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. 2025 Jan 30;20(1):e0288891. doi: 10.1371/journal.pone.0288891

The first complete hand-rearing of two neonatal finless porpoises

Masahiko Kasamatsu 1, Kazuhiro Hasegawa 1, Ikuo Wakabayashi 1, Masami Furuta 1, Hiroki Inoue 2, Hidetomo Iwano 2,*
Editor: Ulrike Gertrud Munderloh3
PMCID: PMC11781668  PMID: 39883650

Abstract

Hand-rearing of marine mammals is an essential technique for the husbandry of orphans in captivity or the wild, especially endangered cetacean species. The purpose of the present study was to establish a method for successful hand-rearing and evaluate the nutritional state of neonatal finless porpoises. Two neonate finless porpoises maternally neglected at 5 days of age (Day 5) (neonate A, animal A) and Day 4 (neonate B, animal B) were hand reared. The amount of each tube feeding and daily number of nursings for animals A and B during the lactation period were gradually increased to 1,355 and 1,120 ml and 16 and 14 times, respectively. The mean daily caloric intake during the lactation period and average increase in body weight of animals A and B were 2,048 ± 207 and 2,206 ± 169 kcal and 65.4 and 66.9 g/day, respectively. Hypoproteinemia and hypertriglyceridemia were observed in the two neonates during the early stage of hand-rearing. The plasma concentrations of 24 free amino acids in the neonatal porpoises were significantly higher compared with adult porpoises. Plasma valine, leucine, and isoleucine levels in the neonates were dramatically higher than those in adults. Hyperlipoproteinemia, characterized by a higher percentage of very-low-density lipoprotein and the appearance of midband, was also observed in the two neonates, along with hypertriglyceridemia. A hand-rearing method for finless porpoises was successfully established in this research. Nutritional evaluation of serum protein, free amino acids, and lipid components is needed to improve the survivability of hand-reared neonatal porpoises. The hand-rearing method established in the present study is an essential technique for the husbandry of finless porpoises and can be applied to the conservation of other members of the porpoise family, including vaquita and Yangtze finless porpoises, which are the most endangered dolphins in the world.

Introduction

Hand-rearing of marine mammals is an essential technique for the husbandry of orphans in captivity or the wild and for their conservation, especially with regard to endangered cetacean species. However, hand-rearing of cetacean calves is difficult. The first recorded successful hand-rearing of an infant harbor porpoise (Phocoena phocoena) occurred at the Point Defiance Zoo and Aquarium, Washington, USA, and that of a bottlenose dolphin (Tursiops truncatus) took place at the Gulfarium, Florida, USA, in 1989 [1]. The difficulty of hand-rearing dolphins can be attributed to the large number of breast feedings necessary and the lack of information regarding artificial formulas.

Artificial formula that matches the physiological characteristics of dolphins, in conjunction with optimizing the amount of formula fed at each lactation and the daily number of nursings, are essential for the successful hand-rearing of dolphins. In order to develop an optimal artificial formula feed for dolphins, information regarding various marine mammal formula feeds that have been reported was organized. Two types of artificial formula have been reported and used in the hand-rearing of bottlenose dolphin, harbor porpoise, killer whale (Orcinus orca), pygmy sperm whale (Kogia breviceps), Risso’s dolphin (Grampus griseus), and spotted dolphin (Stenella attenuata) [1–3]. Safflower oil in the April formula and salmon oil in the Sea World formula have been used for the hand-rearing of dolphins, but whipping cream has only been used in the Sea World formula [1–3]. In these cases, dolphin artificial formulas have been supplemented with herring filets with viscera, oil (salmon oil or safflower oil), and heavy whipping cream to increase the caloric density [1–4]. Although the fat content of bottlenose dolphin milk is 26.5%, Townsend reported that the fat content of artificial milk does not need to match the high fat level of mother’s milk [1,2]. However, there is no indicator regarding how much lipid should be included in artificial milk. Townsend also described methods for the delivery of artificial formula to dolphin calves [1,2]. The optimal method for bottle feeding was for calves to suckle the artificial formula spontaneously from an artificial nipple. A tube feeding technique using a soft rubber stomach tube with a rounded tip was also useful for hand-rearing harbor porpoises.

Feeding frequency depends upon the caloric density of the formula, daily calorie requirements, and maximum volume of formula per feeding [2]. Combined knowledge regarding the approximate caloric density of the formula and the calorie requirements of calves allow calculation of the total daily volume of formula. Daily calorie requirements have been reported for several species of dolphins, harbor porpoises, and whales [2,3].

Hematology and serum chemistry techniques are essential tools for assessing the health status of dolphins [5]. Frequent blood examinations should be performed in neonates because hematological and biochemical values in dolphins are often the earliest indicators of subclinical disease [1]. Twice-weekly blood sampling and daily weighing of calves for 2 weeks are recommended until the calf is medically stable [2]. It is important to understand the physiological aspects of carbohydrate, protein, and lipid metabolism in dolphins for successful hand-rearing.

In the case of hand-rearing of dolphins, monitoring of serum protein, lipid components, and lipoproteins in calves is necessary for nutritional evaluation. Several recent reports have described methods for hand-rearing of dolphins, formula selection and preparation, artificial formula delivery, calorie requirements, and growth of neonates [1–3]. However, other than parameters pertaining to the formula, calorie requirements, and neonatal growth, little is known about analyses that could promote the success of hand-rearing of neonates from a few days after birth to the day of weaning. Few studies have focused on nutritional monitoring to determine health status or the normal growth of neonatal dolphins. Miyaji reported that although plasma amino acid concentrations vary under different physiological conditions, monitoring their concentrations could be a useful tool for assessing the health status of cetaceans [6].

The finless porpoise (Neophocaena asiaeorientalis) is a small member of the toothed whale family and is listed as an endangered species along with all other cetaceans [7,8]. The finless porpoise is distributed throughout tropical and subtropical coastal waters from the Persian Gulf to Japan [7], but it is threatened by habitat degradation, water pollution, and bycatch. Many porpoise orphans rescued from the wild have not survived over the last few decades because no suitable hand-rearing techniques have been established. The establishment of hand-rearing techniques for endangered dolphins, particularly the analysis of artificial formulas and nutritional evaluation of neonatal dolphins, is of paramount importance for the conservation of wild dolphins, including their ex situ conservation in aquariums. The purpose of the present study was to establish a method for successful hand-rearing of finless porpoises and evaluate the nutritional status of neonate porpoises.

Materials and methods

Ethics statement

The objective of the hand-rearing techniques assessed in this study was to enable the rescue of orphaned endangered porpoises, and ethical approval was granted by the director of Toba Aquarium as an emergency measure (permit M-07-2013). The husbandry and animal care protocol used in this research complied with the current laws of Japan, the country in which the study was performed. The study was conducted in accordance with the guidelines of the Japanese Society of Veterinary Science and Japanese Society of Zoo and Wildlife Medicine for the ethical use of animals in research. Ethical considerations and data collection methods were approved by the responsible curator and director of the Toba Aquarium. Procedures for animal husbandry, veterinary care, and welfare of the study species in the Toba Aquarium strictly adhered to JAZA (Japanese Association of Zoos and Aquariums) standards for the accommodation and care of animals in zoos and aquariums.

Animals

Two neonate finless porpoises were born at Toba Aquarium on May 2, 2013, and July 3, 2014. In the case of animal A, the first nursing was started 5 hours after birth. However, maternal neglect occurred at 5 days of age (Day 5). Tube feeding of animal A was started on Day 6. The mother and animal A were kept in the same exhibition pool until Day 6, and then animal A was removed from the mother and hand-reared from Day 7 onward in the medical pool. The body weight and body length of animal A on Day 5 were 9.83 kg and 76.5 cm, respectively. Animal A was force-fed whole fish beginning on Day 88 and then completely weaned by Day 136. The lactation period of animal A was between Days 6 and 87. The pre-weaning period was from the start of force-feeding of whole fish until the calf was weaned.

The first nursing of animal B was started 8 hours after birth. When physical and blood examination of animal B was performed on Day 4, swelling of the palatine mucosa of the maxilla and a comminuted fracture of the maxilla edge were observed. Animal B was removed from the mother and hand-reared from Day 4 in the medical pool. The body weight and body length of animal B on Day 4 were 9.76 kg and 88.5 cm, respectively. Animal B was force-fed beginning on Day 83 and completely weaned by Day 138.

Initial feeding and formula

We tube-fed the two neonates an artificial milk formula through a stomach tube inserted directly into the stomach. A 60-cm stomach tube and 50-ml syringe were used for tube feeding in this study.

The formula was composed of powdered milk substitute (Puppy milk replacers, Pet-AG, Inc., Hampshire, IL 60140), whipping cream, salmon oil, slurry of fish meat (fish paste), multi–amino acids (Amiparen, Otsuka Pharmaceutical Co., Ltd., Chiyoda-ku, Tokyo, Japan), and water (Table 1). Powdered milk substitute and water were mixed in a commercial blender, and then whipping cream, salmon oil, multi–amino acids, multi-vitamins, and lactoferrin (Morinaga Milk Industry Co., Ltd., Minato-ku, Tokyo, Japan) were added to the blended milk substitute right before tube feeding. The slurry of fish meat was made from strained, blended fish meat. Fish meat in which the bones and water had been removed were mixed to a uniform homogenate in a commercial blender and then strained trough a fine-mesh net to remove pieces of bones and scales. The slurry of fish meat was tube-fed to the neonates without mixing into the prepared milk substitute. Each neonate was supplemented with one-half of a multi-vitamin tablet (5M26 Vita-Zu tablet, Mazuri, IN, USA), a lactobacillus-based probiotic (half tablet, BID, Biofermin-R, Takeda Pharmaceutical Co., Ltd.), and 250 mg lactoferrin (SID) per day. Each multi-vitamin tablet contained retinol (10,000 IU), vitamin D (2000 IU), α-tocopherol (40 mg), acetomenaphthone (5 mg), thiamine (120 mg), riboflavin (8 mg), pyridoxin (5 mg), calcium pantothenate (10 mg), ascorbic acid (150 mg), nicotinamide (20 mg), cyanocobalamin (0.015 mg), choline (20 mg), inositol (5 mg), folic acid (1 mg), biotin (0.05 mg), and iron (12 mg).

Table 1. Ingredients in formula for hand-rearing of finless porpoises.

Milk components
Ingredients Initial Lactation Pre-weaning
Day 1―Day 10 Day 11―Day 82 Day 83―Day 137
Milk substitute 30―60 ml a 70―85 ml a 50―150 ml b
Heavy whipping cream 4 ml 4 ml 0―4 ml
Salmon oil 2―4 ml 2 ml 0―3 ml
Multi-amino acid c 0 ml 10 ml 0 ml
Fish meat components
Slurry of fish meat 0 ml 5―45 ml 0―150 ml
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Initial, the first ten days of hand-rearing; Lactation, neonates fed only artificial formula; Pre-weaning, neonates fed artificial formula and whole fishes.

a 100 g powdered milk (Puppy milk replacers, Pet-AG, Inc., 255 Keyes Avenue, Hampshire, Illinois 60140) added into 300 ml hot water.

b added into 400 ml hot water.

c Amiparen (Otsuka Pharmaceutical Co., Ltd., Chiyoda-ku, Tokyo, Japan) added during four and seven weeks of age.

Weaning procedures

The weaning process for animals A and B was started on Day 88 and Day 83, respectively. During the pre-weaning period, the daily number of nursings and quantity of total tube feeding were gradually reduced around Day 90. Animals A and B were force-fed silver-stripe round herring (Spratelloides gracilis) and Japanese jack mackerel (Trachurus japonicus) during the pre-weaning period. Animals A and B were completely weaned by Day 136 and Day 138, respectively, and the total pre-weaning period lasted 88 days and 55 days, respectively. The amount of daily diet was increased from 6 g to 2,024 g in animal A and from 6 g to 2,275 g in animal B during the pre-weaning period.

Blood samples

Blood samples were drawn into evacuated tubes from the tail fluke of the porpoises. Blood was collected into a serum separator tube (TERUMO, Tokyo, Japan) and an ethylenediamine tetraacetic acid tube (TERUMO, Tokyo, Japan) for serum chemistry and complete blood count analysis, respectively. Hematology and serum chemistry values were determined within 12 h. The blood samples from both animals were centrifuged at 1600 × g after coagulation, and the collected serum was immediately used for biochemical analysis.

Analysis

White blood cell count, red blood cell count, hemoglobin level, and hematocrit level were determined using an automated analyzer (CELL-DYN 3200, Abbott, IL, USA). The concentrations of serum chemistry analytes in neonatal samples were determined using a TBA™-c16000 automated analyzer (Toshiba Medical Systems, Tochigi, Japan) and included analyses for total protein, total cholesterol, triglycerides (TGs), phospholipids, free fatty acid (FFAs), glucose, and blood urea nitrogen [5]. Serum lipoproteins were analyzed by polyacrylamide gel electrophoresis using a lipoprotein test kit, as described previously [9,10].

The concentrations of amino acids in plasma were measured using high-performance liquid chromatography–electrospray ionization–mass spectrometry (LC-MS/MS), followed by precolumn derivatization according to the method of the EZ:faast amino acid testing kit (Phenomenex, CA, USA) [11]. LC-MS/MS was carried out using a Prominence high-performance liquid chromatography system with an MS8030 tandem mass spectrometer (Shimadzu, Kyoto, Japan). In the present study, we focused on 40 amino acids: arginine, glutamine, citrulline, anserine, serine, asparagine, prolyl-hydroxyproline, hydroxyproline, 3-methylhistidine (3-MH), 1-methylhistidine, glycine, glycyl-proline, threonine, beta-alanine, alanine, sarcosine, hydroxylysine, gamma-aminobutyric acid, beta-aminoisobutyric acid, alpha-aminobutyric acid, ornithine, carnosine, methionine, proline, lysine, aspartic acid, histidine, thioproline, valine, glutamic acid, tryptophan, alpha-aminoadipic acid, phenylalanine, leucine, isoleucine, aminopimelic acid, cystathionine, cysteine, tyrosine, and kynurenine.

Statistical analysis

The amount of tube feeding, daily calorie intake, hematological and serum biochemistry values, concentrations of serum lipid components, and plasma free amino acid concentrations in the two neonates are presented as mean ± SD. Differences in daily calorie intake and plasma free amino acid levels were analyzed using the Student’s t-test. The null hypothesis was rejected at P<0.05.

Results

To establish a method for successful hand-rearing, the two neglected neonate finless porpoises were hand-reared at 5 days (animal A) and 4 days (animal B) of age. The amount of each tube feeding and the daily number of nursings for animal A were gradually increased to 1,355 ml and 16 times, respectively, by Day 18 (Fig 1). During the lactation period from Days 19 to 87, the daily number of nursings for animal A was 13 or 14, and animal A gradually grew, consuming an average of 1,307 ± 52 ml of formula per day. Because frequent regurgitation was observed in animal A from Day 98, the amount of each tube feeding was increased, and the daily number of nursings was markedly decreased. On Day 100, endoscopy revealed mild esophagitis in the cardia of animal A. The daily number of nursings for animal A was gradually decreased during the pre-weaning period, and animal A was weaned on Day 135.

Fig 1. Amount of each lactation, daily number of nursings, and total daily volume of formula for two hand-reared neonatal porpoises.

Fig 1

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Upper panel shows data for animal A, and lower panel shows data for animal B. White data points indicate the amount of each lactation (vertical axis on the right side of the graph). Black data points indicate daily number of nursings (vertical axis on the left side of the graph). Bars indicate the total daily volume of formula (vertical axis on the left side of the graph).

The amount of each tube feeding and daily number of nursings for animal B were gradually increased to 1,120 ml and 14 times, respectively, by Day 24 (Fig 1). During the lactation period, the daily number of nursings for animal B was between 10 and 14, and animal B gradually grew, consuming an average of 1,193 ± 64 ml of formula per day from Days 25 to 82. The weaning process for animal B was started on Day 83, and the daily number of nursings was gradually decreased during the pre-weaning period. Animal B was weaned on Day 137.

The weights of animal A and animal B are plotted in Fig 2. Animals A and B grew from a Day 5 weight of 9.83 kg to 18.33 kg at weaning (Day 136) and from a Day 4 weight of 9.78 kg to 18.67 kg at weaning (Day 138), respectively, during the hand-rearing period. The average increase in body weight for animals A and B was 65.4 and 66.9 g/day, respectively. The average increase in body weight for the two neonate porpoises during the pre-weaning period was 38.2 g/day, which was lower than that during the lactation period (85.0 g/day) in this study.

Fig 2. Growth of two neonate porpoises during the hand-rearing period.

Fig 2

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Lactation, neonates fed only artificial formula; Pre-weaning, neonates fed artificial formula and whole fish. White data points show data for animal A, and gray data points show data for animal B. Arrowheads indicate the day the weaning process was started.

The daily calorie intake for animal A was gradually increased to 2,594 kcal over the first 14 days. The daily calorie intake for animal A was then decreased from Day 15 to Day 22 due to hyperlipidemia (Fig 3). The mean daily calorie intake for animal A was 2,048 ± 207 kcal during the lactation period from Days 23 to 87. On Day 88, animal A exhibited frequent regurgitation, and therefore, the number of nursings and daily calorie intake were decreased. The daily total calorie intake for animal A during the pre-weaning period was significantly (P<0.05) higher than that during the lactation period.

Fig 3. Daily calorie intake of two neonatal porpoises during the hand-rearing period.

Fig 3

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The upper panel shows data for animal A, and the lower panel shows data for animal B. White bars indicate the daily calorie intake of artificial formula, whereas black bars indicate the daily calorie intake of whole fishes. Lactation, neonates fed only artificial formula; Pre-weaning, neonates fed artificial formula and whole fish. (*) Hyperlipidemia; arrowhead indicates time frequent regurgitation was observed in animal A.

The daily calorie intake for animal B was gradually increased to 2,004 kcal over the first 21 days. The average daily calorie intake for animal B was 2,206 ± 169 kcal from Days 21 to 82 during the lactation period (Fig 3). The daily total calorie intake for animal B during the pre-weaning period was significantly (P<0.01) lower than that during the lactation period.

In this study, the serum concentrations of total protein, lipid and lipoprotein components, and 40 free amino acids were measured as indicators of nutritional status in the two hand-reared neonatal porpoises. Results of hematological and serum biochemistry analyses for the two hand-reared porpoises are shown in Table 2. Hypoproteinemia and hypertriglyceridemia were observed in the two neonate porpoises during the early stage of hand-rearing. The serum total protein concentration in animal A decreased from 6.3 g/dl on Day 5 to 4.5 g/dl on Day 18. Hypoproteinemia in animal A improved, and the serum protein concentration increased to 5.8 g/dl by Day 89. Hypertriglyceridemia was observed in animal A between Days 14 and 18. The number of total peripheral leukocytes in animal A increased markedly (14,833 ± 2,329/μl) between Days 75 and 89, when the animal had esophagitis.

Table 2. Results of hematological and serum biochemical analyses of two hand-reared neonate finless porpoises.

Neonate A Day Week Month HTG
Day 5 2 3 1 2 3 4 Day 14–18
 WBC ( / μl) 9200 7400 8900 8900 13200 17500 7100
 RBC (105 / μl) 576 587 578 564 469 491 504
 Hb (g / dl) 18.5 19.3 18.9 18.2 14.9 14.5 14.4
 Ht (%) 56.9 57.3 56.5 55.4 44.0 44.6 45.7
 TP (g / dl) 6.3 5.7 4.5 5.3 5.6 5.8 6.1
 Tcho mg / dl 157 271 208 195 173 152 213 250
 TG mg / dl 15 870 1027 574 362 300 256 1050
 PL mg / dl 189 401 344 ND 264 243 283 372
 FFA mEq / l 1325 7764 3510 ND 2043 4660 1355 6890
 Glucose mg / dl 127 128 102 119 100 66 132
 BUN mg / dl 37.1 52.8 56.6 64.0 62.5 54.5 81.7
Neonate B Day Week Month HTG
Day 4 2 3 1 2 3 4 Day 11–18
 WBC ( / μl) 6000 8400 5300 13400 13200 16000 17200
 RBC (105 / μl) 549 561 561 515 458 482 492
 Hb (g / dl) 20 20 19.7 18.0 14.2 15.0 14.5
 Ht (%) 60.4 60.6 58.4 52.5 42.9 44.6 43.8
 TP (g / dl) 6.0 5.4 5.2 5.5 6.5 6.4 6.7
 Tcho mg / dl 144 289 231 157 146 174 186 250
 TG mg / dl 60 732 477 161 184 569 159 604
 PL mg / dl 164 362 290 215 233 259 237 330
 FFA mEq / l 985 2958 7301 1701 2083 4063 1143 5129
 Glucose mg / dl 61 89 126 101 72 81 95
 BUN mg / dl 40.5 28.2 42.7 51.5 49.4 59.6 61.0
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WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; Ht, hematocrit; TP, serum total protein; Tcho, total cholesterol; TG, triglyceride; PL, phospholipid; FFA, free fatty acid; BUN, blood urea nitrogen; HTG, hypertriglyceridemia.

Hypoproteinemia was also observed in animal B between Days 11 and 18 and improved by Day 77. Hypertriglyceridemia was also observed in animal B between Days 11 and 18. The number of total peripheral leukocytes in animal B increased markedly after Day 32 (14,044 ± 2,213/μl).

The concentrations of 24 free amino acids in the plasma of the neonate porpoises were significantly (P<0.05) higher than those in adult porpoises, and the concentrations of 2 free amino acids were significantly (P<0.05) lower (Fig 4). The plasma 3-MH level in the neonates was 195.7 ± 74.0 nmol/ml, which was significantly (P<0.05) lower than that of adult porpoises. Plasma valine, leucine, and isoleucine levels in the neonates were 683.4 ± 184.8, 344.9 ± 103.4, and 252.6 ± 79.9 nmol/ml, respectively, which were significantly (P<0.05) higher than those in adult porpoises. The plasma branched-chain amino acid (BCAA) level in the neonates was 1280.8 ± 360.9 nmol/ml, which was three times higher than that of adult porpoises.

Fig 4. Plasma free amino acid concentrations in hand-reared neonatal and adult porpoises.

Fig 4

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ARG, arginine; GLN, glutamine; CIT, citrulline; ANS, anserine; SER, serine; ASN, asparagine; PHP, prolyl-hydroxyproline; HYP, hydroxyproline; 1-MH, 1-methylhistidine; 3-MH, 3-methylhistidine; GLY, glycine; GPR, glycyl-proline; THR, threonine; BALA, beta-alanine; ALA, alanine; SAR, sarcosine; HLY, hydroxylysine; GABA, gamma-amino butyric acid; BAIB, beta-aminoisobutyric acid; ABA, alpha-aminobutyric acid; ORN, ornithine; CAR, carnosine; MET, methionine; PRO, proline; LYS, lysine; ASP, aspartic acid; HIS, histidine; TPR, thioproline; VAL, valine; GLU, glutamic acid; TRP, tryptophan; AAA, alpha-aminoadipic acid; PHE, phenylalanine; LEU, leucine; ILE, isoleucine; APA, aminopimelic acid; CTH, cystathionine; CYS, cysteine; TYR, tyrosine; KNU, kynurenine. *Significant difference (P<0.05).

Hyperlipoproteinemia, characterized by a higher percentage of very-low-density lipoprotein (VLDL) and the appearance of midband, was also observed in the two neonates when they had hypertriglyceridemia (Fig 5). A significant correlation (P<0.01) was found between serum TG and serum FFA concentrations in the two neonates.

Fig 5. Densitometric scans of serum lipoproteins in hand-reared neonatal and adult porpoises determined by polyacrylamide gel electrophoresis.

Fig 5

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A, Hand-reared porpoises with hyperlipidemia; B, normal hand-reared porpoises; C, adult porpoises. Arrowhead, midband.

Discussion

This is the first report of successful complete hand-rearing of endangered finless porpoises. The highest mortality rate in ex situ neonatal finless porpoises typically occurs within the first 15 days following birth. The data obtained in this study are indispensable for determining the conditions under which artificial hand-rearing enables newborn porpoises to grow throughout the hand-rearing period. Lactation in dolphins is characterized by small amounts at each breastfeeding and a large number of daily lactations [4,12–14]. In this study, 9.7-kg and 9.8-kg newborn porpoises grew in the first week through 15 daily nursings comprising a total volume of 773.4 ± 190.4 ml (mean ± SD). In the case of animal A, the number of nursings was markedly reduced on Day 100 because reflux esophagitis was observed in the area near the cardia. It is possible that frequent tube feeding may damage the mucous membranes of the esophagus. The insertion of a stomach tube into the forestomach of a porpoise and the amount of each tube feeding should be closely monitored when tube feeding is used in artificial hand-rearing.

Analyses of neonatal growth in this study showed that the weight gain of the two calves was not consistent throughout the hand-rearing period. The rate of weight gain declined between 3 and 4 months in the two hand-reared calves. In the case of hand-reared bottlenose dolphins, calf weight gain was shown to increase after 2 months of age and to decrease after 4 to 7 months [4,12]. The day when weight gain declined in the porpoise calves was earlier than that reported for bottlenose dolphins. Porpoise calves begin to eat whole fish spontaneously at approximately 90 days of age (unpublished data). A reduction in the growth rate of captive bottlenose dolphin calves would suggest weaning serves as a response to the increase in energy requirement that the calf could not fulfil through nursing [12,15]. The day when the weight gain of calves declined was similar to the day when naturally nursed porpoise calves begin to spontaneously eat whole fish. The tendency for weight gain at approximately 13 weeks of age suggests that the weaning process begins at that age in porpoise calves.

In this study, the relationship between weight gain and daily calorie intake were examined in hand-reared porpoise calves. Daily calorie requirements in hand-reared bottlenose dolphins and harbor porpoises are reportedly 150 and 200 kcal/kg/day, respectively [2,3]. The mean daily calorie requirement in animals A and B in the present study during the lactation period was 155.9 ± 35.6 and 159.9 ± 20.1 kcal/kg/day, respectively, similar to that of hand-reared bottlenose dolphins. The daily calorie intake in the two calves did not differ between the lactation period and pre-weaning period. Also, the growth rate of the calves in the pre-weaning period was lower than that in the lactation period.

Weight gain reportedly begins to decline at approximately 4 months of age in bottlenose dolphin calves that have started eating whole fish [4]. It is thought that porpoise calves need to grow sufficiently before they can spontaneously catch and eat fish. In addition, it is assumed that the growth rate of calves starts to slow at approximately 13 weeks of age when the weaning process begins.

The highest rate of mortality in bottlenose dolphin calves reportedly occurs within the first 30 days following birth [16]. There are only a few reported cases of successful hand-rearing and only limited clinicopathological data available regarding neonatal cetaceans [4,16]. The improvement in survivability can be attributed to better neonatal and maternal monitoring and intervention husbandry techniques [4,16]. Few reports have described complete hand-rearing of cetacean neonates beginning immediately from birth [4,17–19]. Data regarding serum biochemistry parameters assessing nutritional status in neonatal dolphins are needed. Hematology and serum chemistry analyses are essential for assessing health status of not only mother porpoises but also neonates in order to improve the survivability of calves [5].

Hypoproteinemia and hypertriglyceridemia were observed in the neonatal porpoises at between 2 and 3 weeks. These characteristics have not been reported in the limited number of cases of hand-rearing of bottlenose dolphins [4,16]. Decreasing serum protein levels are generally observed with malnutrition, along with protein-losing nephropathies and gastrointestinal diseases such as parasitism and protein-losing enteropathies [20]. Hypoproteinemia in marine mammals has been reported in a few limited cases involving rescued harbor seal (Phoca vitulina) pups, consistent with malnourishment, and in the case of a rehabilitated bottlenose dolphin [21,22]. In our previous research, we found severe hypoproteinemia in a Commerson’s dolphin (Cephalorhynchus commersonii) with protein-losing enteropathy [23]. However, it is unlikely that the hypoproteinemia in the neonatal porpoises in the present study was related to small intestinal or renal protein-losing disease. It has been reported that the protein content of dolphin milk is higher than that of other terrestrial mammals, but the reason for this has not been explained [24]. Hypoproteinemia in newborn porpoises suggests that the source of protein needed for neonatal growth has been lacking.

Adequate dietary intake of amino acids is necessary for the growth, development, and health of animals both in situ and ex situ; thus, more information regarding diets and nutrient profiles of these animals is needed [25]. There are few reports concerning amino acids and their functions in dolphins [6,25,26]. The plasma concentrations of 24 free amino acids in the neonates of the present study were significantly higher than those in adult porpoises. Previous studies have demonstrated that skeletal muscle proteolysis increases in response to energy deficits resulting from malnutrition. Plasma 3-MH levels increase with skeletal muscle proteolysis; thus, 3-MH is generally used as a marker of muscular proteolysis [27,28]. Although there was a decrease in weight gain at approximately 13 weeks of age, the lower plasma 3-MH levels in the neonates compared with adult porpoises indicated that no energy deficit had occurred in the porpoise calves. The temporary hypoproteinemia observed early in the hand-rearing of neonatal porpoises that was not accompanied by significant clinical symptoms is interesting. Serum total protein consists mainly of albumin synthesized in the liver. In rapidly growing newborns, liver function might be overloaded, which in turn can affect protein synthesis. Neonates require a specific balance of amino acids based on growth stage, and amino acid requirements differ between neonatal and adult pigs, for instance [29,30]. Protein synthesis and muscle growth can be stimulated in neonatal pigs by providing amino acids with anabolic properties [29]. These results suggest that neonatal porpoises require protein and supplementation with certain appropriate amino acids in the early stages of hand-rearing. Supporting early muscle growth with an adequate supply of amino acids may be essential for proper growth of neonatal porpoises. We believe that this is an important finding for future case studies and could lead to the development of an ideal artificial milk.

It is worth noting that the plasma BCAA levels of the neonates were 3 times higher than those of adults. Muscle and plasma concentrations of BCAAs are generally elevated by increased physical activity. There is a significant increase in the rate of amino acid catabolism during exercise [31]. BCAAs have been implicated as being involved in energy supply processes in muscle after their release from the liver [32]. It has also been reported that BCAAs are preferentially metabolized by skeletal muscle and that BCAA metabolism in skeletal muscle increases during energy-demanding situations [33]. Increasing BCAA levels during energy restriction can support gluconeogenesis, maintain whole-body and muscle protein synthesis, and attenuate whole-body and muscle proteolysis [33]. Neonatal porpoises must swim to follow their dam immediately after birth. It is possible that skeletal muscle in newborn porpoises effectively utilizes BCAAs during the early stage of the lactation period. The present study suggests that BCAAs are required for the growth of newborn porpoises.

Hypertriglyceridemia and hypoproteinemia were observed in the neonatal porpoises at between 2 and 3 weeks. It is generally accepted that the fat content of dolphin milk collected from the dam is 26.5%, higher than that of many other mammals [2,34,35]. It is necessary to increase the fat content of artificial formula by adding salmon oil or safflower oil [2,3]. However, the oils are not added to artificial formula to the same concentration as in mother’s milk [2]. The fat content of artificial formula is generally 9.0–9.5% [2,3].

Monitoring of serum lipid components and lipoproteins is important for proper nutritional husbandry of captive dolphins [10]. The basis for the addition of oil to an artificial formula and for evaluating neonatal serum lipids have not been established for hand-reared dolphins. Hypertriglyceridemia in bottlenose dolphins has been linked to liver diseases [36]. In this study, midband lipoprotein was observed in serum from the neonates, which developed hypertriglyceridemia in the early stage of hand-rearing. Midband lipoproteins arise from the intermediate products of VLDL catabolism and rarely appear in normal blood [10,37,38]. Hypertriglyceridemia reportedly occurs in human neonatal diabetes and in equine neonates [39,40]. The appearance of midband in blood samples from the hand-reared neonate porpoises with hypertriglyceridemia in the present study suggests the presence of a lipid metabolism disorder. In humans, midband lipoproteins in patients with hyperlipidemia are regarded as indicators of abnormal lipoprotein metabolism in relation to atherosclerosis and cerebrovascular diseases [37,38]. Prolonged hypertriglyceridemia during hand-rearing can cause fatty liver changes and lipid metabolism disorders in neonatal porpoises. These data suggest it is necessary to assess serum protein, free amino acids, lipids, and lipoprotein levels and to feed artificial milk formula to newborn porpoises that is appropriate for their growth stage.

Conclusion

A method for hand-rearing managed finless porpoises was successfully established in this study. The daily number and amount of nursings and details regarding formula preparation were determined in this case of complete hand-rearing of porpoises. Given the observed hyperlipidemia in the early stages of porpoise hand-rearing, it is possible that there is not a high demand for lipid supplementation in the form of fish oil. The results of this study suggest that neonatal porpoises have a high demand for amino acids, and we found that we needed to add BCAAs to artificial formula for the neonatal porpoises. Nutritional evaluations of serum protein, free amino acids, and lipid components are needed to further improve the survivability of hand-reared neonatal finless porpoises.

The hand-rearing method established in the present study is an essential technique for the husbandry of finless porpoises and can be applied to the conservation of other members of the porpoise family, including vaquita and Yangtze finless porpoises, which are the most endangered dolphins in the world.

Supporting information

S1 File

(XLSX)

pone.0288891.s001.xlsx (54.1KB, xlsx)

Acknowledgments

We would like to thank the staff of Toba Aquarium who participated in the hand-rearing of two neonatal finless porpoises. Additionally, we would like to express our gratitude to Atsushi Seko, Yoshihiro Ishihara, and Yoshihito Wakai for professional advice regarding our research.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The authors declare that no specific funding was received for this work.

References

  • 1.Townsend FI. Hand-rearing techniques for neonate cetaceans. In: Fowler ME, Miller ER, editors. Zoo and Wild Animal Medicine Current Therapy 4th Edition. Philadelphia: WB Saunders Company; 1999. pp. 493–497. [Google Scholar]
  • 2.Townsend FI, Gage LJ. Hand rearing and artificial milk formulas. In: Dierauf LA, Gulland FMD, editors. CRC Handbook of Marine Mammal Medicine. 2nd Edition. Boca Raton, Florida: CRC Press; 2001. pp. 829–849. [Google Scholar]
  • 3.Gage LJ, Walsh MT. Hand-rearing and artificial milk formulas. In: Gulland FMD, Dierauf LA, Whitman KL, editors. CRC Handbook of Marine Mammal Medicine 3rd Edition. Boca Raton, Florida: CRC Press; 2018. pp. 739–755. [Google Scholar]
  • 4.Flower JE, Langan JN, Nevitt BN, Chinnadurai SK, Stacey R, Ivančić M, et al. Neonatal critical care and hand-rearing of a bottlenose dolphin (Tursiops truncatus) calf. Aquat Mamm. 2018; 44: 482–490. doi: 10.1578/AM.44.5.2018.482 [DOI] [Google Scholar]
  • 5.Kasamatsu M, Hasegawa K, Wakabayashi I, Seko A, Furuta M. Hematology and serum biochemistry values in five captive finless porpoises (Neophocaena phocaenoides). J Vet Med Sci. 2012; 74: 1319–1322. doi: 10.1292/jvms.11-0407 . [DOI] [PubMed] [Google Scholar]
  • 6.Miyaji K, Nagao K, Bannai M, Asakawa H, Kohyama K, Ohtsu D, et al. Characteristic metabolism of free amino acids in cetacean plasma: cluster analysis and comparison with mice. PLoS ONE. 2010; 5: e13808. doi: 10.1371/journal.pone.0013808 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Kasuya T. Finless porpoise Neophocaena phocaenoides (G. Cuvier, 1829). In: Ridgway SH, Harison R, editors. Handbook of Marine Mammals: The Second Book of Dolphins and The Porpoises. London, UK: Academic Press; 1999. pp. 411–442. [Google Scholar]
  • 8.Reeves RR, Leatherwood S. Dolphins, Porpoises and Whales. In: An 1994–1998 Action Plan for The Conservation of Cetaceans. Gland: IUCN; 1994.
  • 9.Muniz N. Measurement of plasma lipoproteins by electrophoresis on polyacrylamide gel. Clin Chem. 1977; 23: 1826–1833. doi: 10.1093/clinchem/23.10.1826 [DOI] [PubMed] [Google Scholar]
  • 10.Kasamatsu M, Kawauchi R, Tsunokawa M, Ueda K, Uchida E, Oikawa S, et al. Comparison of serum lipid compositions, lipid peroxide, α-tocopherol and lipoproteins in captive marine mammals (bottlenose dolphins, spotted seals and West Indian manatees) and terrestrial mammals. Res Vet Sci. 2009; 86: 216–222. doi: 10.1016/j.rvsc.2008.07.006 . [DOI] [PubMed] [Google Scholar]
  • 11.Šimek, P, Hušek, P. LC/MS and GC/MS amino acid/peptide profiling in human body fluids using a simple sample pretreatment. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics; 2002 Jun 2–6; Orlando, USA. A021599.
  • 12.Peddemors VM, Fothergill M, Cockcroft VG. Feeding and growth in a captive-born bottlenose dolphin Tursiops truncatus. S Afr J Zool. 1992; 27: 74–80. doi: 10.1080/02541858.1992.11448265 [DOI] [Google Scholar]
  • 13.Triossi F, Pace DS, Terranova ML, Renzi P. The development of suckling behavior in two captive-born calves of bottlenose dolphins (Tursiops truncatus). Aquat Mamm. 1998; 24: 75–83. [Google Scholar]
  • 14.Jacobsen TB, Mayntz M, Amundin M. Splitting suckling data of bottlenose dolphin (Tursiops truncatus) neonates in human care into suckling bouts. Zoo Biol. 2003; 22: 477–488. doi: 10.1002/zoo.10094 [DOI] [Google Scholar]
  • 15.Cockroft VG, Ross GJG. Observation on the early development of a captive bottlenose dolphin calf. In: Leatherwood S, Reeves RR, editors. The Bottlenose Dolphin. San Diego, California: Academic Press; 1990. pp. 461–478. [Google Scholar]
  • 16.Sweeney J, Stone R, Campbell M, McBain J, St. Leger J, Xitco M, et al. Comparative survivability of Tursiops neonates from three U.S. institutions for the decades 1990–1999 and 2000–2009. Aquat Mamm. 2010; 36: 248–261. doi: 10.1578/AM.36.3.2010.248 [DOI] [Google Scholar]
  • 17.McKenna LP, Campbell ME. Care and handling of neonate bottlenose dolphins (Tursiops truncatus): A clinical and behavioral approach. Soundings. 2012; 37: 27–31. [Google Scholar]
  • 18.Ramirez K. Training and care of an orphaned beluga whale calf. Soundings. 2001; 26: 24–27. [Google Scholar]
  • 19.Winhall WR. Hand raising and conditioning of neonate beluga whale (Delphinapterus leucas). Soundings. 2012; 37: 1–4. [Google Scholar]
  • 20.Bossart GD, Reidarson TH, Dierauf LA, Duffield DA. Clinical pathology. In: Gulland FMD, Dierauf LA, editors. CRC Handbook of Marine Mammal Medicine 2nd Edition. Boca Raton, Florida: CRC Press; 2001. pp. 383–436. [Google Scholar]
  • 21.Esson D, Nollens H, Schmitt T, Fritz K, Simeone C, Stewart B. Aphakic phacoemulsification and automated anterior vitrectomy, and postreturn monitoring of a rehabilitated Harbor seal (Phoca vitulina Richardsi) pup. J Zoo Wildl Med. 2015; 46: 647–651. doi: 10.1638/2014-0245.1 . [DOI] [PubMed] [Google Scholar]
  • 22.Bruni P, Stanzani LA, Caniglia G, Ubaldi A. Evolution of clinical signs in a baby dolphin (Tursiops truncatus) found in a river. Aquat Mamm. 1990; 16: 23–27. [Google Scholar]
  • 23.Kasamatsu M, Wakabayashi I, Seko A, Hasegawa K. A case report of protein-losing enteropathy in a captive Commerson’s dolphin. Proceedings of the Annual Meeting of Japanese Society of Zoo and Wildlife Medicine; 2010 Sep 1–4; Fukuoka, Japan.
  • 24.West KL, Oftedal OT, Carpenter JR, Krames BJ, Campbell M, Sweeney JC. Effect of lactation stage and concurrent pregnancy on milk composition in the bottlenose dolphin. J Zool. 2007; 273: 148–160. doi: 10.1111/j.1469-7998.2007.00309.x . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Herring CM, Bazer FW, Wu G. Amino acid nutrition for optimum growth, development, reproduction, and health of zoo animals. In: Crusio WE, Dong H, Radeke HH, Rezaei N, Steinlein O, Xiao JX, editors. Advances in Experimental Medicine and Biology. Switzerland: Springer; 2021. pp. 233–253. [DOI] [PubMed] [Google Scholar]
  • 26.Walsh MT, Quinton RR. Taurine levels in cetaceans, a preliminary investigation. Proceedings of the International Association for Aquatic Animal Medicine; 1995; Mystic, CT, USA.
  • 27.Pasiakos S, Carbone J. Assessment of skeletal muscle proteolysis and the regulatory response to nutrition and exercise. IUBMB Life. 2014; 66: 478–484. doi: 10.1002/iub.1291 . [DOI] [PubMed] [Google Scholar]
  • 28.Holeček M. Histidine in health and disease: metabolism, physiological importance, and use as a supplement. Nutrients. 2020; 12: 848. doi: 10.3390/nu12030848 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Posey EA, Davis TA. Review: Nutritional regulation of muscle growth in neonatal swine. Animal. 2023; 17 Suppl 3: 100831. doi: 10.1016/j.animal.2023.100831 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.O’Connor PMJ, Bush JA, Suryawan A, Nguyen HV, Davis TA. Insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonatal pigs. Am J Physiol Endocrinol Metab. 2003; 284: E110–E119. doi: 10.1152/ajpendo.00326.2002 . [DOI] [PubMed] [Google Scholar]
  • 31.Henriksson J. Effect of exercise on amino acid concentrations in skeletal muscle and plasma. J Exp Biol. 1991; 160: 149–165. doi: 10.1242/jeb.160.1.149 . [DOI] [PubMed] [Google Scholar]
  • 32.Moyec LL, Robert C, Triba MN, Billat VL, Mata X, Schibler L, et al. Protein catabolism and high lipid metabolism associated with long-distance exercise are revealed by plasma NMR metabolomics in endurance horses. PLoS ONE. 2014; 9: e90730. doi: 10.1371/journal.pone.0090730 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Carbone JW, McClung J, Pasiakos S. Skeletal muscle responses to negative energy balance: Effects of dietary protein. Adv Nutr. 2012; 3: 119–126. doi: 10.3945/an.111.001792 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ridgway S, Kamolnick T, Reddy M, Curry C, Tarpley RJ. Orphan-induced lactation in Tursiops and analysis of collected milk. Mar Mammal Sci. 1995; 11: 172–182. doi: 10.1111/j.1748-7692.1995.tb00516.x [DOI] [Google Scholar]
  • 35.Pervaiz S, Brew K. Composition of the milks of the bottlenose dolphin (Tursiops trucatus) and the Florida manatee (Trichechus manatus latirostris). Comp Biochem Physiol A Comp Physiol. 1986; 84: 357–360. doi: 10.1016/0300-9629(86)90629-8 . [DOI] [PubMed] [Google Scholar]
  • 36.Venn-Watson S, Smith CR, Jensen ED. Assessment of increased serum aminotransferases in a managed Atlantic bottlenose dolphin (Tursiops truncatus) population. J Wildl Dis. 2008; 44: 318–330. doi: 10.7589/0090-3558-44.2.318 . [DOI] [PubMed] [Google Scholar]
  • 37.Miwa K, Nakagawa K, Suzuki K, Houe H. Detection of the "midband" lipoprotein in patients with coronary artery spasm. Clin Cardiol. 2001; 24: 219–224. doi: 10.1002/clc.4960240309 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kawano M, Shinomiya M, Kanzaki T, Morisaki N, Shirai K, Saito Y, et al. An atherogenic midband lipoprotein: a risk factor for coronary artery disease in diabetes mellitus with hyperlipidemia. Diabetes Res Clin Pract. 1998; 41: 45–48. doi: 10.1016/s0168-8227(98)00057-6 . [DOI] [PubMed] [Google Scholar]
  • 39.Rajadhyaksha SB, Agarwal N, Desai LS. Neonatal diabetes mellitus with severe hypertriglyceridemia. J Pediatr Endocrinol Metab. 1996; 9: 435–437. doi: 10.1515/jpem.1996.9.3.435 . [DOI] [PubMed] [Google Scholar]
  • 40.Waitt LH, Cebra CK. Characterization of hypertriglyceridemia and response to treatment with insulin in horses, ponies, and donkeys: 44 cases (1995–2005). J Am Vet Med Assoc. 2009; 234: 915–919. doi: 10.2460/javma.234.7.915 . [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Ulrike Gertrud Munderloh

13 Dec 2023

PONE-D-23-20946The first complete hand-rearing of two neonatal finless porpoisesPLOS ONE

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Reviewer #1: This paper reported two cases of successful hand-rearing finless porpoise, which provides invaluable technique, information and data on rescuing the orphans of small cetacean species. This paper mainly provided raw data of their work only with t-test for difference analysis. However, it might be more valubale and helpful for other researchers. So I sugges to further improve the statistical analysis for a better quaility of this work. This paper provided the detailed and supportive data and information on hand-rearing the two finless porpoise babies, which include the formula, the way of hand-rearing, the daily intake, and body weight and blood information of the animals, etc. The process of hand-rearing and nutrition status of the animals were all clearly presented in this paper. But the language needs to be further improved by native speakers.

Reviewer #2: This manuscript provides incredibly valuable data about the hand rearing process for porpoise calves, which as the authors say can be extrapolated to endangered species conservation. I commend the authors for sharing as much data and information as they have. Several comments to consider:

-Lines 23 and 108: consider clarifying "neglect" with "maternal neglect"

-Throughout: consider replacing "force-feeding" with "gavage" or "tube feeding".

-Throughout: consider replacing "lactations" with "nursings" or "feedings", as lactations implies the production by the dam as opposed to feeding by calf.

-L 44-45: consider adding locations for aquaria (not sure where Point Aquarium is).

-L 52: two types of formula are listed, but not discussed or detailed further (the differences between the two).

-L 83: This sentence doesn't provide a whole lot of value since all cetaceans are listed. Consider rephrasing as "N asiaorientalis is listed as endangered..."

-L 92: Given that a main purpose of the study is to evaluate the nutritional status, consider strengthening conclusions about what the nutritional status of these two animals is based on the data collected.

-L 108: consider replacing "breastfeeding" with "nursing".

-L 112: I understand that 'neonate A' changes to 'calf A' as the animal ages into that age class, but it may be unclear to readers that this is the same animal; consider changing to 'animal A/B'.

-L 130: The fact that the slurry of fish meat was fed separately from formula is an important detail, and Table 1 may benefit from separating the slurry line out for clarity.

-Table 1: Consider clarifying "Initial" and "Lactation" as those words don't quite capture the differences between the two (perhaps "Day 1-10" and "Day 11-X", etc.)

-L 155: Consider adding total number of days of complete weaning process (I assume 136-88).

-L 199: confirming vomiting (versus regurgitation)

-L 253-255 and 258-260: listing the mean concentrations of all of these parameters is dense and confusing; consider adding column to Table 2 listing mean values there.

-L 301: unsure why 'grow in the first week' is there; this information appears valuable throughout the entire hand-rearing process; recommend clarification.

-L 310 and 312: "13 and 17 weeks" and "2 months and 4-7 months" - consider standardizing units (weeks or months) for clarity.

-L 352: would argue that this is not just necessary for zoo animals, but all animals (including in situ conservation).

-L 355: the authors have not adequately described why higher plasma amino acid concentrations are associated with hypoproteinemia. Also, is there information to cite that this is an unexpected finding for neonates versus adults (e.g. not formula-raised; is this pathologic, or expected)?

-L 362: Apologies, as above, it is not clear why reduced protein synthesis would occur if amino acid levels were elevated.

-L 382: "bases" - I believe this might be "basis".

-L 388: as above, are there citations that suggest that this is a pathologic metabolism disorder based on what is known in other neonate species? E.g. that hypertriglyceridemia is not expected in nursing calves of the same age?

-L 391: citation needed for "fatty liver changes and lipid metabolism disorders in neonatal porpoises"

-L 399: same as with L 355 above: please explain why hypoproteinemia was associated with elevated amino acid levels in the porpoises.

**********

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Reviewer #1: No

Reviewer #2: Yes: Claire A Simeone

**********

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Attachment

Submitted filename: PONE-D-23-20946-Reviewer1.docx

pone.0288891.s002.docx (15.3KB, docx)
PLoS One. 2025 Jan 30;20(1):e0288891. doi: 10.1371/journal.pone.0288891.r002

Author response to Decision Letter 0

10 Mar 2024

Reviewer #1

This paper reported two cases of successful hand-rearing finless porpoise, which provides invaluable technique, information and data on rescuing the orphans of small cetacean species. This paper mainly provided raw data of their work only with t-test for difference analysis. However, it might be more valubale and helpful for other researchers. So I sugges to further improve the statistical analysis for a better quaility of this work. This paper provided the detailed and supportive data and information on hand-rearing the two finless porpoise babies, which include the formula, the way of hand-rearing, the daily intake, and body weight and blood information of the animals, etc. The process of hand-rearing and nutrition status of the animals were all clearly presented in this paper. But the language needs to be further improved by native speakers.

In response to your kind comments and constructive suggestions, we would like to express our sincere appreciation. Your thorough examination of our research and your valuable suggestions are highly appreciated.

I understand and acknowledge the significance of your suggestion regarding the statistical analysis of our research. But it is difficult to obtain additional hand-rearing data sets from neonatal porpoises because the breeding and hand-rearing of porpoises are extremely rare. We would like to increase the number of cases in the future and verify the data as statistically meaningful. Even though the number of cases in this study is small, we consider it important that this report will be valuable in the conservation of endangered animals, and we would like to deliver it to researchers around the world as soon as possible.

Our manuscript has been edited in English by native speakers again.

Thank you again to the reviewers for their comments and constructive advice. We hope that the changes that have been made to the manuscript meet to your satisfaction.

We wish to take this opportunity to thank you for your consideration of our paper for publication in your journal, PLOS ONE.

Reviewer #2

In response to your kind comments and constructive suggestions, we would like to express our sincere appreciation. Your thorough examination of our research and your valuable suggestions are highly appreciated.

Lines 23 and 108: consider clarifying "neglect" with "maternal neglect".

→"neglect" have been changed to "maternal neglect" as suggested (revised manuscript (Rev. M.), line 23 and line 111).

Throughout: consider replacing "force-feeding" with "gavage" or "tube feeding".

→"force-feeding" has been changed to "tube feeding" as suggested (Rev. M., throughout the manuscript).

Throughout: consider replacing "lactations" with "nursings" or "feedings", as lactations implies the production by the dam as opposed to feeding by calf.

→"lactations" have been changed to "nursings" as suggested (Rev. M., throughout the manuscript).

L 44–45: consider adding locations for aquaria (not sure where Point Aquarium is).

→Locations for aquaria have been added as suggested (Rev. M., line 44–45).

L 52: two types of formula are listed, but not discussed or detailed further (the differences between the two).

→The additional information regarding two types of formula has been added as follow: “Safflower oil in the April formula and salmon oil in the Sea World formula have been used for the hand-rearing of dolphins, but whipping cream has only been used in the Sea World formula [1,2]. (Rev. M., line 54).

L 83: This sentence doesn't provide a whole lot of value since all cetaceans are listed. Consider rephrasing as "N asiaorientalis is listed as endangered...".

→This sentence has been rephrased as “is listed as endangered species along with all other cetaceans” as suggested (Rev. M., line 87).

L 92: Given that a main purpose of the study is to evaluate the nutritional status, consider strengthening conclusions about what the nutritional status of these two animals is based on the data collected.

→The sentence strengthening conclusions has been added as suggested (Rev. M., line 411). As follow: “Given the observed hyperlipidemia in the early stages of porpoise hand-rearing, it is possible that there is not a high demand for lipid supplementation in the form of fish oil. The results of this study suggest that neonatal porpoises have a high demand for amino acids, and we found that we needed to add BCAAs to artificial formula for the neonatal porpoises.”

L 108: consider replacing "breastfeeding" with "nursing".

→"breastfeeding" has been changed to "nursing" as suggested (Rev. M., line 112 and 119).

L 112: I understand that 'neonate A' changes to 'calf A' as the animal ages into that age class, but it may be unclear to readers that this is the same animal; consider changing to 'animal A/B'.

→“neonate A”and “neonateB” have been changed to “animal A” and “animal B”, respectively as suggested (Rev. M., throughout the manuscript).

L 130: The fact that the slurry of fish meat was fed separately from formula is an important detail, and Table 1 may benefit from separating the slurry line out for clarity.

→Table 1 has been rearranged as suggested (Rev. M., line 145).

Table 1: Consider clarifying "Initial" and "Lactation" as those words don't quite capture the differences between the two (perhaps "Day 1-10" and "Day 11-X", etc.).

→The specific number of days have been added in Table 1 as suggested (Rev. M., line 145).

L 155: Consider adding total number of days of complete weaning process (I assume 136-88).

→Total number of days of the complete weaning process were have been added as suggested (Rev. M., line 158).

L 199: confirming vomiting (versus regurgitation).

→“vomiting” has been changed to “regurgitation” as suggested (Rev. M., line 202, line 234 and line 247).

L 253-255 and 258-260: listing the mean concentrations of all of these parameters is dense and confusing; consider adding column to Table 2 listing mean values there.

→The mean concentrations of all of these parameters have been deleted and the mean values have been listed in Table 2 (Rev. M., line 261).

L 301: unsure why 'grow in the first week' is there; this information appears valuable throughout the entire hand-rearing process; recommend clarification.

→We agree with the comments from Reviewer #2. The sentences have been added as follows: “The highest mortality rate in ex situ neonatal finless porpoises typically occurs within the first 15 days following birth. The data obtained in this study are indispensable for determining the conditions under which artificial hand-rearing enables newborn porpoises to grow throughout the hand-rearing period.” (Rev. M., line 300).

L 310 and 312: "13 and 17 weeks" and "2 months and 4-7 months" - consider standardizing units (weeks or months) for clarity.

→"13 and 17 weeks" have been changed to "3 and 4 months" as suggested (Rev. M., line 312).

L 352: would argue that this is not just necessary for zoo animals, but all animals (including in situ conservation).

→“of zoo animals” has been changed to “of animals both in situ and ex situ” as suggested (Rev. M., line 355).

L 355, L 362: the authors have not adequately described why higher plasma amino acid concentrations are associated with hypoproteinemia. Also, is there information to cite that this is an unexpected finding for neonates versus adults (e.g. not formula-raised; is this pathologic, or expected)? Apologies, as above, it is not clear why reduced protein synthesis would occur if amino acid levels were elevated.

→We would like to discuss hypoproteinemia and plasma amino acid profiles in neonatal porpoises separately.

The sentences have been added as follows: “The temporary hypoproteinemia observed early in the hand-rearing of neonatal porpoises that was not accompanied by significant clinical symptoms is interesting. Serum total protein consists mainly of albumin synthesized in the liver. In rapidly growing newborns, liver function might be overloaded, which in turn can affect protein synthesis. Neonates require a specific balance of amino acids based on growth stage, and amino acid requirements differ between neonatal and adult pigs, for instance [29,30]. Protein synthesis and muscle growth can be stimulated in neonatal pigs by providing amino acids with anabolic properties [29]. These results suggest that neonatal porpoises require protein and supplementation with certain appropriate amino acids in the early stages of hand-rearing. Supporting early muscle growth with an adequate supply of amino acids may be essential for proper growth of neonatal porpoises. We believe that this is an important finding for future case studies and could lead to the development of an ideal artificial milk. (Rev. M., line 364).

L 382: "bases" - I believe this might be "basis".

→"bases" has been revised to “basis” following your comments (Rev. M., line 393).

L 388: as above, are there citations that suggest that this is a pathologic metabolism disorder based on what is known in other neonate species? E.g. that hypertriglyceridemia is not expected in nursing calves of the same age?

→Yes, the hypertriglyceridemia has not been found in sera from nursing calves of dolphins. Hypertriglyceridemia has been reported to occur in human neonatal diabetes and in equine neonates (Rev. M., line 398).

L 391: citation needed for "fatty liver changes and lipid metabolism disorders in neonatal porpoises".

→The "lipid metabolism disorders" and related citation as suggested have been additionally described from L392 to L403.

L 399: same as with L 355 above: please explain why hypoproteinemia was associated with elevated amino acid levels in the porpoises.

→“Conclusion” section has been revised as suggested and the sentence has been added as follows: “The results of this study suggest that neonatal porpoises have a high demand for amino acids, and we found that we needed to add BCAAs to artificial formula for the neonatal porpoises.” (Rev. M., line 413).

Thank you again to the reviewers for their comments and constructive advice. We hope that the changes that have been made to the manuscript meet to your satisfaction.

We wish to take this opportunity to thank you for your consideration of our paper for publication in your journal, PLOS ONE.

Attachment

Submitted filename: Response to Reviewers_Kasamatsu.pdf

pone.0288891.s003.pdf (147.4KB, pdf)

Decision Letter 1

Ulrike Gertrud Munderloh

28 Mar 2024

The first complete hand-rearing of two neonatal finless porpoises

PONE-D-23-20946R1

Dear Dr. Iwano,

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