus elaphus) singular or irregular EA treatment could only stimulate castrated male, normal or ovariectomised females to grow pedicles, but not antlers. The present study was set out to test whether these EA-induced pedicles in red deer failed to give rise to antlers was because they were constitutively incapable of doing so, or because the plasma T profile naturally exhibited in intact stags was not achieved by the androgen treatment used in these previous studies. Eight castrated red deer stag calves, 3 freemartins (females which were born co-twin to males), and 3 normal female red deer were used in the present study and treated with EA, either as biweekly injections for the castrates or as implants for freemartin and females until the late stage of pedicle growth. Blood sampling was carried out biweekly for the analyses of plasma T and IGF1 concentration. The results showed that the natural plasma T profile in the experimental deer was successfully mimicked through regular EA treatment and subsequent withdrawal at late pedicle growth stage. All castrated males, 2 out of 3 freemartin, and 1 out of 3 normal female red deer formed not only pedicles, but also antlers. Based on these results, we conclude that EA-induced pedicles at least in red deer of the genus Cervus, like those in the genus Odocoileus, are constitutively capable of giving rise to antlers, if they are of sufficient height.

Tissue collection methods for antler research.

The rapid growth of deer antlers makes them potentially excellent models for studying tissue regeneration. In order to facilitate this, we have developed and refined antler tissue sampling methods through years of antler research. In the study, antler tissues were divided into three main groups: antler stem tissue, antler blastema and antler growth centre. For sampling stem tissue, entire initial antlerogenic periosteum (around 22 mm in diameter) could be readily peeled off from the underlying bone using a pair of rat-toothed forceps after delineating the boundary. Apical and peripheral periosteum/ perichondrium of pedicle and antler could only be peeled off intact when they were cut into 4 quadrants and 0.5 cm-wide strips respectively. Antler blastema included blastema per se, and potentiated and dormant periostea. Blastema per se was sampled after it was divided into 4 quadrants using a disposable microtome blade. Potentiated and dormant periostea were collected following the same method used for sampling peripheral periosteum of pedicle and antler. The antler growth centre was divided with a scalpel into 5 layers according to distinctive morphological markers. The apical skin layer could be further separated into dermis and epidermis using enzyme digestion for the study of tissue interaction. We believe that the application of modern techniques coupled with the tissue collection methods reported here will greatly facilitate the establishment of these valuable models.

Modification of standard freezing media to limit capacitation and maximise motility of frozen-thawed equine spermatozoa.

OBJECTIVE: To investigate cryopreservation-induced capacitation-like changes in equine spermatozoa frozen in three different media using chlortetracycline (CTC) fluorescence staining analysis. PROCEDURE: Semen collected from three stallions was diluted in one of three centrifugation media and, after centrifugation and removal of supernatant, extended in corresponding freezing media containing additional egg yolk, glycerol, lactose and Equex paste. The semen was frozen in 5 mL straws and the spermatozoa assessed for motility and membrane quality after thawing. RESULTS: Following centrifugation, spermatozoa diluted with modified Kenney's Centrifugation Medium (MKCM) displayed a higher percentage of (normal) F pattern (94.3%) compared with spermatozoa in Kenney's Centrifugation Medium (KCM) (84.9%) and Glucose-EDTA Centrifugation Medium (GECM) (85.2%). Conversely, the percentage of spermatozoa displaying the (capacitated) B pattern was higher in the KCM (14.1%) and GECM (13.8%) than in the MKCM (5.0%). Following freezing-thawing, there were lower percentages of spermatozoa displaying the AR (acrosome reacted) pattern in modified Kenney's Freezing Medium (MKFM) (45.6%) compared with Kenney's Freezing Medium (KFM) (61.4%) and lactose-EDTA Freezing Medium (LEFM) (61.1%). There was a correspondingly higher percentage of spermatozoa displaying the B pattern in MKFM (52.3%) compared with KFM (37.9%) and LEFM (38.6%). There was no significant difference between the freezing media in the percentage of spermatozoa displaying the F pattern. The percentage of progressively motile spermatozoa was also influenced by the type of freezing medium (P < 0.001). Post-thaw percentages of progressively motile spermatozoa, frozen in MKFM, KFM, and LEFM, were 31.4, 25.8 and 23.3%, respectively. CONCLUSION: MKFM was the preferred medium for cryopreservation of equine spermatozoa due to its superior protection against changes in motility and membrane quality compared with the other freezing media studied.

Capacitation-like changes in equine spermatozoa throughout the cryopreservation process.

Chlortetracycline (CTC) fluorescence staining analysis was used to investigate cryopreservation-induced capacitation-like changes in equine spermatozoa. Freshly ejaculated spermatozoa were found to display a high proportion of F pattern cells (uncapacitated; 93.6%) and a lower proportion of B pattern (capacitated; 5.4%) and AR pattern (acrosome-reacted; 1%) cells. Following cryopreservation in modified Kenney's medium, capacitation-like changes were observed. There was a significant increase in the proportion of spermatozoa displaying the B pattern (64.8%; P<0.001) and AR pattern (32.8%; P<0.001), with a corresponding decrease in the proportion of spermatozoa displaying the F staining pattern (2.5%; P<0.001). Further analysis of CTC fluorescence staining patterns showed that there was a major decrease in the proportion of F pattern spermatozoa corresponding to an increase in B pattern spermatozoa following removal of seminal plasma after centrifugation and resuspension in freezing medium. There was a further decline in the proportion of F pattern spermatozoa, corresponding to increases in B and AR pattern spermatozoa, after the freezing and thawing steps. Resuspension of centrifuged spermatozoa in homologous seminal plasma did not induce capacitation-like changes. These data indicate that the process of freezing and thawing stallion semen induces capacitation-like changes in spermatozoa and that most of the change is brought about by removal of seminal plasma, with further changes induced by the actual freezing and thawing step.

Vitamin K supplementation does not affect ovariectomy-induced bone loss in rats.

Vitamin K may be important in bone metabolism. Notably, high-dose menaquinone-4 (menatetrenone, MK4) has been reported to reduce ovariectomy (ovx)-induced bone loss in rats and to decrease osteoporotic fracture in postmenopausal women. However, it is unclear whether these beneficial effects reflect a physiologic effect of vitamin K, or indicate direct pharmacologic activity of MK4. To further evaluate this, 60 6-month-old nulliparous Sprague-Dawley rats were randomized by distal femur bone mineral density (BMD) in a 3:1 ratio to ovx or sham groups. The sham and one ovx group's diet contained 1% calcium and 1300 microg/kg of vitamin K1, phylloquinone. Diets of the other two ovx groups were supplemented with 882 mg phylloquinone or MK4 per kilogram chow. Distal femur bone mineral density (DFBMD) in an 8 mm region of interest was measured at baseline, 1 and 3 months postoperatively, utilizing dual-energy X-ray absorptiometry (DXA). All animals were killed at 3 months, their right femurs excised, ex vivo BMD measured by DXA, and biomechanical testing performed. No effect of phylloquinone or MK4 supplementation on ovx-induced bone loss was observed. Specifically, DFBMD declined 10.5%, 9.2%, and 11.2% at 1 month and 14.4%, 10.6%, and 13.9% at 3 months in the ovx control, high phylloquinone, and high MK4 groups, respectively. In addition, serum osteocalcin was elevated by ovx; this was not altered by phylloquinone or MK4. Finally, femoral biomechanical properties were not affected by phylloquinone or MK4. To conclude, in this study, neither high-dose phylloquinone nor MK4 reduced the ovx-associated increase in bone turnover or decline in DFBMD.

Effects of testosterone either alone or with IGF-I on growth of cells derived from the proliferation zone of regenerating antlers in vitro.

Deer antlers are male secondary sexual characters and are the fastest growing mammalian tissue. As such, both androgens and growth factors play a major role in antler development. The timing of the antler cycle is controlled by the seasonal fluctuations of testosterone, and the actual growth of antlers is mainly stimulated by growth factors including insulin-like growth factor-1 (IGF-I). However, whether or not testosterone at low levels plays a growth-promoting role during antler formation is controversial. In the present study, we took an in vitro approach to investigate whether testosterone either alone or with IGF-I had mitogenic effects on mesenchymal or cartilaginous cells derived from the proliferation zone of regenerating antlers. In addition, a binding assay was carried out to determine whether the specific binding sites for testosterone were preserved after cell disaggregation. The results showed that testosterone either in physiological concentrations or at low levels did not exert direct mitogenic effects on antler cells derived from the proliferation zone in serum-free medium in vitro (P>0.05), even if the specific binding sites for testosterone in these cells were well preserved. Likewise, testosterone in a very wide range of concentrations not only failed to enhance (P>0.05), but at certain levels (0.1-5 nM) impaired the mitogenic effects of IGF-I on these antler cells in vitro (P<0.001). Therefore, these results support neither a conclusion that low level testosterone has growth-promoting effects on antler formation nor the hypothesis that testosterone effects may be achieved through sensitizing these antler cells to the mitogenic effects of IGF-I.

Vitamin K status of premature infants: implications for current recommendations.

OBJECTIVE: Newborn infants are vitamin K deficient. Vitamin K status in full-term infants after intramuscular vitamin K supplementation at birth has been described. Similar information in growing premature infants has not been reported. The objective of this study was to assess vitamin K status in premature infants by measuring plasma vitamin K and plasma protein-induced in vitamin K absence (PIVKA II) from birth until 40 weeks' postconceptional age. METHODS: Premature infants (/=1000 g) via total parenteral nutrition. After hyperalimentation, most received vitamin K-fortified enteral feedings with the remainder receiving unfortified breast milk. Blood was obtained for PIVKA II in cord blood and for PIVKA II and vitamin K at 2 weeks and 6 weeks after birth and at 40 weeks' postconception. RESULTS: Of the 44 infants enrolled, 10 infants in each gestational age group completed the study. The patient characteristics for groups 1, 2, and 3 were as follows: gestational age, 26.3 +/- 1.7, 30.3 +/- 1.3, and 33.9 +/- 1.1 weeks; birth weight, 876 +/- 176, 1365 +/- 186, and 1906 +/- 163 g; and days of hyperalimentation, 28.9 +/- 16, 16.8 +/- 12, and 4.3 +/- 4 days, respectively. At 2 weeks of age, the vitamin K intake and plasma levels were highest in group 1 versus group 3 (intake: 71.2 +/- 39.6 vs 13.4 +/- 16.3 microg/kg/day; plasma levels: 130.7 +/- 125.6 vs 27.2 +/- 24.4 ng/mL). By 40 weeks' postconception, the vitamin K intake and plasma levels were similar in all 3 groups (group 1, 2, and 3: intake, 11.4 +/- 2.5, 15.4 +/- 6.0, and 10.0 +/- 7.0 microg/kg/day; plasma level, 5.4 +/- 3.8, 5.9 +/- 3.9, and 9.3 +/- 8.5 ng/mL). None of the postnatal plasma samples had any detectable PIVKA II. CONCLUSIONS: Premature infants at 2 weeks of age have high plasma vitamin K levels compared with those at 40 weeks' postconceptional age secondary to the parenteral administration of large amounts of vitamin K. By 40 weeks' postconception, these values are similar to those in term formula-fed infants. Confirming "adequate vitamin K status," PIVKA II was undetectable by 2 weeks of life in all of the premature infants. With the potential for unforeseen consequences of high vitamin K levels, consideration should be given to reducing the amount of parenteral vitamin K supplementation in the first few weeks of life in premature infants.vitamin K, PIVKA II, premature, total parenteral nutrition, enteral nutrition.

No direct mitogenic effect of sex hormones on antlerogenic cells detected in vitro.

Deer pedicles, antecedents of antlers, develop from a specialized periosteum (antlerogenic periosteum) which overlies the lateral crest of the deer frontal bone. The initiation of pedicle growth is triggered by androgen hormones. Thus far, it is not known whether pedicle initiation is caused by direct stimulation of androgen hormones on the antlerogenic periosteum or whether some intermediate mechanisms are necessary. The present study took an in vitro approach to investigate whether sex hormones have direct mitogenic effects on primary cultured antlerogenic periosteal cells (antlerogenic cells). Antlerogenic cells were obtained from two 5-month-old red deer calves. The cells were passaged twice and then treated with testosterone, dihydrotestosterone, and estradiol. The proliferation assays showed that no direct mitogenic effects on the second passage antlerogenic cells could be detected with any of the sex hormone treatments (P > 0.05). Testosterone-binding studies showed that at the second passage, specific testosterone-binding sites were present in the antlerogenic cells. Therefore, we conclude that androgens do not have mitogenic effects on antlerogenic cells in vitro. Our results suggest that pedicle formation may not be the result of direct stimulation of androgen hormones on antlerogenic tissue. Instead, androgen hormones may only allow the process to proceed by increasing the sensitivity of antlerogenic cells to mitogens, e.g., some growth factors.

Tissue interactions and antlerogenesis: new findings revealed by a xenograft approach.

Tissue interactions play a pivotal role in organogenesis. Here we describe a xenograft approach to investigate how heterotypic tissue interactions control antler formation in deer. Deciduous antlers grow from the apices of permanent protuberances, called pedicles. Histogenesis of pedicles depends on the antlerogenic periosteum (AP). Pedicles and growing antlers are made up of interior osseocartilage (a mixture of bone and cartilaginous tissue) and exterior skin. In a previous study we hypothesised that pedicle growth may result from mechanical interactions between the interior and exterior components whereas antler generation from a pedicle would involve molecules communicating between the interior and exterior components. To test this hypothesis, we subcutaneously transplanted AP of red deer (Cervus elaphus), either alone or with future pedicle skin, onto nude mice. The results showed that under the nude mouse skin, subcutaneously xenografted AP alone not only could form pedicle-shaped protuberances but also could differentiate into well-organised pedicle-like structures. The overlying mouse skin accommodated the expansion of the grafted AP by initial mechanical stretching and subsequent formation of new skin. Nude mouse skin was not capable of participating in antler tissue formation. However, grafted deer skin together with AP may have successfully rescued this failure after wounding, which highlights the necessity of the specificity of the overlying skin for antler tissue generation. Therefore, we conclude that it is the interaction between the antlerogenic tissue and the overlying skin that results in antlerogenesis: reciprocal mechanical interactions cause pedicle formation, whereas reciprocal instructive interactions induce first antler generation.

Deer antlerogenic periosteum: a piece of postnatally retained embryonic tissue?

This article reviews the research findings on the piece of periosteum overlying the lateral crest of prepubertal deer frontal bone, known as antlerogenic periosteum (AP). AP was initially discovered by Hartwig and Schrudde in 1974 when searching for the tissue that gives rise to antlers. In their experiment, when AP was transplanted elsewhere on the deer body it formed ectopic antlers. This clearly shows that AP possesses full self-differentiating ability, an attribute that can only be paralleled by embryonic tissue in mammals, like lateral plate mesoderm (LPM). Studies along this line by Goss in the 1980s further demonstrated that AP also holds the patterning information for antler formation. In the 1990s, our group carried out a series of studies on this unique tissue. The results showed that some of the critical features of AP resemble those of embryonic tissues, such as the astonishing growth potential in vivo and in vitro, and rich glycogen content. Histological observations and cell lineage tracing using a genetic marker convincingly demonstrate that pedicles and antlers are the derivatives of AP. Based on these findings, we advanced a hypothesis that AP is a piece of postnatally retained embryonic tissue. Morphological and histological examinations on the presumptive antler growth regions in deer prenatal life showed that the growth of primordial pedicles is initiated in the early pregnant stage (about 55 days) but then ceases (about 100 days) and is subsequently repressed at the late stage of pregnancy. The epidermis overlying the primordial pedicles resembles the apical ectoderm ridge (multicellular layer). These results strongly support our hypothesis. The results from the specific comparison between deer antler formation (from AP in postnatal) and mammalian limb development (from LPM in prenatal) showed that the ontogeny of antlers and limbs are comparable, and that deer antler has the same level of regulative properties as mammalian limbs. We believe that revealing the mechanism underlying the retention of embryonic tissue properties by AP until deer postnatal life will have important implications in biomedical research. Antler formation from AP offers an ideal model to work with in investigating how a self-differentiating system functions.

Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker.

A selectable marker system for plant transformation that does not require the use of antibiotics or herbicides was developed. The selectable marker consists of the manA gene from Escherichia coli under the control of a plant promoter that encodes for phosphomannose isomerase, pmi. Only transgenic plants were able to metabolize the selection agent, mannose, into a usable source of carbon, fructose. Transgenic plants were produced efficiently after delivery by Biolistics™ of the pmi gene into maize and wheat tissues, with mean transformation frequencies of 45% for maize and 20% for wheat. Adjustment of the sucrose and mannose levels in the selection medium essentially eliminated escapes. Transgenic events can be identified as early as 2 months for wheat and 4 months for maize. A simple test, a modified chlorophenol red assay, was used for early identification of transgenic events expressing the pmi gene. Transformation frequencies for both crops exceeded those obtained with the bar and pat genes with selection on either Basta® or bialaphos.

Vitamin K supplementation reduces serum concentrations of under-gamma-carboxylated osteocalcin in healthy young and elderly adults.

BACKGROUND: Subclinical vitamin K insufficiency, manifested by under-gamma-carboxylation of the bone matrix protein osteocalcin, may be common. OBJECTIVE: Our objective was to delineate the prevalence of submaximal gamma-carboxylation as assessed by response to phylloquinone supplementation and to evaluate the effect of this intervention on skeletal turnover in healthy North American adults. DESIGN: Healthy subjects (n = 219), approximately equally distributed by sex and age (18-30 y and >/=65 y), received daily phylloquinone (1000 microg) or placebo for 2 wk. Serum undercarboxylated osteocalcin (ucOC) and total osteocalcin, N:-telopeptides of type I collagen (NTx), bone-specific alkaline phosphatase (BSAP), and phylloquinone concentrations were measured at baseline and after weeks 1 and 2. RESULTS: At baseline, the mean serum phylloquinone concentration was lower in the young than in the old group; there was no effect of sex. Concomitantly, baseline %ucOC was highest in the young and lowest in the old men (P: < 0.0001) but did not differ significantly by age in women. After supplementation, serum phylloquinone concentration increased approximately 10-fold (P: < 0.0001) at week 1 (from 0.93 +/- 0.08 to 8.86 +/- 0.70 nmol/L, x+/- SEM); this was sustained through week 2. Among all supplemented groups, mean %ucOC decreased from 7.6% to 3. 4% without significant differences by age or sex; 102 of 112 subjects had a >1% decrease. Phylloquinone supplementation reduced serum osteocalcin but did not alter NTx or BSAP concentration. CONCLUSIONS: Usual dietary practices in this population did not provide adequate vitamin K for maximal osteocalcin carboxylation. Phylloquinone supplementation reduced serum osteocalcin concentration but did not alter other markers of serum bone turnover.

Toxicological evaluation of New Zealand deer velvet powder. Part I: acute and subchronic oral toxicity studies in rats.

Potential toxic effects of acute and subchronic dosage regimens of deer velvet powder have been assessed in rats following OECD guidelines. In the acute study, rats of both sexes were exposed to a single dose of 2 g/kg body weight. There was no mortality or other signs of toxicity during 14 days' observation. Furthermore, no significant alteration either in relative organ weights or their histology was discernible at terminal autopsy. In the 90-day subchronic study, deer velvet was administered in 1 g/kg daily doses by gavage to rats. A control group of rats received water only. There was no effect on body weight, food consumption, clinical signs, haematology and most parameters of blood chemistry including carbohydrate metabolism, liver and kidney function. No significant differences were seen between the mean organ weights of the adrenal, kidney and brain in rats treated with deer velvet and control rats. However, there was a significant difference (P<0.05) in the group mean relative liver weight (3.52 +/- 0.30 vs 3.81 +/- 0.26 g/100 g body weight) of deer velvet-treated and control male rats. The gross necropsy and pathological examination of rats treated with deer velvet did not reveal any abnormalities in tissue morphology. Based on these results, it may be concluded that rats had no deer velvet treatment-related toxicological and histopathological abnormalities at the doses administered, despite the observed minor changes in liver weight.

Histological studies of pedicle skin formation and its transformation to antler velvet in red deer (Cervus elaphus).

Deer antlers and their antecedent pedicles are made up of two components, interior osseocartilage and exterior integument. In a previous study, we described that histogenesis of the interior osseocartilage proceeds through four ossification stages. These are intramembranous (IMO), transition (OPC), pedicle endochondral (pECO), and antler endochondral (aECO). In the present study, we used histological techniques to examine pedicle skin formation and its transformation to antler velvet. The results showed that pedicle skin initiated from the apex of a frontal lateral crest and was formed through three distinctive stages. These stages are 1) compression of the subcutaneous loose connective tissue at the OPC stage, 2) stretching of the undulated epidermis at the early pECO stage, and 3) neogenesis of the skin and its associated appendages at the mid pECO stage. Transformation into antler velvet, which occurs at the late pECO stage, is mainly associated with alteration in the skin appendages. This alteration includes the loss of arrector pili muscle and sweat glands, and the gain of the large bi- or multi-lobed sebaceous glands. These results suggest that pedicle skin expansion occurs to release the mechanical tension created by underlying forming antlerogenic tissue, initially in response to it by mechanical stretch, and then by neogenesis of skin. In turn, the stretched pedicle skin may exert mechanical pressure on the underlying antlerogenic tissue causing it to change in ossification type. Antler velvet generation may be accomplished by both mechanical stimulation and chemical induction from the underlying pECO stage antlerogenic tissue. If this hypothesis is correct it is likely that mechanical stimulation would drive skin formation and chemical induction then determine skin type. Furthermore, asynchronous transformation of the interior and exterior components during pedicle formation and antler generation may result from the delayed chemical induction and the way antler velvet initially generates. The results from both mitotic cell labelling of the basal layer and ultrastructure of the basement membrane of the apical skin in the study support these hypotheses.