[Show abstract][Hide abstract] ABSTRACT: Growth in temperate and arctic deer is seasonal, with higher growth rates in spring and summer while growth rates are low or negative in autumn and winter. We have measured IGF1 concentrations in the plasma of reindeer calves exposed to a manipulated photoperiod, indoors, of either 16 hours light followed by 8 hours dark each day (16L:8D) (n = 3) or 8L:16D (n = 3) from about the autumnal to the vernal equinox, to determine whether the seasonal growth spurt normally seen in spring is associated with changes in the circulating level of IGF1. A high quality concentrate diet was available ad libitum. The animals were weighed, and bled every 2 weeks and plasma samples assayed for IGF1 by radioimmunoassay. 6-8 weeks after the start of the study those calves exposed to 16L.-8D showed a significant increase in plasma IGF1 concentration which was maintained until the close of the experiment, 24 weeks after the start. In contrast IGF1 plasma concentrations in those calves exposed to a daylength of 8L:16D did not significantly alter during the study. The elevated IFG1 in the 16L:8D group was associated with rapid weight gain compared with the 8L:16D group. We have shown that the seasonal growth spurt is preceded by an elevation in plasma IFG1 concentration. Further, this elevation in IGF1 is daylength dependent. For comparison IGF1 and growth rate seasonal profiles from temperate and tropical deer are included. This comparison reveals that seasonal increases in IGF1 take place only in animals with a seasonal growth spurt. Thus IGF1 plasma level elevations seem most closely associated with the resumption of rapid growth in spring following the winter.
[Show abstract][Hide abstract] ABSTRACT: Heterotypic tissue interactions play an indispensable role in organ generation and regeneration. In contrast to the classic examples of tissue interactions prevailing in the formation of tetrapod limbs or pectoral fins that can only take place when the interactive tissues are in intimate contacts, the interactions in deer antler formation are novel in that the inducer and the responder are separated by a distance of 1-2 mm. This feature offers a unique opportunity to explore the mechanism underlying tissue interactions by permitting membrane insertion between the two interactive tissues. Four experiments were conducted in this study. The results showed that the impermeable membranes inhibited antler formation. In contrast, the permeable membrane (0.45 microm in pore size) substantially slowed pedicle growth and antler initiation but did not stop them. Interestingly, the impermeable membrane/sheath only slightly retarded antler elongation. Overall, our results demonstrate that interactions between the two interactive tissues, antlerogenic tissue and the overlying skin, are indispensable for first antler initiation and are achieved through diffusible molecules rather than direct physical contact. As the heterotypic tissue interactions are only required during antler initiation but not elongation, they must be transient in nature, and thus differ from those operating in limb/fin formation that can only be sustained by continuous interactions. A system in which organ development is achieved only through transient tissue interactions must be novel, if not completely unique. Understanding this system will undoubtedly enrich the knowledge in the field of tissue interactions and organogenesis.
Journal of Experimental Zoology Part B Molecular and Developmental Evolution 06/2008; 310(3):267-77. · 2.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Deer antlers are the only mammalian organs that can fully regenerate each year. During their growth phase, antlers of red deer extend at a rate of approximately 10 mm/day, a growth rate matched by the antler nerves. It was demonstrated in a previous study that extracts from deer velvet antler can promote neurite outgrowth from neural explants, suggesting a possible role for Nerve Growth Factor (NGF) in antler innervation. Here we showed using the techniques of Northern blot analysis, denervation, immunohistochemistry and in situ hybridization that NGF mRNA was expressed in the regenerating antler, principally in the smooth muscle of the arteries and arterioles of the growing antler tip. Regenerating axons followed the route of the major blood vessels, located at the interface between the dermis and the reserve mesenchyme of the antler. Denervation experiments suggested a causal relationship exists between NGF mRNA expression in arterial smooth muscle and sensory axons in the antler tip. We hypothesize that NGF expressed in the smooth muscle of the arteries and arterioles promotes and maintains antler angiogenesis and this role positions NGF ahead of axons during antler growth. As a result, NGF can serve a second role, attracting sensory axons into the antler, and thus it can provide a guidance cue to define the nerve track. This would explain the phenomenon whereby re-innervation of the regenerating antler follows vascular ingrowth. The annual growth of deer antler presents a unique opportunity to better understand the factors involved in rapid nerve regeneration.
[Show abstract][Hide abstract] ABSTRACT: Deer antlers represent a unique model of mammalian regeneration in that they cast and fully regenerate every year. The deer antler thus provides a fascinating model of both rapid angiogenesis and chondrogenesis and the opportunity to investigate unique growth regulatory processes. One such phenomenon is the presence of vascularized cartilage in the growing antler tip-unlike other cartilage, which is typically avascular. The mechanisms by which blood vessels grow in the cartilage as well as the factors that drive antler extension at approximately 1 cm a day have been hitherto largely unknown. The aim of this study was to determine the expression of VEGF and pleiotrophin within the growing antler tip. We isolated cervine VEGF121 and VEGF165 from deer antler and found that mRNA is produced for VEGF in the precartilage and cartilage regions. By in situ hybridization, we examined whether the VEGF receptors Flt-1 and KDR are present in deer antler and found only KDR mRNA within the endothelial cells of the precartilage region. This finding is compatible with VEGF having an angiogenic effect within antler. Pleiotrophin mRNA was found in the vascular smooth muscle cells of the dermis, thus supporting a possible role in vascular growth. High levels of pleiotrophin mRNA were also detected in the precartilage region with possible implications for both angiogenesis and chondrogenesis. This is the first report of cervine angiogenic growth factors within the growing antler tip.
The Anatomical Record Part A Discoveries in Molecular Cellular and Evolutionary Biology 01/2007; 288(12):1281-93.
[Show abstract][Hide abstract] ABSTRACT: The process of angiogenesis is of interest because of the significant clinical benefits associated with controlling vascular growth. Within the antler, chondrogenesis and antler elongation are occurring at the rate of 1-2 cm per day and thus blood vessels are growing at this same rapid pace. We demonstrate that the process of angiogenesis in the antler is controlled at various tissue locations. The findings clearly differentiate the spatial location of the stem cells that drive chondrogenesis from the proliferation process driving the angiogenesis. Vessels within the lateral dermis contained BrdU-positive cells, suggesting that these vessels were elongating. Within the precartilage region, proliferating vessels were detected in bundles of complex structure evenly distributed throughout this tissue layer. The support cells within these bundles of vessels were detected by staining with alpha-smooth muscle actin, while the endothelial cells were negative. Additionally, the alpha-smooth muscle actin staining was found in association with the cartilage cells of the antler. The marked proliferation of the vascular associated cells in the precartilage region identified this area as a major region of vascular growth in the antler. We propose that within the precartilage region, the most likely mechanisms to explain the observed vascular morphology are that of vascular extension of the existing vessels and intussusceptive angiogenesis or sprouting to generate the small bundles of vessels. Wiley-Liss, Inc.
The Anatomical Record Part A Discoveries in Molecular Cellular and Evolutionary Biology 10/2006; 288(9):973-81.
[Show abstract][Hide abstract] ABSTRACT: Annual antler renewal presents the only case of epimorphic regeneration (de novo formation of a lost appendage distal to the level of amputation) in mammals. Epimorphic regeneration is also referred to as a blastema-based process, as blastema formation at an initial stage is the prerequisite for this type of regeneration. Therefore, antler regeneration has been claimed to take place through initial blastema formation. However, this claim has never been confirmed experimentally. The present study set out to describe systematically the progression of antler regeneration in order to make a direct histological comparison with blastema formation. The results showed that wound healing over a pedicle stump was achieved by ingrowth of full-thickness pedicle skin and resulted in formation of a scar. The growth centers for the antler main beam and brow tine were formed independently at the posterior and anterior corners of the pedicle stump, respectively. The hyperplastic perichondrium surmounting each growth center was directly formed in situ by a single type of tissue: the thickening distal pedicle periosteum, which is the derivative of initial antlerogenic periosteum. Therefore, the cells residing in the pedicle periosteum can be called antler stem cells. Antler stem cells formed each growth center by initially forming bone through intramembranous ossification, then osseocartilage through transitional ossification, and finally cartilage through endochondral ossification. There was an overlap between the establishment of antler growth centers and the completion of wound healing over the pedicle stump. Overall, our results demonstrate that antler regeneration is achieved through general wound healing- and stem cell-based process, rather than through initial blastema formation. Pedicle periosteal cells directly give rise to antlers. Histogenesis of antler regeneration may recapitulate the process of initial antler generation.
The Anatomical Record Part A Discoveries in Molecular Cellular and Evolutionary Biology 03/2005; 282(2):163-74.
[Show abstract][Hide abstract] ABSTRACT: Deer antler offers a unique opportunity to explore how nature solves the problem of mammalian appendage regeneration. Annual antler renewal is an example of epimorphic regeneration, which is known to take place through initial blastema formation. Detailed examination of the early process of antler regeneration, however, has thus far been lacking. Therefore, we conducted morphological observations on antler regeneration from naturally cast and artificially created pedicle/antler stumps. On the naturally cast pedicle stumps, early antler regeneration underwent four distinguishable stages (with the Chinese equivalent names): casting of previous hard antlers (oil lamp bowl), early wound healing (tiger eye), late wound healing and early regeneration (millstone), and formation of main beam and brown tine (small saddle). Overall, no cone-shaped regenerate, a common feature to blastema-based regeneration, was observed. Taken together with the examination on the sagittal plane of each regenerating stage sample, we found that there are considerable overlaps between late-stage wound healing and the establishment of posterior and anterior growth centers. Observation of antler regeneration from the artificially created stumps showed that the regeneration potential of antler remnants was significantly reduced compared with that of pedicle tissue. Interestingly, the distal portion of a pedicle stump had greater regeneration potential than the proximal region, although this differential potential may not be constitutive, but rather caused by whether or not pedicle antlerogenic tissue becomes closely associated with the enveloping skin at the cut plane. Antler formation could take place from the distal peripheral tissues of an antler/pedicle stump, without the obvious participation of the entire central bony portion. Overall, our morphological results do not support the notion that antler regeneration takes place through the initial formation of a blastema; rather, it may be a stem cell-based process.
Journal of Morphology 01/2005; 262(3):731-40. · 1.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
General and Comparative Endocrinology 04/2003; 131(1):21-31. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
European Journal of Morphology 03/2003; 41(1):23-30.
[Show abstract][Hide abstract] ABSTRACT: The utilization of a deer antler model to study gene expression in tissues undergoing rapid growth has been hampered by an inability to sample the different tissue types. We report here a standardized procedure to identify different tissue types in growing antler tips and demonstrate that it can help in the classification of expressed sequence tags (ESTs). The procedure was developed using observable morphological markers within the unstained tissue at collection, and was validated by histological assessments and virtual Northern blotting. Four red deer antlers were collected at 60 days of growth and the tips (top 5 cm) were then removed. The following observable markers were identified distoproximally: the dermis (4.86 mm), the subdermal bulge (2.90 mm), the discrete columns (6.50 mm), the transition zone (a mixture of discrete and continuous columns) (3.22 mm), and the continuous columns (8.00 mm). The histological examination showed that these markers corresponded to the dermis, reserve mesenchyme, precartilage, transitional tissue from precartilage to cartilage, and cartilage, respectively. The gene expression studies revealed that these morphologically identified layers were functionally distinct tissue types and had distinct gene expression profiles. We believe that precisely defining these tissue types in growing antler tips will greatly facilitate new discoveries in this exciting field.
[Show abstract][Hide abstract] ABSTRACT: 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.
Anatomy and Embryology 12/2001; 204(5):375-88. · 1.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
General and Comparative Endocrinology 10/2001; 124(1):75-81. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Food and Chemical Toxicology 11/2000; 38(11):985-90. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Coopworth sheep selected for low backfat (lean genotype) have been shown to have heavier pituitary glands than those selected for high backfat (fat genotype). This paper investigated whether this difference was due to an increase in pituitary cell number or cell size and whether the relative proportions of different pituitary cell types differed between the genotypes. In three separate trials, ram lambs aged 6 to 8 months were slaughtered and the pituitary glands were processed for stereological or immunocytochemical studies. The pituitary glands of lean genotype sheep were between 30 and 60% heavier than those of the fat sheep. Lean sheep had a significantly (P<0.05) larger cross-sectional area of the pituitary fossa (96.6 vs. 81.7 mm(2)) than fat genotype sheep. The pituitaries from lean sheep contained significantly more total cells than fat sheep (Trial 1: 290 vs. 183 million cells, P<0.01; Trial 2: 353 vs. 239 million cells, P <0.05). The volume of individual cells did not differ between the genotypes. Trial 3 showed that there was no difference between lean and fat sheep in the percentage of cells staining positive for the five pituitary hormones studied. It is concluded that the larger pituitary glands of lean compared to fat genotype sheep are a result of a nonspecific increase in the size of the whole gland through increased cell numbers, with no change in cell size or the relative proportion of different cell types.
[Show abstract][Hide abstract] ABSTRACT: Asian markets for velvet antler perceive the colour of the core as a primary indicator of quality. The factors which influence colour are not known, but the market preference in Korea is for an even mid‐red colour. The aim of the present study was to determine whether removal technique and post‐removal handling influence velvet colour. Investigations took place at AgResearch Invermay and at Mount Hurt Station in mid Canterbury, New Zealand. The influences on velvet antler colour of sedative drug, mild stress, local anaesthetic administration, timing of tourniquet application, and restraint of the stags in a crush or workroom for velvet antler removal were investigated. The effects of antler orientation post‐removal and post‐removal environmental temperature on velvet antler colour were also investigated. In all studies, velvet antler was frozen and held at ‐20°C before being dried either by freeze drying or commercially. In all trials, a consistent pattern of both lightness and hue angle was shown from the tip of the velvet antler stick to the base; the tip was lighter and browner, the mid section was darker and redder, and the base was lighter and browner. There were no significant overall effects of drug treatment on colour, but there were significant differences among sections. Specifically, sedative drug treatments resulted in less red velvet antler than in control antlers removed using local analgesic only. Mild stress and method of local analgesic administration had no effect on any aspect of velvet antler colour. Placing the velvet at an angle of 15° (tip down) gave a darker and redder antler than the typical fully inverted position. There were no significant differences in colour whether the velvet antler was frozen immediately after removal or held at 4°C or ambient temperature for up to 6 hours prior to freezing. Overall, the use of sedative drugs produces velvet antler that is lighter and less red, and post‐removal handling technique can influence colour.
New Zealand Journal of Agricultural Research 01/2000; 43(2):207-225. · 0.84 Impact Factor