This study is the first report on circulating testosterone and inhibin levels in a species of whales, the beluga. Circulating testosterone and immunoreactive (ir-) inhibin levels in two captive male belugas ("Nack", originally from Canada and "Duke", from the Okhotsk Sea) were measured every month for 9 years between 1995 and 2003. Assuming that clearly increased testosterone levels in the circulation indicates that the belugas had reached sexual maturity, at the ages of 10 ("Nack") and 11 years old ("Duke"). Their testosterone levels before the significant increase (pre-pubertal) were 0.42 ± 0.07 ng/ml (n=18) and 0.35 ± 0.10 ng/ml (n=18) and, those of after the increase (maturity) were 1.65 ± 0.14 ng/m l (n=74) and 2.06 ± 0.14 ng/ml (n=74). Circulating ir-inhibin levels before sexual maturity were 0.78 ± 0.04 ng/ml (n=18) and 0.64 ± 0.04 ng/ml (n=15) and, after sexual maturity were 0.52 ± 0.02 ng/ml (n=56) and 0.43 ± 0.02 ng/ml (n=67). Seasonal changes were observed in the testosterone levels after sexual maturity and the levels increased during March and April in Canadian origin "Nack", and peaked in February in Okhotsk origin "Duke". Circulating ir-inhibin level gradually decreased as they aged. A negative correlation between the circulating testosterone and ir-inhibin was observed. No seasonal changes were observed in the ir-inhibin levels after sexual maturity. These data will surely correspond to clarification of endocrinology and the successful reproduction of the beluga.

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鴨川シーワールドで長期飼育されていたシロイルカ

が腎機能障害により死亡した。腎臓には多数の結節が認められ，結節内部に石灰化した線虫虫体が集簇していた。右腎尿管には大型の線虫の寄生が認められ，形態観察および分子生物学的解析によりCrassicauda giliakianaと同定された。シロイルカの導入時に既に線虫が寄生しており，長期飼育中に腎臓内で死滅した成虫に対する反応として結節が形成され,腎機能障害が発生した可能性が考えられた。

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The relationship between basal body temperature and circulating progesterone levels were investigated in a female beluga. Body temperature and serum concentrations of progesterone were measured daily and at 2-4 week intervals respectively, in a female beluga that was in captivity for 7 years between 1996 and 2003. The beluga first ovulated in April, 2000 (13 years old). Thereafter, serum concentrations of progesterone showed cyclic changes, indicating that the ovulatory cycle had started. Serum concentrations of progesterone ranged from 0.1 ng/ml to 15.7 ng/ml. Body temperature also showed cyclic changes during the estrous cycle. Body temperature ranged from 34.9 to 35.9 C, and tended to reach the peak during the high progesterone phase. Mating behavior was observed during the low body temperature phase. The changes in body temperature positively correlated with the circulating progesterone levels. The length of the estrous cycle was 36.7 ± 3.9 (mean ± SEM) days based on the intervals between the days of mating behavior. This is the first report demonstrating that body temperature clearly changes during the estrous cycle in a captive female beluga. The present finding suggests that measurement of body temperature is a useful method for detecting the ovarian cycle of the beluga in captivity.

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Safe sedation doses for performing minor procedures such as bronchoscopy, endoscopy, and tooth extraction for beluga whales (

) require elucidation. This study aimed to provide suggestions for determining appropriate midazolam and butorphanol doses to adequately sedate beluga whales to complete procedures and minimize the risk of side effects. We administered midazolam and butorphanol to six captive beluga whales (9–44 years old). Topical lidocaine anesthesia was administered during bronchoscopy. The sedation doses for the beluga whales varied from 0.020 to 0.122 mg/kg for midazolam and from 0.020 to 0.061 mg/kg for butorphanol. In beluga whales, optimal midazolam and butorphanol doses were lowest in old whales. These findings contribute to knowledge regarding appropriate sedation and prevention of overdose accidents during minor procedures in beluga whales.

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飼育下のシロイルカが食欲不振,下痢および嘔吐を示し死亡した。様々な寄生虫,すなわち小腸にHadwenius sp.,気管支内にHalocercus sp.ならびに尿管内にCrassicauda giliakianaが認められた。また,肺では,肺胞腔内に寄生虫卵が認められ,肺胞壁の著明な線維化を伴う間質性肺炎が認められた。

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Putative nucleotide sequences for DQB exon 2 alleles of the major histocompatibility complex (MHC) were amplified and determined in the 16 cetacean species using the PCR technique. The 172 bp sequences amplified showed no multiple alleles more than two in each of the examined individuals. The sequences of the 31 cetacean DQB alleles detected were monophyletic with the HLA-DQB1, and were separated from the lineages of HLA- DQB2 and DQB3. These results suggest that the locus is the homolog to the human DQB1 gene. The observed frequency of nonsynonymous substitutions in the cetacean DQB sequences was significantly higher than that of synonymous substitutions. The amino acid variation at the putative peptide binding region (PBR) was considerably high. These results imply that positive selection has promoted its variability at the cetacean DQB gene as other mammalian MHC. The DQB gene tree showed that four Mysticeti alleles branched off from the clade consisted of Mysticeti alleles only and were included in the clade consisted of Odontoceti alleles. This suggests trans-species polymorphism in the cetacean MHC gene.

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Cortisol is a member of the corticoid family and is known as a stress hormone. In cetaceans, cortisol is frequently measured as a stress indicator. However, inappropriate estimation of cortisol concentration frequently occurs due to a lack of fundamental knowledge of corticoids in cetaceans. We carried out several experiments to establish standard methods of cortisol measurement in cetaceans. 1) Cortisol biosynthesis was confirmed in Antarctic minke whales Balaenoptera bonaerensis. Corticoids including cortisol, progestins, adrenal androgens and many other intermediates were synthesized from ¹⁴C-pregnenolone. 2) Serum cortisol concentrations in 15 cetaceans species were measured. Average cortisol concentrations in each species showed a wide range from 2.9 ng/ml in killer whale Orcinus orca to 39.9 ng/ml in striped dolphin Stenella coeruleoalba. A negative correlation between cortisol and body length was confirmed. 3) Diurnal changes in cortisol concentrations were investigated in well-trained Indo-Pacific bottlenose dolphins Tursiops aduncus and in killer whales. Cortisol concentrations in both species exhibited episodic fluctuations with decreases in the evening and increases in the early morning. Annual cortisol changes in killer whales were also investigated, and pattern varied according to sex. 4) Changes in serum cortisol concentrations following transport into captivity in a male rough-toothed dolphin Steno bredanensis were investigated. Cortisol concentrations were high on the day of transport, and continued to decline until stabilizing about four months later. 5) Twenty-one-deoxycortisol concentrations increased with time after transport, and a significant negative correlation between cortisol and 21 -deoxycortisol was demonstrated in a rough-toothed dolphin. This suggested that 21 -deoxycortisol is useful as a negative indicator of stress.

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Dolphins are expected to possess a highly developed visual information processing system. However, there are few studies investigating how they perceive the outer world by vision. In the present study, we examined the capability of solving complex problems that were visually guided, in terms of counting ability of items. A male beluga was trained to use Arabic numerals (1, 2) to name the number of items such as one or two fins and balls. The subject was able to discriminate the number of objects and he could name them using those numerals. Though it was not verified that those numerals were used as symbols in the present study, our results revealed that the dolphin was capable of solving complex problem visually guided and supported that it had a highly developed visual information processing system.

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To better understand cetacean immunology, it is important to develop markers that identify specific leukocyte populations. We created a monoclonal antibody (mAb) library against leukocytes of the beluga whale (

), and established five hybridoma clones that produce mAbs. Three of these mAbs (ID: BW-3C3, BW-2G4, and BW-2G6) react with mononuclear leukocytes including lymphocytes and monocytes. mAb BW-3C3 react to a fraction of the lymphocytes. The mAb-positive cells were identical to cells that also stained with polyclonal anti-whale IgM antibodies, indicating that the mAb BW-3C3 specifically reacts to B lymphocytes. mAb BW-2G4 specifically binds to monocytes that possess a reniformed nucleus. mAb BW-2G6 was found to bind to heterogeneous lymphocytes, namely, anti-whale IgM antibody-positive and -negative lymphocyte populations. This indicates that this mAb reacts with B and non-B lymphocyte fractions. The other two mAbs (ID: BW-4B10 and BW-4G12) react with polymorphonuclear granulocytes. Double staining with Giemsa-eosin showed that mAb BW-4B10 and mAb BW-4G12 specifically identify neutrophils and eosinophils, respectively. This panel of mAbs will be a useful tool for classifying leukocytes and for determining their localization in different tissues, which in turn would contribute to our understanding of cetacean immunology and allow evaluation of leukocyte function in infectious diseases.

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　本研究では飼育下で分娩した太平洋カマイルカの母乳を搾乳し，乳成分分析を分娩後約3か月間，熱量及び色調変化を5カ月間調査した。

　乳成分変動は，脂質9～32.5%，蛋白質9.9～13.3%，糖分1.1～2.1%，灰分0.33～0.84%の範囲であり，高乳脂質，低糖質な乳であった。乳脂質は分娩後0日と1日で大きく変動し，蛋白質と糖質は変動が見られなかった。母乳熱量は2.3～3.5 kcal/gであり，泌乳期が進むにつれ増加した。乳熱量は飼育下のバンドウイルカより高い傾向にあり，この違いは生息域や生活様式が関与している可能性が考えられた。子の成長は出生時体長105 cm，体重12.9 kgであり，出生後15週で，129 cm，27.8 kgに直線的に増加した。

　母乳の色調は，約2週目ごろより緑白色に変化した。この現象は他の鯨類においてもいくつか報告されており，鯨類の特徴の一つである可能性が示唆された。

　本研究で得られた情報は1個体に基づくもので，今後他個体によるデータも集める必要はあるが，カマイルカの生態への理解や飼育下での人工哺乳の基礎データとして重要な意義を有する。

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本研究は，ミナミハンドウイルカ（Tursiops aduncus） における生理学的知見の蓄積を目的として，餌の消化管通過速度を測定する実験を行った。実験は飼育下のミナミハンドウイルカのオス1頭を対象とし，3日間1日1回行われた。赤色色素を粉のままゼラチン製の薬用カプセルに梱包し，餌のカラフトシシャモの体内に挿入し給餌した。着色餌の給餌後，水中観察窓から連続観察を行った。着色餌の給餌から，初めて糞に赤色が確認されるまでの期間をIPT （Initial Passage Time） と定義した。着色糞は3日間全ての実験で確認された。着色糞が最初に観察されるまでの経過時間（IPT） は平均254 ± 20.4 分（n＝3）であった。先行研究における同属のハンドウイルカの平均IPTと比べ近い値となった。本研究により，ミナミハンドウイルカは摂餌から最短で4時間から6時間程度で排泄し，加えて餌が消化管内に20時間以上留まる可能性があることが通常色の糞の観察から示唆された。

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The aim of the present study was to investigate whether changes in body temperature are related to the ovarian cycle in elephants. Rectal, tongue or fecal temperature was measured for 2 Asian and 5 African elephants using an electric thermometer. Evaluation of ovarian cycles was based on the changes in serum or fecal progestin. The mean ± SD values of the rectal, tongue, and fecal temperatures were 36.3 ± 0.3 (2 Asian), 36.2 ± 0.5 (1 African) and 36.5 ± 0.3 C (4 African), respectively; the fecal temperature was the highest of the 3 temperatures (P<0.01). The longitudinal changes in body temperatures correlated with the ovarian cycle, with higher temperatures occurring during the luteal phase. The fecal temperatures of one acyclic African elephant did not change cyclically. These results suggest that measurement of body temperature can be used to easily evaluate the ovarian cyclicity of an individual animal, although it might not be able to determine the ovarian cycle length.

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We observed two tongues of Pontoporia blainvillei (body length 98.5 and 171 cm, females) macro- and light microscopically. The Pontoporia's tongue is divided into two different parts: the front and the rear. The front part, the anterior one-third in the adult and the anterior one-fourth in the young, is nearly semicircular in outline having a completely smooth dorsum and a marginal free surface without an anterior free tip. The lateral free edges are scalloped in appearance but in the young many projections, about 3 mm long, can be seen at that position. The rear part continues from the front part at nearly the same level with irregular fissures and a remarkable longitudinal fissure present on the mid-line. But these features were unclear in the young. No trace of the sulcus terminalis and foramen caecum can be found on the tongue. Neither lingual papillae nor taste buds exist over the entire tongue. Many small pores are found on the dorsum from the mid-region of the front part to the base of the epiglottis. These pores also can be seen at the caudo-lateral free surface of the front part and on the continuing oral wall of the rear part. The pores are the orifices of the excretory canals of well developed salivary glands which are especially crowded at the rear part and are mucous in nature. Very scarce lymphatic nodules are observed in the lamina propria mucosae of the tongue and the neighboring part. Lingual tonsils can The tongue of Franciscana (La Plata Dolphin), Pontoporia blainvillei 85not be observed in Pontoporia. These findings obtained from our Pontoporia were compared with those of the other two kinds of Platanistidae (Platanista and Inia) we observed.

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A serologic survey of Brucella infection was performed in Caspian seals (Pusa caspica, n=71), Baikal seals (P. sibirica, n=7), ringed seals (P. hispida hispida, n=6), and beluga whales (

, n=4) inhabiting Russian waters, by enzyme-linked immunosorbent assay (ELISA) using Brucella abortus and B. canis as antigens. The sera of 4 Caspian seals (4%) tested positive for B. abortus. The same sera samples demonstrated weaker yet detectable affinity for B. canis antigens. Several discrete bands against B. abortus and B. canis antigens were detected on Western blot analysis of the ELISA-positive seal sera; the bands against B. canis were weaker than those against B. abortus. The sera of 3 beluga whales (75%) were positive for B. abortus antigens but showed no binding to B. canis antigens in the ELISA. The positive whale sera showed a strong band appearance only against B. abortus antigens in the Western blot analysis. Many detected bands were discrete, while some of them had a smeared appearance. The present results indicate that Brucella infection occurred in Caspian seals and beluga whales inhabiting Russian waters, and that the Brucella strains infecting the seals and the whales were antigenetically distinct.

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ミナミバンドウイルカ（ミナミハンドウイルカ）Tursiops aduncusの成熟オス4個体を対象として，日長が大きく異なる冬至，春分，夏至に3時間間隔で24時間の連続採血を行い，血液学的検査7項目（冬至，春分，夏至）および血液生化学的検査17項目（冬至，夏至）の日内変動を調べた．ヘマトクリット（HT），尿素窒素（BUN），尿酸（UA），中性脂肪（TG），ヘモグロビン濃度（HGB），赤血球数（RBC），白血球数（WBC），総コレステロール（T-CHO），アルカリフォスファターゼ（ALP），カリウム（K）に日内リズムが認められ，このうちHT，BUN，UA，TGのリズムは特に明瞭であり（P＜0.01），HTは18時に低値を示し，BUN，UA，TGはいずれも夕方から夜間にかけて高値を示した．これらの日内リズムの頂点平均時刻は，いずれの季節においても日長とは無関係にほぼ同時刻であったことから，そのリズムは内因性の概日時計に制御されている可能性が示唆された．他の項目の日内変動は，不規則なパルス状（好酸球分画，Eos；アルブミン，Alb；グルタミン酸オキサロ酢酸トランスアミナーゼ，GOT；グルタミン酸ピルビン酸トランスアミナーゼ，GPT；総ビリルビン，T-Bil：クレアチンフォスフォキナーゼ，CPK；ナトリウム，Na；クロール，Cl），経時的上昇あるいは下降（クレアチニン，Cre；血糖，Glu），ほぼ不変（好中球分画，Neut；リンパ球分画，Lym；総タンパク，TP；乳酸脱水素酵素，LDH）に区別できた．

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Seven Longman’s beaked whales mass stranded in New Caledonia in November 2013, of which 4 ultimately died, in a first worldwide event reported for this poorly known Ziphioid species. Teeth were extracted, collected and thoroughly cleaned of gum tissue from 3 females ranging from juvenile to adult and one adult male. These were sectioned (crown-root) and prepared using two different methods and examined under microscope magnification when Growth Layer Groups (GLGs) in both dentine and cement were successfully identified. The methods employed for aging included 1) sectioning centrally at approx. 150 µm through crown and root on an Isomet circular diamond saw and examining under a microscope using both transmitted polarised light and plain light; and, 2) thick sectioning (wafering) at approx. 2.5 mm and subsequent decalcifiation in RDO™ (a proprietory brand, Illinois, USA) and then thin sectioning the wafer at 10–25 µm and staining with Ehrlich’s acid haematoxylin. GLGs were investigated in both dentine (25 micron) and cementum (10–15 µm). Layering was evident in both tissues but higher counts were more evident in thin stained sections of cementum. Although dentinal GLGs in untreated tooth sections have been used successfully for aging in Ziphioid species Hyperoodon ampullatus (Christensen 1973, Feyrer et al., 2020), it is believed this is the first time that teeth have been used for estimating age from GLGs in this tropical species.

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Dolphins in a captive environment are exposed to various kinds of stresses. Handling and transportation are stressful events for terrestrial mammals, and such stress may affect immune system function and increase susceptibility to infectious diseases. The same phenomenon could occur in dolphins, however, few studies have reported this in dolphins. The objective of this study was to evaluate the relationship between stress and polymorphonuclear (PMN) cell function of dolphins during transportation. Four bottlenose dolphins (Tursiops truncatus) were transported for 6 hr by truck. Serum cortisol levels, leukograms, phagocytosis, and superoxide production of PMN cells were evaluated during handling and transportation compared to resting values. The mean serum cortisol level was significantly increased during handling and transportation (p<0.05) when compared with the resting values. White blood cell (WBC) counts, eosinophil counts, phagocytosis, and superoxide production of PMN cells during handling and transportation stages decreased significantly in comparison with the resting stage (p<0.05). The concentration of serum cortisol was significantly correlated with the results of the WBC counts, eosinophil counts, superoxide production, and phagocytosis (p<0.01, p<0.05, p<0.05, and p<0.001, respectively). The present results indicate that handling and transportation are stressful events for dolphins and could affect their PMN cell functions, thereby leading to the impairment of the immune system.

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Legend to Table 1 should have appeared.

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