长鳍吻卵巢发育组织学及周年变化的初步研究

采用常规解剖和组织学切片的方法,对长江上游宜宾江段长鳍吻卵巢发育、卵子发生以及成熟系数的周年变化进行了研究。研究结果表明,长鳍吻卵巢发育分为Ⅵ期,卵母细胞发育分为6个时相,第2时相中期出现卵黄核,2时相晚期迁移至皮质层破碎、消失;第3时相早期卵黄核消失位置开始出现卵黄泡,第3时相中后期放射带出现后卵黄颗粒开始积累;第5时相卵母细胞体积最大,胞径(1805±60.99)μm。卵巢成熟系数周年变化范围为0.78%~18.22%,一年内仅在4月份出现一个峰值,4—5月大部分卵巢处于Ⅳ、Ⅴ期,综合成熟系数测定和性腺组织学观察结果,初步推测长鳍吻繁殖期为3—5月,产卵盛期为4月上旬至5月上旬。     

养殖金乌贼卵子发生和卵巢发育的组织学观察

为了深入了解金乌贼(Sepia esculenta Hoyle)的生殖生理特性,本研究采用解剖学方法及组织切片技术,观察和描述了金乌贼卵巢形态和卵子各个时相结构特征及其变化规律。研究结果表明,金乌贼卵子发生过程的主要特征表现为,卵子成熟之前滤泡细胞一直存在且伴随着其结构变化,滤泡细胞解体与卵黄物质出现同步发生,卵黄物质从无到有再到大量积累直至充满整个卵母细胞,细胞核则随着滤泡细胞内折逐渐发生偏移直至最后消失。研究还发现金乌贼卵巢内卵子发生不同步且形态差异显著,未成熟的卵子粘连在卵巢基质上,依次环布于卵巢基质周围,越靠近中心,卵母细胞发育程度越低;成熟后的卵子游离在卵巢腔中,经过输卵管排出体外。根据卵巢内卵子的大小和形态、滤泡细胞形态及卵黄形成情况,将金乌贼卵子发生分为增殖期(卵原细胞期)、小生长期(无滤泡期,单层滤泡期,双层滤泡期)、大生长期(滤泡内折期,卵黄形成早期,卵黄形成晚期)、成熟期、退化吸收期共5个时期9个时相。卵巢发育分期则是以卵子时相为基础,根据卵巢的外观形态、性腺系数以及切面上优势卵子的时相,把卵巢分为Ⅰ~Ⅵ共6个时期。金乌贼卵巢发育和卵子发生的动态观察与描述,对丰富头足类繁殖生物学理论及提升人工繁殖技术都具有重要意义。     

大鳞副泥鳅卵巢发育的组织学研究

采用组织切片方法,对大鳞剐泥鳅的卵巢发育进行了组织学研究,系统地描述了卵巢的形态结构、组织学特征.2个卵巢序有成对,位于体腔中线两侧和消化管的背面,以肠系膜与体壁相连.大鳞副泥鳅卵巢的发育分为Ⅵ个时期,第Ⅱ期卵巢出现产卵板,卵母细胞明显增多,原牛质增长,胞质嗜碱性增强,早期排列紧密,后期排列疏松;第1II期卵巢体积显著增大,扁圆,浅黄色,卵黄颗粒出现在皮质M,单层滤泡层形成,放射带…现;第IV期卵巢体积更加增大,圆筒形,深黄色,能见许多血管分枝;第V期卵巢一般只在繁殖季节达到性成熟的可自然排卵产出体外的雌鳅体内出现;第VI期卵巢呈现排空的滤泡.卵母细胞的发育分为5个时相,在第2时相晚期列第3时相早期,卵母细胞中具有卵黄核.     

孔雀鱼雌性生殖器官组织结构及卵子发生的研究

应用光镜技术研究了孔雀鱼（Poecilia reticulata）雌性生殖器官组织结构和卵子发生的特点,以期为孔雀鱼的良种繁育工作提供基础资料。结果表明,孔雀鱼雌性生殖器官由卵巢、卵子输送管和泄殖孔构成。卵子发生过程经历了6个时相：第Ⅰ时相主要为卵原细胞;第Ⅱ时相是处于小生长期的初级卵母细胞,形成生长环、卵黄核和单层扁平滤泡细胞膜;第Ⅲ时相是进入大生长期的初级卵母细胞,卵黄开始积累,形成2层滤泡细胞膜和2层卵包膜;第Ⅳ时相为发育晚期的初级卵母细胞,细胞发生了极化,卵包膜发育完善;第Ⅴ时相发育成熟,卵黄颗粒凝结呈块状,卵黄泡主要分布于细胞膜内缘,滤泡细胞膜萎缩;第Ⅵ时相是闭锁卵泡,被滤泡细胞消化和吸收转变为卵巢内结缔组织。     

葛氏鲈塘艚卵子发生的组织学观察

采用组织切片技术系统观察和描述了葛氏鲈塘鱧（Perccottus glenii Dybowski）卵巢各时相卵母细胞的形态结构、特征及变化。卵巢切面显示：卵母细胞发育分为6个时相：第Ⅰ时相卵母细胞由处于原始分化阶段的卵原细胞构成;第Ⅱ时相卵母细胞进入滤泡细胞期,胞外形成滤泡细胞膜：第Ⅲ时相卵母细胞由质膜向核膜逐渐积累皮质液泡：第Ⅳ时相卵母细胞主要形成卵黄颗粒：第Ⅴ时相卵母细胞为成熟的卵子,胞内含有丰富的卵黄;第Ⅵ时相卵母细胞是未排出的处于退化吸收阶段的卵细胞,卵膜破裂,卵黄被吸收。根据卵巢切片及性腺系数变化推测葛氏鲈塘鱧属于一次产卵类型。     

四川华鳊卵子发生的显微结构观察

为探究四川华鳊(Sinibrama taeniatus)卵子的发生规律,对其卵子发生进行了显微结构观察和描述。将卵子发生分为卵原细胞(Ⅰ时相)、单层滤泡时相(Ⅱ时相)、卵黄泡出现时相(Ⅲ时相)、卵黄充满时相(Ⅳ时相)和成熟卵子(Ⅴ时相) 5个时相。观察发现,四川华鳊卵原细胞存在2种形态,分别为早期卵原细胞和有丝分裂过程中的卵原细胞;同时将早期初级卵母细胞划入Ⅱ时相,此阶段细胞核膜边缘出现多个小核仁,将其作为小生长期开始的标志;Ⅲ时相卵黄泡出现,当卵黄泡积累至3～5层时,卵黄物质即开始积累,此时多以卵黄颗粒的形式存在;进入Ⅳ时相后,卵黄物质沉积形成卵黄小板,卵黄泡被挤压至卵周,形成皮层小泡,卵子成熟后,原皮层小泡所在区域呈块状或颗粒状,经苏木精-伊红(HE)染色后呈橘红色。     

池养鲻的卵巢发育和卵子发生过程

通过卵巢切片的组织学分析揭示，幼鲻在土池养殖3个月后可见线状卵巢，大约5个月后卵原细胞进入第一次成熟分裂前期的双线期转变为早期初级卵母细胞。接着卵母细胞生发泡(核)和胞质体积增加，核质比从3．5：1减少至2：1。此后卵巢中卵母细胞停滞发育持续至养殖的第3年。在第3年卵巢切片看出卵母细胞进入脂肪泡时相，在第3年秋季进入卵黄发生时相。但在人工养殖条件下卵母细胞仅能发育到卵黄发生后期，即卵母细胞胞质充满卵黄颗粒，生发泡居中而不移位。这些结果对于用人工养殖鲻为亲鱼开展人工繁殖提供重要的科学依据。并讨论了卵子发生6个时相的生物学特点及其重要的细胞器在卵黄发生中可能的生理作用。     

嘉陵江粗唇鮠卵巢组织学的周年变化

对嘉陵江粗唇鮠(Leiocassis crassilabris Günther)的卵巢发育、雌鱼成熟系数的周年变化进行研究。结果显示:嘉陵江粗唇鮠的卵巢发育划分为6期,其卵母细胞相应地划分为6个时相,第Ⅰ时相的卵母细胞呈原始分化状态,细胞外具一层细胞质膜,第Ⅱ时相卵母细胞外不仅具质膜,而且还有滤泡膜和结缔组织膜,第Ⅲ和第Ⅳ时相的卵母细胞分化明显,由散在的卵黄沉积逐渐形成大的卵黄颗粒。第Ⅴ时相和第Ⅵ时相分别为卵子形成和退化吸收时相。繁殖季节中雌鱼的成熟系数可达13.4%,而在其它季节有所下降。     

金钱鱼性腺发育及其组织结构观察

采用常规石蜡切片,H.E染色研究了金钱鱼性腺发育及组织结构特征.结果显示,2+龄的金钱鱼性腺发育成熟.金钱鱼精巢为管型,可分为:精原细胞增殖期、精母细胞生长期、精母细胞成熟期、精子细胞变态期、精子成熟期等5个时期.从Ⅱ期精巢起,金钱鱼精巢的成熟系数(GSI)为0.2％～1.5％,精巢成熟系数在发育到精子细胞成熟期(Ⅴ期)达到峰值,肝重指数(HSI)在精子细胞变态期(Ⅳ期)达到峰值.金钱鱼卵巢的卵母细胞发育过程可分为5个时相,相对应的卵巢发育亦分为5个时期.从Ⅱ期卵巢起,金钱鱼卵巢的成熟系数为1.2％～14.9％,在发育到Ⅴ期时达到峰值,HSI在Ⅳ期达到峰值.Ⅱ时相卵母细胞出现卵黄核和滤泡膜.Ⅲ时相卵母细胞中开始出现油滴,卵黄颗粒.Ⅳ时相卵母细胞中卵黄颗粒与油滴的数量迅速增多.Ⅴ时相卵母细胞中卵黄颗粒融合成片,在卵母细胞中卵黄颗粒与油球之间在数量上没有明显的差异.根据切片观察,Ⅴ期卵巢中,除了Ⅴ时相卵母细胞外,还有一定数量的Ⅱ、Ⅲ和Ⅳ时相卵母细胞,卵母细胞发育呈现非同步型.并且发现在多数产完卵后的卵巢中,除空的滤泡外亦存在一定数量的Ⅱ、Ⅲ和Ⅳ期卵母细胞,因此推测,金钱鱼的产卵类型为分批非同步产卵类型.     

黄海高眼鲽卵巢发育特征及卵径分布

本研究通过组织学观察,描述了黄海高眼鲽(Cleisthenes herzensteini)卵母细胞发育特征及其退化过程。高眼鲽卵母细胞发育分为5个时相:第1时相为卵原细胞,细胞体积小,细胞质少,细胞核明显;第2时相卵母细胞细胞核附近出现卵黄核;第3时相由胞质外缘向内层逐渐产生液泡并生成卵黄颗粒,出现双层滤泡膜;第4时相卵母细胞内充满卵黄,细胞核向动物极移动,放射膜增厚;第5时相细胞核溶解,卵母细胞从滤泡膜中释放出来并发生水合作用;产卵期过后,卵巢发生退化,卵黄颗粒逐渐被吞噬,放射膜溶解断裂。通过比较卵巢中各时相卵母细胞组成比例,表明卵母细胞发育具有非同步性。Ⅳ、Ⅴ、Ⅵ-Ⅳ'期卵巢内卵径(长径)呈单峰分布,优势粒径组分别为0.45~0.55 mm、0.60~0.65 mm和0.40~0.50 mm;Ⅴ'期卵巢,卵径分布呈双峰型,峰值分别为0.50~0.55 mm和0.90~0.95 mm,水合卵母细胞与卵径较小的小生长期卵母细胞比例增大,呈现出明显的双峰分批产卵型特征。     

不同比例复合益生菌对凡纳滨对虾生长、免疫及抗氨氮能力的影响

对从凡纳滨对虾(Litopenaeus vannamei)肠道中分离出的鲍鱼希瓦氏菌(Shewanella haliotis)、蜡样芽孢杆菌(Bacillus cereus)和双壳气单胞菌(Aeromonas bivalvium)3株有益菌株,利用正交设计得到9种复合比例,通过饲料中添加上述9种比例混合的菌体(菌数总量为109 cfu/g)饲喂凡纳滨对虾,经过28 d养殖实验,评价其对凡纳滨对虾生长、免疫指标的影响。随后,利用氯化铵调节水体氨氮浓度至26.67 mg/L,经过16 d的氨氮毒性实验,研究不同比例复合益生菌对凡纳滨对虾抗氨氮能力的影响。研究结果表明,9个复合益生菌实验组的增重率和特定生长率均显著高于对照组(P0.05),并且以3株菌菌数6︰1︰3比例效果较好;不同配比的复合益生菌能够显著提高酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、总超氧化物歧化酶(T-SOD)活力(P0.05),并表现出了不同的影响效果,其中,3株菌菌数(菌数总量为109 cfu/g,下同)2︰3︰3、4︰2︰3及6︰1︰3比例对ACP活力具有显著促进作用(P0.05),3株菌菌数4:2︰3和6︰1︰3比例对ALP活力具有显著促进作用(P0.05);3株菌菌数2︰1︰1比例对T-SOD活力具有显著促进作用(P0.05);各比例的复合菌对溶菌酶活力的影响不显著(P0.05);氨氮浓度26.67 mg/L条件下,不同比例复合菌组对虾累计存活率显著高于对照组(P0.05),其中以3株菌菌数4︰3︰1和6︰3︰2比例组累计存活率较高,即抗氨氮效果较好。     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Movement of the fluvial form of masu salmon, <Emphasis Type="Italic">Oncorhynchus masou masou</Emphasis>, in a mountain stream in Kyushu,Japan

Using mark-recapture methods, the movements of the fluvial form of masu salmon (Oncorhynchus masou masou) in a mountain stream on the island of Kyushu, Japan, were studied. Most (78%) of the masu salmon were recaptured in the pool in which they had been originally caught and tagged. Of those that moved between pools, the proportion of individuals that moved during the breeding period was not significantly higher than the proportion that moved during the non-breeding period. However, during the breeding period, a higher proportion of larger salmon moved than did smaller fish. The proportion of mobile large males during breeding period was higher than that for small males. Also, it was found that a few individuals showed long-range movement in the autumn. As a long-term movement, 78 individual fish (65%) that were recaptured more than three times showed high sedentary tendencies. Sixteen individual mobile fish (13%) moved and returned to the original pool. Fluvial form of masu salmon in Kyushu show a high sedentary nature; however, large mature males seem to actively move in search of female during breeding period.     

Cloning, expression, and characterization of a novel anti-HIV lectin from the cultured cyanobacterium, Oscillatoria agardhii

OAA, the potent anti-HIV protein from Oscillatoria agardhii NIES-204 belongs to a new lectin family, shows strict binding specificity for high-mannose N-glycans, and has an extremely high association constant in the picomolar range for recombinant gp120, an envelope protein of HIV. In this study we have cloned the gene encoding OAA from the genomic DNA of the cyanobacterium, and efficiently expressed the recombinant lectin (rOAA) in Escherichia coli. The rOAA expressed as a His-tagged fusion protein was recovered in a soluble form and purified in high yield (48 mg/1 l-culture) by metal chelate chromatography. The fusion protein was cleaved with factor Xa, and the resulting rOAA was isolated in a final yield of 14.8 mg/1 l-culture by reversed-phase HPLC. Both the N-terminal sequence and the molecular mass of rOAA were found to be identical with those of OAA. The rOAA was fully functional with the same properties as OAA, as evidenced by hemagglutination activity, hapten-inhibition test, and binding specificity for high-mannose-type N-glycans. This rOAA should be applicable as a specific probe for high-mannose N-glycans and should contribute to elucidation of the molecular basis of its strict carbohydrate-binding specificity and potent anti-HIV activity.     

Mortality of scallop spat in cultivation, infested with tube dwellingbristle worms, Polydora sp.

Between 1995 and 1997, a traditional Norwegian oyster poll with an attachednursery was used for the production of scallop (Pecten maximus) spat.During summer 1997, four batches of 2 mm scallop spat were placed in thenursery. In June, hydroids (Obelia) caused problems with fouling andaccumulation of sediment in the nursery. Thereafter, the scallop spat wereheavily infested with tube dwelling polychaets (Polydora sp.). Thepolychaets were both introduced with poll water and reproduced inside thenursery. Infested spat suffered high mortalities. Although the spat could have been affected by the hydroids, as well as shortage of food, Polydorainfestations were considered the main cause of the mortalities. Dead andinfested spat were continuously sorted out and discarded, but the infestationproblems persisted. As a result, the scallop spat did not represent anycommercial value for the company, and in September, the nursery wasemptied. In total, approximately one million spat – representing onethird of Norway's intensive scallop spat production in 1997, was lost.     

Parasites, pathological conditions and mortality in QX-resistant and wild-caught Sydney rock oysters, Saccostrea glomerata

Differences in the survival of QX-resistant fifth generation (QXR₅) and wild-caught (Wild-Caught) Sydney rock oysters were assessed over the QX-disease risk period in the Pimpama River, SE Queensland. Cumulative mortality of Wild-Caught oysters (31.7%) was significantly greater than QXR₅ oysters (0.0%) over the 118 days of the experiment. PCR and histological results showed that Wild-Caught oyster did not die from QX disease. Histology revealed oysters were infected with disseminating hemocytic neoplasia, a Steinhausia-like infection, a Rickettsia-like organism infecting epithelial cells of the gill, digenean flukes encysted in the gonadal tissue and a gill response to an unknown toxin. The cause of mortality is attributed to disseminating hemocytic neoplasia.     

Enrichment of Artemia nauplii in PUFA, phospholipids, and water-soluble nutrients using liposomes

Different liposome formulations, includingseveral combinations of membrane composition,type of vesicle (multilamellar and largeunilamellar vesicles), preparation method, andvehiculated nutrient, have been assayed asbioencapsulation products to enrich Artemia nauplii with nutrients for feeding fish larvae.The stability of the liposome preparationsunder conditions of use as enrichment producthas been tested using water soluble fluorescentmarkers as leakage indicators. The content ofthe fatty acids and lipid classesbioencapsulated in Artemia nauplii withliposomes has been analyzed by gas and thinlayer chromatography, respectively, andcompared with other enrichment products. Theeffect of the liposome enriched Artemianauplii used as food for fish larvae has beenevaluated in sea bass cultures. Liposomes withhigh content in polyunsaturated fatty acidsleak out more than 50% of their aqueous phasein less than 2 hours, unless they arestabilized with cholesterol and formed as largeunilamellar vesicles. Such vesicles hold70% of the encapsulated material for 8 hours.Liposome enriched nauplii in this study reflectthe influence of the enrichment products,however, they are far from the commercialemulsion (Super Selco) in terms ofdocosahexaenoic acid content, except for thenauplii enriched with liposomes made of purekrill phospholipid extract by the method ofdetergent solubilization. The liposome enrichednauplii show a higher amount of polar lipids incontrast to the feed enriched with emulsions.The larvae fed liposome enriched nauplii haveonly a slightly lower docosahexaenoic acidcontent than those fed emulsion enrichednauplii. The results obtained confirm thesuitable potential use of liposomes as foodsupplement in larviculture. Problems andadvantages are discussed.     

鳢流行性溃疡综合征病原分离鉴定与病理形态学观察

为寻找冬春季鳢(Channa maculata)烂身病的病原,应用组织压片、组织切片、扫描电镜技术、HE常规染色法和Grocott六胺银染色法、霉菌分离纯化及ITS的序列分析等对冬春季患疑似溃疡综合征的烂身病鳢进行了病原学与病理学研究。病变组织压片观察到大量直径10～20μm,分枝较少、纤细菌丝。扫描电镜观察到肌肉组织中延伸出大量的纤细的丝状真菌。患病鳢皮肤和肌肉表现为变性、坏死与炎性细胞浸润,溃疡灶肌肉内可见大量的慢性肉芽肿结节和炎性细胞浸润。结节基本结构由类上皮细胞和多核巨细胞、炎性细胞、霉菌菌丝(横断面呈圆形,斜断或纵断呈丝状)组成,霉菌位于结节中央。Grocott六胺银染色观察到结节中有大量棕色菌体。无菌分离培养可观察到典型丝状霉菌;霉菌在灭菌池塘水20℃过夜, 12 h后可观察原代孢子群形成,真菌ITS序列分析表明其与侵袭丝囊菌(Aphanomyces invadans)同源性为100%。侵袭丝囊霉菌(A. invadans)是杂交鳢溃疡综合症病的主要病原,为鱼类流行病学调查和疾病防治的深入研究奠定基础。     

Feeding of scleractinian coral, Galaxea fascicularis, on Artemia salina nauplii in captivity

Satisfying nutrient requirement of corals is still a major constraint for maintaining corals in marine aquariums. Corals are polytrophic in nature. Heterotrophic feeding on zooplankton is one of the corals’ strategies to overcome nutrient deficiency. Artemia salina nauplii are commonly used as biocarriers for many fish larvae in aquaculture and can also serve as a biocarrier for coral in aquariums, provided coral acceptability, optimal feeding rate, and digestibility of the nauplii are well understood. Feeding rate and digestibility of coral fed on A. salina nauplii at 100, 2,000, 4,000, 6,000, and 10,000 ind. l⁻¹ under light and dark conditions was assessed in this study. The maximum feeding rates of Galaxea fascicularis under light and dark conditions was 113.6 ind. polyp⁻¹ h⁻¹ and 76.9 ind. polyp⁻¹ h⁻¹, respectively. The daily feeding rates of G. fascicularis varies and depends on nauplii density. Light plays an important role in coral feeding. Nevertheless, the quantity of A. salina nauplii consumed by the coral under light and dark conditions was not significantly different (P > 0.05). A. salina nauplii are well accepted by G. fascicularis. Complete nauplii digestion was observed after 180 min. Digestibility of A. salina nauplii by G. fascicularis was positively correlated with digestion time.     

Comparison of age estimates from scale, opercular bone, otolith, vertebrae and dorsal fin ray in Labeo rohita (Hamilton), Catla catla (Hamilton) and Channa marulius (Hamilton)

The present study was undertaken with a view to compare the precision and reliability of the age readings obtained from different bony structures of some important freshwater teleosts viz., Labeo rohita (Hamilton), Catla catla (Hamilton) and Channa marulius (Hamilton). Standard procedures were followed to prepare and study the age structures. In L. rohita and C. marulius percent agreement between reader's age estimates was highest for scales, i.e. 96.3% and 90.5%, respectively and in C. catla percent agreement was highest (93.3%) for opercular bone. When scale ages were compared with other alternative structures viz., otoliths, opercular bone, vertebral centra and dorsal fin rays, percent agreement was found highest between scale and opercular bone age estimates (77.8%) in L. rohita and between scale and otoliths (94.8%) in C. marulius. In case of C. catla highest percent agreement was found between opercular bone and scale age estimates. In L. rohita each of the ageing structure showed significant (P < 0.05) underestimation of age in comparison to scales. In C. catla mean age estimates from opercular bone were comparable (P > 0.05) to the values obtained from all other structures except dorsal fin rays. In C. marulius mean age estimates from scales were comparable (P > 0.05) to those from all other structures except from dorsal fin rays. Results indicated scales to be the most suitable structure for ageing L. rohita and C. marulius and opercular bone for C. catla. However, in C. catla also scales may be used as a non-destructive method of age estimation with satisfactory results.