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Protein nutrition in the alligator
Camp. Biochem. Physiol. Printed in Great Britain
Vol. 87A,
No. 2, pp. 449459,
1987 0
PROTEIN
NUTRITION
0300-9629/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
IN THE ALLIGATOR
ROLAND A. COULSON, THOMAS D. COULSON, JACK D. HERBERT and MARK A. STATON* Department of Biochemistry, Louisiana State University Medical Center, New Orleans, LA 70119, USA; *Department of Poultry Science, University of Georgia College of Agriculture, Athens, GA 30602, USA
(Received 18 August
1986)
Abstract-l.
Fourteen different protein-containing diets were fed to small alligators. Their rates of digestion and their nutritional values were determined by following changes in free amino acids in the plasma. 2. Fish, chicken and nutria were digested rapidly and all their component essential amino acids disappeared quickly and at the same rate. When given in the dry, fat-free form the amino acids were released and assimilated about 50% faster than when fat was included. 3. None of the isolated proteins tested (casein, gelatin, edestin, gliadin, corn gluten and soy) proved nutritionally adequate and all but gelatin digested slowly and incompletely. 4. One diet compounded of salts, vitamins and mixed commercial animal products was tested. It showed promise but it was lacking in methionine and isoleucine. 5. It was concluded that dry, powdered, preparations from whole animals could prove a satisfactory, stable diet for alligator husbandry. 6. Prolonged force-feeding of an animal diet increased the percent of nitrogen excreted as NH, over that excreted in urates.
INTRODUCTION Over the past 15 years the once threatened alligator (Alligator mississipiensis) has multiplied and they are often encountered in the wetlands of the southeastern
United States. Along with the population increase has come renewed interest in their economic potential, for not only are their hides valued, but there is increasing demand for the meat as well. In several states licensed alligator farms have been established with the aim of producing hides and meat, much as traditional livestock is raised. For the husbandry of this reptile to be practical in an economic sense, diets are required that are nutritionally adequate and inexpensive. Although, in theory, one could raise them by feeding their usual diet of crustaceans, fish, other reptiles, small mammals, etc., in practice this would be too expensive. If fresh food is fed, it must either be collected daily or collected in great amounts and stored frozen. The initial cost of the food is not a serious problem, but the cost of refrigeration is almost prohibitive. Not much is known about reptile nutrition compared with the wealth of information on small mammals, ruminants, and poultry. Coulson and Hernandez (1974) were able to determine indirectly which amino acids are essential for crocodilians and it came as no surprise that they were the same as those for mammals. Joanen and McNease (1987) conducted long term feeding experiments in which an exclusive fish diet was compared with a diet of nutria (Myocastor coypu). Nutria-fed animals not only grew faster than those fed fish, but at maturity the females also laid more hatchable eggs. The reason for the different effects of the two apparently adequate foods is not entirely understood (Lance er al., 1983). Coulson and Hernandez (1970) determined the rate of digestion (at 28°C) of raw fish in small caimans (Caiman crocodihs crocodilus). Since free amino
acids did not accumulate in the gut lumen at any time after feeding, the rate of free amino acid increase in the plasma proved a reliable index of the rate of digestion. Force-feeding several vegetable proteins did not result in an increase in plasma amino acids, and it was therefore concluded that the alligator was incapable of utilizing them for food. However, in recent experiments conducted at 31”C, less protein appeared in the feces and plasma amino acids increased considerably (see below). It may seem remarkable that a temperature rise of only 3°C could have such an effect, but it should be noted that this small rise doubles the alligator’s metabolic rate (Coulson and Hernandez, 1964). At any rate, this temperature effect illustrates one problem to be reckoned with in nutritional studies in reptiles. Efforts are being made to formulate a diet with commercially available feed ingredients that will give good growth and not require refrigeration. One of us (M.S.) has spent several years in the attempt and some of the results of feeding one of the formulations appears here. Since alligators are carnivorous, the composition of the dietary protein is most important and therefore the present study concerns protein nutrition, albeit in experiments that were usually of short duration. More detailed results of ongoing long-term experiments on formulated diets will be published later. Considering the present state of our knowledge, the results and conclusions detailed here should be considered preliminary. However, enough information is available to permit a few generalizations. Although the metabolic rate of the alligator is so low that their use in a study of vitamin requirements would prove an exercise in patience, they are excellent for a study in protein nutrition since one has days in which to observe the rates of digestion, amino acid absorption, protein synthesis from those amino acids, etc.
449
ROLAND A. COULSON et al.
450 MATERIALS
Experimental Alligators
AND METHODS
animals were obtained from the Rockefeller
Refuge of the Louisiana Department of Wildlife and Fisheries within a week or so after they were hatched in late August. They were placed in outside tanks containing water at about 3 1“C with exposure to sunlight for at least part of the day. Ground nutria were placed on elevated platforms giving them access to food for five consecutive days each week. Growth was rapid (Coulson et al., 1974) exceeding that in the wild more than IO-fold. They were not used in the diet test experiments until after their weights exceeded 500 g. The nutria diet was not supplemented with vitamins. Temperature Although most of the previous experiments on alligators in this laboratory had been conducted at 28°C the much faster rate of digestion at 31°C (Coulson and Coulson, 1986) led us to adopt the higher temperature. Where the outside tank temperatures varied somewhat, during the forcefeeding experiments, the temperature was kept constant (+0.3’C) in a controlled temperature chamber.
Water Since regurgitation was less frequent when the force-fed animals had access to water, and since ammonia excretion is limited by the degree of hydration (Coulson and Hernandez, 1964; Herbert, 1981), the animals were kept in water for the duration of each experiment. To reduce evaporation, the tops of the receptacles were covered loosely. This meant that the alligators were in the dark at all times except when blood or urine was being collected. One advantage of isolation and darkness was that this encouraged them to remain motionless most of the time.
Diet composition Nutria. Skinned, eviscerated nutria carcasses were ground (bones included) in an electric grinder to a coarse mince for some of the experiments, and fresh nutria muscle from the legs and back were used in others. In the dry diet experiments, ground nutria was dehydrated in a warmed desiccator with concentrated sulfuric acid as the desiccant. When dry, the residue was ground again in a laboratory blender and force-fed in a water slurry directly, or defatted and then fed. Fat was removed in the dry “fat-free” diets by extraction with dry ether three times, using five volumes of ether each time. The lipid content (see below) was determined by weight, and the steroid content by weighing the nonsaponifiable fraction. The crude de-fatting procedure left a lipid residue of less than 1% of the dry weight of the powder. The fresh eviscerated carcasses were analyzed and found to have the following composition: water, 70.9%; total ether-extractable lipid, 2.3%; non-saponifiable lipid, 0.12%; ash, 5.91%; and protein, 20.3%. On a dry weight basis the composition was: total lipid, 7.9%; non-saponifiable lipid, 0.43%; ash, 20.3%; and protein, 71.8%. The values as determined are only approximate since the actual composition varied with the age, size, and nutritional state of the nutria. We considered the protein content of the dry, defatted product to be about 70% for practical purposes. Bone was responsible for most of the high ash content. The second grinding reduced the bone fragments to a fine powder, making the components easy to assimilate. Chicken andfish. “Chicken” consisted of necks, hearts, livers, and gizzards as obtained from a local distributor. The mixed pieces were ground, dried, reground, and either fed with the fat in it, or defatted as described above and then fed. Compared with ground nutria, the chicken pieces that
were analyzed were higher in both fat and water and about equal in ash. The dry, defatted, chicken meal had about the same protein content as nutria prepared similarly. Again, we assumed the protein content to represent about 70% of the dry, non-fat weight. In experiments where chicken powder containing fat was given, the lipid content of the dry preparation was 37%, or over four times that of the fat content of the dry nutria powder. The one species of fish (CJ~oscion nebulosis, called locally “speckled trout”) that was fed was ground whole (head included), and then reground and defatted as above. The ash content of this fish was low at only about 7% on a dry, non-fat basis, but the protein content was again close to 70%. The amino acid composition of the hydrolyzed proteins of both fish and nutria was more variable than we would have liked, perhaps reflecting problems with our method of hydrolysis. The average of the individual essential amino acids in both was close enough for us to consider them for practical purposes to be identical. The somewhat rough -.approximations (in moles/l00 moles) were as follows: Asx, 7.27; Glx. 18.72; Thr. 3.75; Ser. 5.70: Pro. 7.32: Glv. 7.08: Ala, 3.09; Val, 5113; Cys-Cys, 0.38; Met, 2.38; Ile, 5.<8; Leu, 7.13; Tyr, 6.51; Phe, 4.18; Lys, 8.32; His, 2.61; Trp 1.81; and Arg, 3.42. This composition must have been close to the correct value since feeding a mixture of free amino acids in this ratio gave a plasma amino acid pattern almost identical to that found after feeding raw fish (Hebert and Coulson, 1976). The amino acid composition of nutria eaten by an alligator in the wild would differ from that above as a consequence of the ingestion of the skin containing collagen rich in glycine, proline and hydroxyprohne. Casein. Obtained from milk, this was a “feed grade” product obtained from the Sigma Chemical Company, St. Louis, Missouri, USA. Gelatin. This was a “laboratory” grade product supplied by Fisher Scientific Company, Fair Lawn, New Jersey, USA. Edestin. This was a preparation derived in this instance from hemp seed (Sigma), but similar in composition to the principal protein in garden peas. G&din. Obtained from wheat gluten, this was obtained from Sigma. This protein was fed with difficulty. In water it formed a sticky paste even more viscous than that obtained when wheat flour is mixed with water. As attempts to get it into alligator stomachs met with no success, it was necessary to introduce it packed in gelatin capsules. Unfortunately, this meant that the results reported for the gliadin experiments actually represented those for gliadin contaminated with about 7% of gelatin. Corn gluten meal. This was a “feed grade” product obtained from the University of Georgia Poultry Science feed mill. Unlike gliadin from wheat, it did not form a sticky paste when wet, and it was fed without difficulty. Soy. Isolated soybean protein, this was obtained from Ralston Purina Co., St. Louis, Missouri. Staton mix. Referred to as “mix” in the figures and tables, this formulation was a mixture of various commercial feed ingredients having the following composition: casein, 20%; blood meal, 25%; fish meal (menhaden), 15%; feather meal, 10%; meat and bone meal, 17%; poultry by-products meal, 10%; carboxymethyl cellulose, 1.j%; mixed-vitamins, 1%; and NaCl. 0.5%. The calculated (ether-extractable) fat content of the dry preparation was 5.2%, and the protein content, 72%. The calculated amino acid content of the hydrolyzed mix (in moles/l00 moles of those amino acids listed) appears below, compared with our analyses (*) for both the mix and for fish muscle, expressed in the same units. (For ease of comparison, the molar % of the 13 listed amino acids was given a value of 100.) In our analyses of the mix, Glx (glutamate + glutamine) was much lower than the value found for either nutria or fish, in the ratio of 18.7 to 8.7.
Protein nutrition in the alligator
Thr Val Met Be Leu Phe Lys His Trp Arg Ser Tyr GUY
Fish *
Mix
5.89 8.05 3.74 8.13 11.19 6.56 13.06 4.10 2.84 5.31 8.95 10.23 11.11
6.93 11.89 2.24 4.95 14.50 6.30 10.03 4.30
1.06 5.96 10.66 4.22 15.94
SUM 99.20 99.90 **Not determined by us.
Mix * 5.16 10.69 3.06 5.21 13.27 7.1 I 11.42 5.16 (1.06)** 8.74 6.58 4.31 18.22
451
Amino acid analyses
The analyzer was the original type designed by Spackman er al. (1958), but modified to make it capable of analyzing three samples in 6 hr. To complete this many analyses in that length of time, only 570 pm filters were used in the calorimeters. This meant that neither hydroxyproline nor proline were estimated routinely. (The detailed procedure has been described previously (Coulson and Hernandez, 1968).] Two amino acids (aspartate and tryptophan) were present in the plasma in such low concentrations, even after feeding, that changes in their concentrations could not be followed with any degree of accuracy, and therefore they are not included in the data.
99.99
RATIONALE OF THE EXPERIMENTS Staton mix plus methionine. This was the mix as shown
Feeding an alligator a diet deficient in a single or several essential amino acids would of course result in a serious nutritional deficiency over a period of time. Growth would cease in immature specimens and eventually they would actually lose weight. The Feeding procedures problem with such experiments is the length of time Dry preparations. Each was given at 0600 hr in an amount needed to detect the effects. In theory, it should be calculated to supply about IO g of protein/kg. The powders possible to test each protein’s biological value more were suspended in a water slurry and poured into the stomach through a long-stemmed funnel. When the funnel quickly by determining changes in the free amino had drained it was rinsed with a small amount of water and acids in the plasma after a single feeding. Should one withdrawn slowly. To prevent the alligators from biting the amino acid prove proportionately low in the plasma, funnel, the jaws were kept apart by means of a flat plywood presumably it was present in the digesting protein in board with a hole in the center through which the funnel was inadequate amounts. Such tests are difficult in most inserted. In most feedings the slurry was intubated without small mammals where the amount of the increase in incident, but in a few cases the food was regurgitated at once free amino acids in the plasma is low and the small and it was necessary to abandon the attempt. increase is of short duration. However, in a large Fresh nutria muscle. Pieces of nutria muscle were wetted and then pushed down the esophagus by means of a long reptile, plasma amino acids rise considerably during glass stirring rod. protein digestion and they remain elevated for days. Gliadin and gelatin. These were both given enclosed in It is important to note that this type of experiment large gelatin capsules. To make sure the capsules dissolved, measures the demand for each amino acid, and that about 50ml of water was poured into the stomach after the demand in one species may differ from that in introduction of the capsules. others. A simple chemical analysis of a protein will reveal which amino acids are present, and in what amounts, but the information often cannot be used to Blood collection Blood was collected from the tip of the tail by means of predict how satisfactory the protein will be when it is eaten. It may be digested incompletely, or its coma Pasteur pipette, placed in heparinized 0.4ml microcentrifuge tubes, and then centrifuged at 13,000 rpm for position may not match the demand in that particular 20 sec. Plasma was removed and 0.2 ml was added to a tube species. containing 3.8 ml of 95% ethanol to precipitate the proteins. The capacity of a small, growing alligator to A clear, colorless supernatant was obtained by centrifuging tolerate massive amounts of protein at a single feedat 2500rpm for five min. The supernatant was stored at ing makes this reptile well suited for such research. In -20’C until it was used for the amino acid analysis. previous experiments (Coulson and Hernandez, 1970) small caimans were force-fed meat containing as much as 15 g of protein/kg of body wt at a single Urine collection and analysis feeding. This contrasts with the 1 g of protein eaten The animals were kept dry at 31°C during the collection period. Urine was obtained by inserting a fire-polished glass per kg by an average man over a 24 hr period. The problem of deciding which amino acid is proportube (6 mm O.D.) into the cloaca and draining the urine into a flask containing 1 ml 1 N HCI (to trap NH,). To prevent tionally low in the plasma may be solved in part by loss, It was necessary to collect urine at 30 min intervals as use of a standard. If one assumes that an alligator the flow rate often exceeded 10 ml/kg per hr. After analysis could thrive on a diet of whole mammals or fish, then of ammonia by the method of Conway and Byrne (1933), the plasma free amino acid content after such a meal the urine was boiled briefly with a small amount of L&CO, may be considered to be close to the ideal, and (Z IOOmg) to solubilize the urates, then analyzed for uric deviations from that following feeding a lower qualacid by the method of Archibald (1957). Urine collection usually began about 4 hr after feeding, when the rate of ity protein could be apparent. In consideration of the above, in the experiments urine flow was near maximal, and continued for 2 or more reported here, alligators weighing from 0.5 to 2 kg hours. Blood samples obtained at the end of the collection were force-fed, blood was removed at 4, 7, 10, 14, 24, period were also analyzed for uric acid and for free amino acids. 30, 37, 48 and, in those cases where digestion was
above, except that it had been supplemented with methionine in an amount 73% in excesss of that in the mix.
452
ROLAND
A. COLJLSON et al.
slow, at 54, 72 and 96 hr. Changes in the concentration of each of the absorbed amino acids were followed by analyzing the plasma. RESULTS
“Essential” and “non-essential” amino acids
From an examination of the results given below, it could be argued that all amino acids except alanine and glutamine are “essential” for maximal growth rates in a small alligator. For example, if the rate at which a non-essential amino acid (such as glutamate) can be incorporated into body protein after a meal exceeds the rate of its gain in the plasma, the amino acid should be essential for maximal growth. It is true that the amino acid can be synthesized, but if that rate is slower than the maximal rate of its incorporation into body protein, a deficiency in it would be growth limiting. Therefore, the data indicate apparent deficiencies without regard for the traditional division between essentials and non-essentials. Peak times
The time required for each free amino acid to reach its maximum concentration in the plasma (peak time) after feeding is determined both by the rate of digestion of the protein, and by the rate of removal of its component amino acids from the plasma. When a protein digests slowly, free amino acids rise slowly in the plasma, and vice versa. However, should any one amino acid be proportionately low in the absorbed mixture, incorporation of that amino acid into body protein may remove it almost as fast as it is absorbed and its peak will occur before those of the others in more plentifu1 supply. (A “good” protein that digests slowly could be adequate for maintenance but not for maximum growth promotion since less could be fed per week.) On the basis of the average peak time, dried, defatted, powdered nutria, fish and chicken were not only digested more rapidly than the other diets, but the rate of removal of each of the absorbed amino acids was more nearly uniform (Table 1). In other words, each amino acid reached its peak at about the same time, and all disappeared at about the same percentage rate. Fresh nutria muscle digested more slowly than the non-fat, dry nutria, as would be expected, and the presence of fat delayed digestion of both nutria and chicken (see below). Edestin was the slowest to digest. Solely on the basis of peak times, one would predict that the animal diets were adequate, and that all the other diets were deficient in one or more amino acids. Those amino acids that appeared to reach a peak well before the average time for the others in each diet are marked with an asterisk in Table 1. Glutamine, glycine and alanine maxima were usually several hours late for the probable reason that their initial rises in the plasma were the result of absorption from the gut, while the prolonged elevations were the result of synthesis (see below). Peak concentrations
Table 2 shows the change (PM) in the concentrations of the individual amino acids (above the
$ 2 ?
Ala
Gly
Gla
Glu
Tyr
ser
Arg
His
LYs
Phe
Leu
Ile
Met
Vd
Thr
-
-.-__Peak 30 hr Peak 30hr Peak 30 hr Peak 3Ohr Peak 30 hr Peak 30hr Peak 3Ohr Peak 30 hr Peak 30 hr Peak 30hr Peak 30hr Peak 30 hr Peak 30 hr Peak 30 hr Peak Mhr
173&29 12Ok8 316 & 21 316k21 103&li 103fII 112rt_23 98+11 157k32 157&32 94+12 78 +_9 11X*23 104+23 92k 15 34+ 12 92_+14 74+ 13 109+45 109+45 46+ 14 441t: II 96& 10 96+ 10 604+ 127 438+_ 112 333 + 36 251 + 55 1004~50 788 _+70
Fresh nutria muscle
100*10 14+4 465f8 144*5 100+11 1424 177k25 16+9 355-661 90 It 37 137+20 s4+ 17 1622 17 39 f 18 127&27 46* 12 74_+13 30+11 113+31 18k21 63%- 19 12 i 13 71 & 18 22+ 13 248+M) 152 f 56 498 _+ 12 498 L- 12 1050 + 120 7702 II0
-
in fm
207 zt 56 187 +45 S40+ 153 399+ 116 155+57 127+_44 175_+ 16 116_+29 367 +_ 137 262 + 51 186 + 53 852 13 182 +48 85+18 196+_74 133 _+42 932 18 77+_ 14 251 + 74 249 + 49 Ill + 17 51 +6 167 + 51 109+35 4695 100 389 2 80 973 + 234 902 _+201 1722 + 245 1722 _+245
Fat nutria, dry
Table 2. Increase
Non-fat nutria, dry
&M)
65 + 9 -20_+8 231 F 30 230 + 59 152+30 140+35 53+ 11 32+ 10 104+_24 77 + 24 74_t 15 74+ 15 172 +36 64+ 19 116+23 104_C32 23 + 5 4+_2 235k40 95+2S 174 +29 109+41 509+52 357 & 78
109+20 70+ 13 374 +_83 335+64 -
acid concentrations
123+35 90+ 17 491 t_ 18 356 + 27 95+ 16 7+_5 S6+ 13 -15+5 354 f 97 263 + 37 217rt27 135+_5 74+20 51* 19 180-124 138+24 62 + 8 41* 14 148 + 36 98 + 23 127&20 7.5+9 45+8 6+_5 346+55 346 f 55 402f59 275 It 76 8002 114 _._ .._ 562 F 13U
amino
325 145 695 321 267 115 210 90 423 196 267 84 188 102 244 166 189 118 312 197 161 58 203 94 674 402 1830 1190 2300 ._._ IYltJ
Non-fat
in plasma
’
254 rl: 95 191 * 74 962 ?r 97 473 i 184 433 Ifr 61 223 C 56 446+30 171&93 792 + I42 228 rf: 128 336 + 52 69rfi 16 170227 73+26 289 + 116 176+67 i44i22 93*31 332 & 53 158 rt 35 289f48 36 F 24 243 + 25 133+_50 1393 _+282 576 f 222 665 +_ 16 511+ 135 2463 + 671 ..__ _~_ 1 IKZ * 355
dry
Non-fat chicken. 315 230 1072 517 418 420 405 405 688 591 203 172 213 213 180 130 159 132 234 144 134 80 192 130 838 645 558 313 1602 1262
dry
Fat chicken, 171 154 740 740 113 113 227 134 47t 449 155 150 118 104 248 240 60 21 274 214 287 136 13s 95 571 463 86 73 997 892
Casein
at the peak and at 30 hr after feeding
3014 2214 2124
4400
69 26 296 152 4 -2 113 43 208 114 130 34 6 -12 89 107 241 145 239 186 55 17 74 66 221 121
Gelatin
various
130 49 550 550 77 17 83 183 294 294 147 147 55 23 143 143 265 265 110 110 197 197 so 50 351 351 397 397 586 496
Edestin
foods corn G&din gluten ~____ 48 91 -7 67 305 280 192 190 23 40 8 32 178 171 177 165 279 867 190 617 137 loo 70 94 21 i __6 -20 231 54 138 IS 326 32 238 32 49 95 34 27 171 104 72 a2 212 40 114 36 4459 309 2439 241 163l 5 I255 -8 1616 293 1451 164
~._~ SOY 163 & 48 163 k48 456 f 35 4S6*35 10+4 -4&l 211&40 204?,11 413 *66 413 +_66 184_t25 184 + 25 48k 15 401t: II 51 *4 47 & 27 177t_33 l46+27 250 +- 57 212 &42 155 &70 89t I1 91 If: 13 71 I5 452 f 58 452 + 58 283 rt 68 245 & 74 663 * 152 663 f 152
454
ROLAND
A. COWSON el ai.
control values) at the time each was at its peak. Where the number of alligators in an experiment were adequate, the standard error of the mean (SEM) is shown. Arbitrarily, the concentration at a second time (30 hr) appears beneath the peak value. This type of data presentation represents a compromise between including all the data for 4,7, 10, 14,24, 30, 37, 48, 54 and 72 hr, and only showing the peak concentration. Where the value at 30 hr was well below that at the peak, the concentration was declining rapidly, and where both values were about equal, removal was slow. Apparent biological value of the diets summarized
Table 3 summarizes the apparent deficiencies in the several diets based both on the peak concentrations in the plasma relative to those found after feeding the animal diets, and on the relative time at which the peaks were reached. Some comments on the diets other than nutria, fish and chicken appear below. Casein. This protein was slow to digest but nutritionally complete with the exception of arginine and glycine. It was well tolerated and little appeared in the feces. Gektin. Perhaps the poorest protein available, it proved quite easy to digest. Not only is it lacking several amino acids, but those present are in such an improper ratio as to render body protein synthesis from its component amino acids impossible. Failure to remove the absorbed amino acids by incorporating them into protein forces the alligators to resort to the slower catabolic routes for their disposal. Feeding large amounts (at 28°C) to alligators and turtles in previous experiments (Herbert and Coulson, 1976) caused a number of deaths. Edestin. Only a single alfigator was fed this protein. Aithou~h it digested slowly, and was deficient in several amino acids, none of the deficiencies were serious. Gliadin and corn gluten meal. Gliadin, an important protein of wheat flour, digested slowly at 31°C and much more slowly (if at all) at 28°C (Coulson and Hernandez, 1983). Although of limited nutritional value, its paste-like consistency when wet should make it an excellent binder for making diet pellets. In contrast, corn gluten meal, which contains a protein of the same class as gliadin, appeared to have less
satisfactory binding properties (if its consistency when wet is a valid measure). Isolated soybean protein. As far as the alligator is concerned, this vegetable protein, commonly used in animal and human foods, is not a satisfactory substitute for animal protein. It was deficient in some amino acids, slow to digest, and considerable amounts appeared in the feces. Staton mix. Its composition (see above) was designed to provide a mixture of acceptable feed grade proteins with high stability at room temperature in the dry state. The results reported here were derived from tests on only one such mix, and they are of value principally for illustrating the single feeding approach to determine biological value. Changes in individual amino acids following force-feeding dry nutria powder are compared with those following feeding the mix (Fig. 1). For some unknown reason, the curves are somewhat irregular with a notch or shoulder appearing a few hours after feeding. It is possible that this is an artifact caused by dilution of the plasma amino acids by absorption of a large amount of water from the stomach between the seventh and tenth hours after feeding. Numbers appearing inside the curves refer to peak values (PM). Where an amino acid was present in sub-optimal amounts in the protein, the peak concentration was low and the total area was also small compared with that for other amino acids in plentiful supply. To the right are bar graphs comparing the relative areas of the curves. When nutria was fed, the area of each amino acid curve was about the same (excluding the nitrogen carriers, glycine, alanine and glutamine). When the mix was fed, the amino acid areas varied, those that were deficient being smaller. Deficiencies in methionine, isoleucine and glutamate are apparent. e~~tarn~ne, glycine, alun~ne, and glutamate
The first three are the principal distributors of nitrogen, accounting for about half the plasma total. Glutamate (like aspartate) is never a major constituent in plasma (Coulson and Hernandez, 1983). Glutamine. That an alligator can digest a protein rich in glutamine and absorb this amino acid unchanged may be seen from the results of the gliadin experiment where peak glutamine exceeded 4 mM, or 46% of the total free amino acids. It may then be
Table 3. Apparent amino acid deficiencies in various foods as determined in the alligator -_.--..Mix Mix + Met Casein Gelatin Edestin Gliadin Corn gluten SOY
__ _
Thr ~~
~~~Val_
__Met._ x*
x
_tie _ x x
_Leu
Phe
x ix
x *x
x x x
l x = deficiency. ** t x = possible or marginal deficiency. Fresh nutria muscle 1 Non-fat. drv nutria 1 Fat. dry n&a No deficiencies evident Non-fat, dry fish Non-fat, dry chicken Fat, dry chicken
IX
His Lys + x ** *x x x x x x
Arg
Ser
x
GUY
x x
x
Glu x x
ix x
TY~
ix zkx x
x ,x
+x x x
x
Protein nutrition in the alligator
455
Rolatlvo
20
40
amar
60
Time
(hrl
Fig. 1. The effects of feeding 10 g of protein/kg in the form of dry, non-fat nutria powder compared with those seen after feeding an equal amount of protein in the mix. So that the relative changes could be compared more easily, in each case the peak concentration was given an arbitrary value of 100, and the amount of the changes before and after the peak are plotted as % of the peak value. Numbers inside the curves represent the increase in each amino acid in the plasma @M) above that of the non-fed controls. For SEM at the peak and at 30 hr, see Table 2. Bars to the right indicate the relative areas of the amino acid curves. Where the areas of the curves were nearly uniform in the nutria-fed group (glycine, alanine and glutamine excepted), they are quite irregular in those fed the mix. The reason for the consistent notching of the curves at about 10 hr after feeding is unknown, but it could be the result of dilution of the plasma from absorption of the large amount of water given with the food.
assumed that a low peak after feeding would indicate a low glutamine content in the protein fed. Glycine. Evidently, peak concentration of this amino acid also reflects its content in the protein fed.
It was very high in the gelatin experiment and almost absent after corn gluten meal feeding. In common with glutamine and alanine, synthesis from other amino acids was responsible for its more prolonged elevation (Table 1, Fig. 1). Alunine. The range in peaks was from 293 p M after corn gluten to 2463 PM in those fed chicken. Even where the rise was small, adequate amounts for protein synthesis seem to have been available during digestion of all 14 diets. Glutamate. Most of the glutamic acid in food protein is apparently degraded in the gut wall in the dog (Neame and Wiseman, 1957). However, our results seem to indicate that in the alligator at least, the plasma content after feeding was related to the content in the protein. For example, the Staton mix had only about 40% of the glutamine plus glutamate content of fish, an amount apparently below that needed to prevent a deficit in the plasma. One might expect glutamine to serve as an adequate substitute, but it did not seem to. There was some correlation between the plasma glutamine concentration and that of glutamate, but the significance is questionable.
Relationship between the amino acid content of proteins and peak values
The reported amino acid content of the various proteins was related in most cases to the height of the peak value, but there were some notable exceptions. Plasma alanine concentrations were usually considerably higher than would have been predicted from the protein alanine content, and in part, this was true for glycine. In all cases where the protein content of an essential amino acid was half or less of what would be considered ideal (using the composition of fish or nutria as an “ideal” standard), the peak plasma concentration was very low, and in the case of both methionine and lysine, a modest deficit in the fed protein always led to a major shortage in the plasma. Unfortunately, the exact content of glutamine and glutamate in most of the proteins tested was unknown. Hydrolysis in either acid or alkali converts glutamine to glutamate and therefore the combination in the protein can only be designated as Glx, the symbol for the combination of the two. Where the peak glutamine concentration was very high, it would be reasonable to assume that most of the Glx was glutamine, and where it was very low, it was probably principally in the form of glutamic acid (see above).
ROLAND A. COULSON
456
et al.
Effect of fat on the rate of digestion As noted above, fat delayed the digestion of nutria and chicken. The difference between the fat and non-fat experiments is shown in Fig. 2. Long-term growth rates of alligators fed non-fat nutria powder Two alligators fed 10 g of protein/kg per day in the form of nutria powder on 5 consecutive days each week grew rapidly (Fig. 3). They were weighed weekly at the conclusion of the Z-day fast for a total of 14 weeks. The plotted weights gave a remarkably straight line. The gain in per cent per week fell as the alligators grew, while the actual gain in grams remained constant. At the conclusion of the feeding experiment, the alligators appeared in good health. Considering the metabolic rate of the alligator, even 14 weeks should be judged a short-term experiment, and had the feeding been continued for several more months, deficiencies in vitamins, essential fatty acids, etc., might have been observed. NITROGEN EXCRETION PATTERNS
Since alligators raised in captivity sometimes develop gout (Coulson et al., 1973) nitrogen excretion patterns and plasma urate levels were monitored in groups of animals force-fed different diets or forcefed the same diet at different temperatures. In some experiments a single meal was fed to fasted animals; in others, the animals were fed 5 days a week for l-14 weeks. E#ect of iemperatare Groups of three alligators were force-fed lean nutria muscle, 2.5% of body weight, while being maintained at 25, 28 or 31”C, and nitrogen excretion, plasma urate, and plasma amino acid levels monitored daily for 7-8 days. Blood was obtained at 0 hr (just before feeding), and at 20, 44, 69, 92, 116, 140, and 165 hr after feeding. The animals were kept dry during collection of timed urine samples for 4-6 hr each day. At ail other times they were kept in water in covered containers.
Time
ihr)
Fig. 2. The effect of fat on the rate of digestion and assimilation of dry, powdered nutria. Both groups (six animals in each) were fed 10 g of nutria protein per kg. For SEM at the peaks and at 30 hr, see Table 2. The fat content was about 8%.
700:
I 20
I
I
40 60 Time (days)
I 80
I 100
Fig. 3. Increase in body weight in two alligators fed dry, powdered, defatted nutria for several weeks. No vitamin supplements were given, but they were exposed to sunlight for about 2 hr/day, starting at week 5. At 25°C a temperature at which captive animals ordinarily will not eat much voluntarily, digestion was incomplete, and some animals regurgitated portions of undigested meat 2-3 days after feeding. At this temperature (N = 6), plasma urate was high after feeding [1.02 & 0.19 mM (mean + SEM) at 20 hr], uric acid excretion was increased about six-fold (to 6.13 + 0.68 mmol nitrogen~kg per day at 92 hr), but ammonia excretion increased only a little (to 2.98 + 0.51 mmol/kg per day at 92 hr). Since feeding at th% temperature was obviously unphysiological, further experiments at this temperature were not conducted. Although digestion is somewhat slower at 28 than at 31°C (Coulson and Coulson, 1986), there was no apparent difference in nitrogen excretion patterns, changes in plasma urate, or uric acid clearance between the two groups (see Table 4).
Fish powder us nutria powder Table 2 indicates that feeding fish powder resulted in greater increases in plasma glycine and alanine than did feeding nutria powder. These amino acids are important precursors of uric acid in many animals, and gout was a perennial problem when our alligators were routinely fed a diet of ground marine fish (Coulson et al., 1973). Therefore, three alligators were force-fed a single meal of defatted fish powder, 10 g protein/kg body wt, and their nitrogen excretion patterns monitored for 1 week. When plasma and urine values had returned to control levels, the same three animals were fasted for five days, fed defatted nutria powder (10 g protein/kg) and nitrogen excretion and plasma monitored as before. Although these experiments confirmed that plasma glycine and alanine rose significantly higher in the fish-fed group than in those fed nutria (Fig. 4) there was no apparent difference in plasma urate levels or in nitrogen excretion patterns in the two groups, at least in this short-term, single-meal experiment. Effect of long-term feeding Nitrogen excretion patterns and plasma urate were monitored in two alligators force-fed defatted nutria
Protein
nutrition
in the alligator
457
Table 4. Changes in nitrogen excretion, plasma orate, and uric acid clearance in alligators fed lean nutria muscle at 28 and 31°C (mean + SEM) Hours after feeding 44
Temp. I”C)
N
0
20
28 3t
6 9
0 + 0.03 0 f 0.05
0.25 it 0.04 0.27 2 0.08
0.21 rf:0.06 0.22 * 0.06
0.14rto.04 0.15 iO.08
28 31
6 9
O&O.31 0 2 0.48
3.89 f 0.68 5.88 * 0.92
5.07 rf 1.44 6.97 & 1.67
4.03 * 1.52 6.18 f 2.19
3.34* 1.31 3.10 4 1.65
28 31
6 9
020.31 0 i: 0.45
5.83 & 1.87 5.36 rt 1.14
7.39+ 1.15 6.50 t 0.88
3.62 + 0.50 3.29 + 0.77
2.39 + 0.36 0.76 + 0.65
28 31
6 9
0+0.19 0 2 0.23
1.46$0.16 2.09 _+0.20
2.14_+0.31 2.54 & 0.26
1.80f 0.46 2.46 L-0.53
2.19 +0.74 2.44 + 1.05
Plasma urate (mM) Uric acid nitrogen excretion (mmolikg per day) Ammonia nitrogen excretion (mmol/kg per day) Uric acid clearance (l/kg per day)
92
69
0.06 & 0.01 -0.01 f 0.05
0.6
-ktl=; 1 E
Plosmo
0.4
02
/
I/
‘p-
urote
4-
R2;
_ _
a
I
Ez E
I
Uric acid nltroqut
1
: I
-~
20
acid
40 I7m6
amino ’
acid6
\ t \ \
0
nittqllt
Amonio
Uric
Ementlol
II’
3
0
0
T
c16oronce
60 (hr)
80
loo Time
lhrf
Fig. 4. Plasma mate, nitrogen excretion, urine volumes, uric acid clearance, and changes in plasma amino acid levels (Mean + SD) in three alligators force-fed a single meal of dried, defatted fish powder, 10 g protein/kg body wt, (-A--), or dried, defatted nutria powder, 10 g protein/kg (-0-J The “essential amino acid” segment represents the change (from fasting control levels) in the sum of six amino acids: 7’hr, Val, Met, He, L_euand Phe. Uric acid excretion is plotted as uric acid nitrogen (mmoi uric acid x 4) to facilitate comparison of the two routes of nitrogen excretion.
ROLANDA. COULSONet al.
458
Time (days)
Fig. 5. Mean nitrogen excretion, plasma urate, and urine volumes in two alligators force-fed dried, defatted nutria powder 5 days a week for 14 weeks. Blood and urine samples were collected 46 hours after the 5th feeding
each week. Individual
values varied
powder (10 g protein/kg body wt) 5 days a week for 14 weeks. Two-hour timed urine samples were collected 4-6 hr after feeding on day 5 of each week; blood was taken for uric acid analysis at the end of the urine collection period. Figure 5 shows ammonia and uric acid nitrogen excretion levels, plasma urate concentrations and urine volumes through 96 days on this diet. Although ammonia nitrogen excretion was only about twice the uric acid nitrogen excretion in the early stages, continued feeding resulted in an increase in ammonia and a decrease in uric acid nitrogen excretion until ammonia exceeded uric acid nitrogen by about six-fold. Plasma urate levels fell steadily from an initial value of 0.53 mM to about 0.2 mM after 82 days of feeding. Uric acid clearance ranged from about 8.5 l/kg per day in the first week to about 10.5 l/kg per day at the end of the experiment. Urine volumes are included to show the close correlation with ammonia excretion levels. The urine ammonia concentration was therefore nearly constant at 149 + 2 mM (N = 26). There was no obvious ill effect of feeding this diet on either growth rates (Fig. 3), or on nitrogen excretion rates or plasma urate levels (Fig. 5). At the termination of this experiment, the same animals were fed the Staton mix diet for 7 days, then the “Mix + Met” formulation for 5 days. Blood and urine were collected on the last day of feeding each diet and analyzed. The values obtained in these short-term, multiple-feeding experiments were as follows: plasma urate, 0.22 mM (Mix) and 0.23 mM (Mix + Met); ammonia nitrogen excretion, 42 and 44 mmol/kg per day; uric acid nitrogen excretion, 10 and 11 mmol/kg per day; urine volumes, 282 and 302 ml/kg per day; uric acid clearance, 11 and 12 l/kg per day. These values are comparable to those obtained after long-term nutria feeding (Fig. 5) and indicate no obvious problem for nitrogen excretion mechanisms in the short term at least. CONCLUSIONS
Although some progress has been made toward an understanding of nutrition in the alligator, much
less than
10% from the mean values
shown.
remains to be done. The amount of fat that should be included in the diet is unknown. Too much fat leads to steatitis, a condition in which the liver may be destroyed by a massive intrusion of fat. Too little fat might limit the maximum rate of growth by forcing the alligators to rely on protein for all of the energy needs, and for the gluconeogenesis so necessary to prevent hypoglycemia. Although fat is not usually considered to be “protein sparing”, in an animal with a small brain and a low metabolic rate, the glycerol in fat can provide the nervous system with an adequate amount of glucose without the need for glycogenic amino acids. Presumably, if the animals would use only fat for their energy needs, more protein would be available for growth. In the experiments reported here, 10 g of protein were fed per kg. That quantity was not adopted for any valid scientific reason, but rather as a convenient amount for the preliminary experiments. Should this amount have exceeded the ability of the alligator to synthesize body protein, the excess would be converted to fat, which would increase the weight of the animal but not affect its length. So long as the alligator lengthens, the animal may be considered to be growing, in contrast to just gaining weight through fat deposition. The amount of each of the probably large number of vitamins required by a growing alligator is also unknown. It may be assumed that the vitamin requirement is a function of the metabolic rate, as that rate will govern the rate of their excretion, metabolic degradation, etc. Certain vitamins given in excess are toxic to mammals, and this would probably be true in an alligator. The stability of vitamins in the dry state at room temperature, when blended with other components of the diet, may also be a problem. Deficiencies in the Staton mix should not be considered proof that it would not be of value as an alligator feed. If those amino acids present are in a combination that will lead to growth at a near maximal rate if eaten in sufficient amounts, then the problem would be less one of nutrition than one of economics. Improvements in the balance could then decrease the amount needed and lower the cost.
Protein nutrition in the alligator Results
of ongoing
feeding
trials
with formulated
feeds will be presented elsewhere. Acknowledgements-We thank the Louisiana Department of Wildlife and Fisheries for alligators and alligator food. The Louisiana Board of Regents Research and Development Program and the Louisiana Department of Wildlife and Fisheries provided financial support. REFERENCES
Archibald R. M. (1957) Calorimetric measurement of uric acid. Clin. Chem. 3, 102-105. Conway E. J. and Byrne A. (1933) An absorption apparatus for the micro-determination of certain volatile substances. 1, The micro-determination of ammonia. Biochem. J. 27, 419429.
Coulson R. A. and Coulson T. D. (1986) Effect of temperature on the rates of digestion, amino acid absorption and assimilation in the alligator. Camp. Biochem. Physiol. 83A, S-588.
Coulson T. D., Coulson R. A. and Hernandez T. (1973) Some observations on the growth of captive alligators. Zoologica 58, 47-52.
Coulson R. A. and Hernandez T. (1964) Biochemisrry of the Alligator, A Study of Metabolism in Slow Motion. Louisiana State University Press, Baton Rouge. Coulson R. A. and Hernandez T. (1968) Amino acid metabolism in chameleons. Comp. Biochem. Physiol. 25, 861-872.
459
Coulson R. A. and Hernandez T. (1970) Protein digestion and amino acid absorption in the caiman. J. Nurr. 100, 81&826. Co&on R. A. and Hernandez T. (1974) Intermediary metabolism of reptiles. In Chemical.Zooiogy (Edited by Florkin M. and Scheer B. T.). Vol. 9. DD. 217-247. AA Academic Press, New York. ” Coulson R. A. and Hernandez T. (1983) Alligator Merabolism. Studies on Chemical Reactions in vivo. Pergamon Press, Oxford. Herbert J. D. (1981) Nitrogen excretion in “maximally-fed” crocodilians. Comp. Biochem. Physiol. 69B, 499-504. Herbert J. D. and Coulson R. A. (1976) Plasma amino acids in reptiles after feeding protein or amino acids and after injecting amino acids. J. Nurr. 106, 1097-l 101. Joanen T. and McNease L. (1987) Alligator farminn research in Louisiana. Proceedings of- a Technic> Conference on Crocodile Conservation and Management,
January 1985, Darwin, Australia (in press). Lance V., Joanen T. and McNease L. (1983) Selenium, vitamin E, and trace elements in the plasma of wild and farm-reared alligators during the reproductive cycle. Can. J. Zool. 61, 17441751. Neame K. D. and Wiseman G. (1957) Transamination of glutamic and aspartic acids during absorption by the small intestine of the dog in uivo. J. Physiol., Land. 135, 442450.
Spackman D. N., Stein W. and Moore S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyr. Chem. 30, 119&1206.
Vol. 87A,
No. 2, pp. 449459,
1987 0
PROTEIN
NUTRITION
0300-9629/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
IN THE ALLIGATOR
ROLAND A. COULSON, THOMAS D. COULSON, JACK D. HERBERT and MARK A. STATON* Department of Biochemistry, Louisiana State University Medical Center, New Orleans, LA 70119, USA; *Department of Poultry Science, University of Georgia College of Agriculture, Athens, GA 30602, USA
(Received 18 August
1986)
Abstract-l.
Fourteen different protein-containing diets were fed to small alligators. Their rates of digestion and their nutritional values were determined by following changes in free amino acids in the plasma. 2. Fish, chicken and nutria were digested rapidly and all their component essential amino acids disappeared quickly and at the same rate. When given in the dry, fat-free form the amino acids were released and assimilated about 50% faster than when fat was included. 3. None of the isolated proteins tested (casein, gelatin, edestin, gliadin, corn gluten and soy) proved nutritionally adequate and all but gelatin digested slowly and incompletely. 4. One diet compounded of salts, vitamins and mixed commercial animal products was tested. It showed promise but it was lacking in methionine and isoleucine. 5. It was concluded that dry, powdered, preparations from whole animals could prove a satisfactory, stable diet for alligator husbandry. 6. Prolonged force-feeding of an animal diet increased the percent of nitrogen excreted as NH, over that excreted in urates.
INTRODUCTION Over the past 15 years the once threatened alligator (Alligator mississipiensis) has multiplied and they are often encountered in the wetlands of the southeastern
United States. Along with the population increase has come renewed interest in their economic potential, for not only are their hides valued, but there is increasing demand for the meat as well. In several states licensed alligator farms have been established with the aim of producing hides and meat, much as traditional livestock is raised. For the husbandry of this reptile to be practical in an economic sense, diets are required that are nutritionally adequate and inexpensive. Although, in theory, one could raise them by feeding their usual diet of crustaceans, fish, other reptiles, small mammals, etc., in practice this would be too expensive. If fresh food is fed, it must either be collected daily or collected in great amounts and stored frozen. The initial cost of the food is not a serious problem, but the cost of refrigeration is almost prohibitive. Not much is known about reptile nutrition compared with the wealth of information on small mammals, ruminants, and poultry. Coulson and Hernandez (1974) were able to determine indirectly which amino acids are essential for crocodilians and it came as no surprise that they were the same as those for mammals. Joanen and McNease (1987) conducted long term feeding experiments in which an exclusive fish diet was compared with a diet of nutria (Myocastor coypu). Nutria-fed animals not only grew faster than those fed fish, but at maturity the females also laid more hatchable eggs. The reason for the different effects of the two apparently adequate foods is not entirely understood (Lance er al., 1983). Coulson and Hernandez (1970) determined the rate of digestion (at 28°C) of raw fish in small caimans (Caiman crocodihs crocodilus). Since free amino
acids did not accumulate in the gut lumen at any time after feeding, the rate of free amino acid increase in the plasma proved a reliable index of the rate of digestion. Force-feeding several vegetable proteins did not result in an increase in plasma amino acids, and it was therefore concluded that the alligator was incapable of utilizing them for food. However, in recent experiments conducted at 31”C, less protein appeared in the feces and plasma amino acids increased considerably (see below). It may seem remarkable that a temperature rise of only 3°C could have such an effect, but it should be noted that this small rise doubles the alligator’s metabolic rate (Coulson and Hernandez, 1964). At any rate, this temperature effect illustrates one problem to be reckoned with in nutritional studies in reptiles. Efforts are being made to formulate a diet with commercially available feed ingredients that will give good growth and not require refrigeration. One of us (M.S.) has spent several years in the attempt and some of the results of feeding one of the formulations appears here. Since alligators are carnivorous, the composition of the dietary protein is most important and therefore the present study concerns protein nutrition, albeit in experiments that were usually of short duration. More detailed results of ongoing long-term experiments on formulated diets will be published later. Considering the present state of our knowledge, the results and conclusions detailed here should be considered preliminary. However, enough information is available to permit a few generalizations. Although the metabolic rate of the alligator is so low that their use in a study of vitamin requirements would prove an exercise in patience, they are excellent for a study in protein nutrition since one has days in which to observe the rates of digestion, amino acid absorption, protein synthesis from those amino acids, etc.
449
ROLAND A. COULSON et al.
450 MATERIALS
Experimental Alligators
AND METHODS
animals were obtained from the Rockefeller
Refuge of the Louisiana Department of Wildlife and Fisheries within a week or so after they were hatched in late August. They were placed in outside tanks containing water at about 3 1“C with exposure to sunlight for at least part of the day. Ground nutria were placed on elevated platforms giving them access to food for five consecutive days each week. Growth was rapid (Coulson et al., 1974) exceeding that in the wild more than IO-fold. They were not used in the diet test experiments until after their weights exceeded 500 g. The nutria diet was not supplemented with vitamins. Temperature Although most of the previous experiments on alligators in this laboratory had been conducted at 28°C the much faster rate of digestion at 31°C (Coulson and Coulson, 1986) led us to adopt the higher temperature. Where the outside tank temperatures varied somewhat, during the forcefeeding experiments, the temperature was kept constant (+0.3’C) in a controlled temperature chamber.
Water Since regurgitation was less frequent when the force-fed animals had access to water, and since ammonia excretion is limited by the degree of hydration (Coulson and Hernandez, 1964; Herbert, 1981), the animals were kept in water for the duration of each experiment. To reduce evaporation, the tops of the receptacles were covered loosely. This meant that the alligators were in the dark at all times except when blood or urine was being collected. One advantage of isolation and darkness was that this encouraged them to remain motionless most of the time.
Diet composition Nutria. Skinned, eviscerated nutria carcasses were ground (bones included) in an electric grinder to a coarse mince for some of the experiments, and fresh nutria muscle from the legs and back were used in others. In the dry diet experiments, ground nutria was dehydrated in a warmed desiccator with concentrated sulfuric acid as the desiccant. When dry, the residue was ground again in a laboratory blender and force-fed in a water slurry directly, or defatted and then fed. Fat was removed in the dry “fat-free” diets by extraction with dry ether three times, using five volumes of ether each time. The lipid content (see below) was determined by weight, and the steroid content by weighing the nonsaponifiable fraction. The crude de-fatting procedure left a lipid residue of less than 1% of the dry weight of the powder. The fresh eviscerated carcasses were analyzed and found to have the following composition: water, 70.9%; total ether-extractable lipid, 2.3%; non-saponifiable lipid, 0.12%; ash, 5.91%; and protein, 20.3%. On a dry weight basis the composition was: total lipid, 7.9%; non-saponifiable lipid, 0.43%; ash, 20.3%; and protein, 71.8%. The values as determined are only approximate since the actual composition varied with the age, size, and nutritional state of the nutria. We considered the protein content of the dry, defatted product to be about 70% for practical purposes. Bone was responsible for most of the high ash content. The second grinding reduced the bone fragments to a fine powder, making the components easy to assimilate. Chicken andfish. “Chicken” consisted of necks, hearts, livers, and gizzards as obtained from a local distributor. The mixed pieces were ground, dried, reground, and either fed with the fat in it, or defatted as described above and then fed. Compared with ground nutria, the chicken pieces that
were analyzed were higher in both fat and water and about equal in ash. The dry, defatted, chicken meal had about the same protein content as nutria prepared similarly. Again, we assumed the protein content to represent about 70% of the dry, non-fat weight. In experiments where chicken powder containing fat was given, the lipid content of the dry preparation was 37%, or over four times that of the fat content of the dry nutria powder. The one species of fish (CJ~oscion nebulosis, called locally “speckled trout”) that was fed was ground whole (head included), and then reground and defatted as above. The ash content of this fish was low at only about 7% on a dry, non-fat basis, but the protein content was again close to 70%. The amino acid composition of the hydrolyzed proteins of both fish and nutria was more variable than we would have liked, perhaps reflecting problems with our method of hydrolysis. The average of the individual essential amino acids in both was close enough for us to consider them for practical purposes to be identical. The somewhat rough -.approximations (in moles/l00 moles) were as follows: Asx, 7.27; Glx. 18.72; Thr. 3.75; Ser. 5.70: Pro. 7.32: Glv. 7.08: Ala, 3.09; Val, 5113; Cys-Cys, 0.38; Met, 2.38; Ile, 5.<8; Leu, 7.13; Tyr, 6.51; Phe, 4.18; Lys, 8.32; His, 2.61; Trp 1.81; and Arg, 3.42. This composition must have been close to the correct value since feeding a mixture of free amino acids in this ratio gave a plasma amino acid pattern almost identical to that found after feeding raw fish (Hebert and Coulson, 1976). The amino acid composition of nutria eaten by an alligator in the wild would differ from that above as a consequence of the ingestion of the skin containing collagen rich in glycine, proline and hydroxyprohne. Casein. Obtained from milk, this was a “feed grade” product obtained from the Sigma Chemical Company, St. Louis, Missouri, USA. Gelatin. This was a “laboratory” grade product supplied by Fisher Scientific Company, Fair Lawn, New Jersey, USA. Edestin. This was a preparation derived in this instance from hemp seed (Sigma), but similar in composition to the principal protein in garden peas. G&din. Obtained from wheat gluten, this was obtained from Sigma. This protein was fed with difficulty. In water it formed a sticky paste even more viscous than that obtained when wheat flour is mixed with water. As attempts to get it into alligator stomachs met with no success, it was necessary to introduce it packed in gelatin capsules. Unfortunately, this meant that the results reported for the gliadin experiments actually represented those for gliadin contaminated with about 7% of gelatin. Corn gluten meal. This was a “feed grade” product obtained from the University of Georgia Poultry Science feed mill. Unlike gliadin from wheat, it did not form a sticky paste when wet, and it was fed without difficulty. Soy. Isolated soybean protein, this was obtained from Ralston Purina Co., St. Louis, Missouri. Staton mix. Referred to as “mix” in the figures and tables, this formulation was a mixture of various commercial feed ingredients having the following composition: casein, 20%; blood meal, 25%; fish meal (menhaden), 15%; feather meal, 10%; meat and bone meal, 17%; poultry by-products meal, 10%; carboxymethyl cellulose, 1.j%; mixed-vitamins, 1%; and NaCl. 0.5%. The calculated (ether-extractable) fat content of the dry preparation was 5.2%, and the protein content, 72%. The calculated amino acid content of the hydrolyzed mix (in moles/l00 moles of those amino acids listed) appears below, compared with our analyses (*) for both the mix and for fish muscle, expressed in the same units. (For ease of comparison, the molar % of the 13 listed amino acids was given a value of 100.) In our analyses of the mix, Glx (glutamate + glutamine) was much lower than the value found for either nutria or fish, in the ratio of 18.7 to 8.7.
Protein nutrition in the alligator
Thr Val Met Be Leu Phe Lys His Trp Arg Ser Tyr GUY
Fish *
Mix
5.89 8.05 3.74 8.13 11.19 6.56 13.06 4.10 2.84 5.31 8.95 10.23 11.11
6.93 11.89 2.24 4.95 14.50 6.30 10.03 4.30
1.06 5.96 10.66 4.22 15.94
SUM 99.20 99.90 **Not determined by us.
Mix * 5.16 10.69 3.06 5.21 13.27 7.1 I 11.42 5.16 (1.06)** 8.74 6.58 4.31 18.22
451
Amino acid analyses
The analyzer was the original type designed by Spackman er al. (1958), but modified to make it capable of analyzing three samples in 6 hr. To complete this many analyses in that length of time, only 570 pm filters were used in the calorimeters. This meant that neither hydroxyproline nor proline were estimated routinely. (The detailed procedure has been described previously (Coulson and Hernandez, 1968).] Two amino acids (aspartate and tryptophan) were present in the plasma in such low concentrations, even after feeding, that changes in their concentrations could not be followed with any degree of accuracy, and therefore they are not included in the data.
99.99
RATIONALE OF THE EXPERIMENTS Staton mix plus methionine. This was the mix as shown
Feeding an alligator a diet deficient in a single or several essential amino acids would of course result in a serious nutritional deficiency over a period of time. Growth would cease in immature specimens and eventually they would actually lose weight. The Feeding procedures problem with such experiments is the length of time Dry preparations. Each was given at 0600 hr in an amount needed to detect the effects. In theory, it should be calculated to supply about IO g of protein/kg. The powders possible to test each protein’s biological value more were suspended in a water slurry and poured into the stomach through a long-stemmed funnel. When the funnel quickly by determining changes in the free amino had drained it was rinsed with a small amount of water and acids in the plasma after a single feeding. Should one withdrawn slowly. To prevent the alligators from biting the amino acid prove proportionately low in the plasma, funnel, the jaws were kept apart by means of a flat plywood presumably it was present in the digesting protein in board with a hole in the center through which the funnel was inadequate amounts. Such tests are difficult in most inserted. In most feedings the slurry was intubated without small mammals where the amount of the increase in incident, but in a few cases the food was regurgitated at once free amino acids in the plasma is low and the small and it was necessary to abandon the attempt. increase is of short duration. However, in a large Fresh nutria muscle. Pieces of nutria muscle were wetted and then pushed down the esophagus by means of a long reptile, plasma amino acids rise considerably during glass stirring rod. protein digestion and they remain elevated for days. Gliadin and gelatin. These were both given enclosed in It is important to note that this type of experiment large gelatin capsules. To make sure the capsules dissolved, measures the demand for each amino acid, and that about 50ml of water was poured into the stomach after the demand in one species may differ from that in introduction of the capsules. others. A simple chemical analysis of a protein will reveal which amino acids are present, and in what amounts, but the information often cannot be used to Blood collection Blood was collected from the tip of the tail by means of predict how satisfactory the protein will be when it is eaten. It may be digested incompletely, or its coma Pasteur pipette, placed in heparinized 0.4ml microcentrifuge tubes, and then centrifuged at 13,000 rpm for position may not match the demand in that particular 20 sec. Plasma was removed and 0.2 ml was added to a tube species. containing 3.8 ml of 95% ethanol to precipitate the proteins. The capacity of a small, growing alligator to A clear, colorless supernatant was obtained by centrifuging tolerate massive amounts of protein at a single feedat 2500rpm for five min. The supernatant was stored at ing makes this reptile well suited for such research. In -20’C until it was used for the amino acid analysis. previous experiments (Coulson and Hernandez, 1970) small caimans were force-fed meat containing as much as 15 g of protein/kg of body wt at a single Urine collection and analysis feeding. This contrasts with the 1 g of protein eaten The animals were kept dry at 31°C during the collection period. Urine was obtained by inserting a fire-polished glass per kg by an average man over a 24 hr period. The problem of deciding which amino acid is proportube (6 mm O.D.) into the cloaca and draining the urine into a flask containing 1 ml 1 N HCI (to trap NH,). To prevent tionally low in the plasma may be solved in part by loss, It was necessary to collect urine at 30 min intervals as use of a standard. If one assumes that an alligator the flow rate often exceeded 10 ml/kg per hr. After analysis could thrive on a diet of whole mammals or fish, then of ammonia by the method of Conway and Byrne (1933), the plasma free amino acid content after such a meal the urine was boiled briefly with a small amount of L&CO, may be considered to be close to the ideal, and (Z IOOmg) to solubilize the urates, then analyzed for uric deviations from that following feeding a lower qualacid by the method of Archibald (1957). Urine collection usually began about 4 hr after feeding, when the rate of ity protein could be apparent. In consideration of the above, in the experiments urine flow was near maximal, and continued for 2 or more reported here, alligators weighing from 0.5 to 2 kg hours. Blood samples obtained at the end of the collection were force-fed, blood was removed at 4, 7, 10, 14, 24, period were also analyzed for uric acid and for free amino acids. 30, 37, 48 and, in those cases where digestion was
above, except that it had been supplemented with methionine in an amount 73% in excesss of that in the mix.
452
ROLAND
A. COLJLSON et al.
slow, at 54, 72 and 96 hr. Changes in the concentration of each of the absorbed amino acids were followed by analyzing the plasma. RESULTS
“Essential” and “non-essential” amino acids
From an examination of the results given below, it could be argued that all amino acids except alanine and glutamine are “essential” for maximal growth rates in a small alligator. For example, if the rate at which a non-essential amino acid (such as glutamate) can be incorporated into body protein after a meal exceeds the rate of its gain in the plasma, the amino acid should be essential for maximal growth. It is true that the amino acid can be synthesized, but if that rate is slower than the maximal rate of its incorporation into body protein, a deficiency in it would be growth limiting. Therefore, the data indicate apparent deficiencies without regard for the traditional division between essentials and non-essentials. Peak times
The time required for each free amino acid to reach its maximum concentration in the plasma (peak time) after feeding is determined both by the rate of digestion of the protein, and by the rate of removal of its component amino acids from the plasma. When a protein digests slowly, free amino acids rise slowly in the plasma, and vice versa. However, should any one amino acid be proportionately low in the absorbed mixture, incorporation of that amino acid into body protein may remove it almost as fast as it is absorbed and its peak will occur before those of the others in more plentifu1 supply. (A “good” protein that digests slowly could be adequate for maintenance but not for maximum growth promotion since less could be fed per week.) On the basis of the average peak time, dried, defatted, powdered nutria, fish and chicken were not only digested more rapidly than the other diets, but the rate of removal of each of the absorbed amino acids was more nearly uniform (Table 1). In other words, each amino acid reached its peak at about the same time, and all disappeared at about the same percentage rate. Fresh nutria muscle digested more slowly than the non-fat, dry nutria, as would be expected, and the presence of fat delayed digestion of both nutria and chicken (see below). Edestin was the slowest to digest. Solely on the basis of peak times, one would predict that the animal diets were adequate, and that all the other diets were deficient in one or more amino acids. Those amino acids that appeared to reach a peak well before the average time for the others in each diet are marked with an asterisk in Table 1. Glutamine, glycine and alanine maxima were usually several hours late for the probable reason that their initial rises in the plasma were the result of absorption from the gut, while the prolonged elevations were the result of synthesis (see below). Peak concentrations
Table 2 shows the change (PM) in the concentrations of the individual amino acids (above the
$ 2 ?
Ala
Gly
Gla
Glu
Tyr
ser
Arg
His
LYs
Phe
Leu
Ile
Met
Vd
Thr
-
-.-__Peak 30 hr Peak 30hr Peak 30 hr Peak 3Ohr Peak 30 hr Peak 30hr Peak 3Ohr Peak 30 hr Peak 30 hr Peak 30hr Peak 30hr Peak 30 hr Peak 30 hr Peak 30 hr Peak Mhr
173&29 12Ok8 316 & 21 316k21 103&li 103fII 112rt_23 98+11 157k32 157&32 94+12 78 +_9 11X*23 104+23 92k 15 34+ 12 92_+14 74+ 13 109+45 109+45 46+ 14 441t: II 96& 10 96+ 10 604+ 127 438+_ 112 333 + 36 251 + 55 1004~50 788 _+70
Fresh nutria muscle
100*10 14+4 465f8 144*5 100+11 1424 177k25 16+9 355-661 90 It 37 137+20 s4+ 17 1622 17 39 f 18 127&27 46* 12 74_+13 30+11 113+31 18k21 63%- 19 12 i 13 71 & 18 22+ 13 248+M) 152 f 56 498 _+ 12 498 L- 12 1050 + 120 7702 II0
-
in fm
207 zt 56 187 +45 S40+ 153 399+ 116 155+57 127+_44 175_+ 16 116_+29 367 +_ 137 262 + 51 186 + 53 852 13 182 +48 85+18 196+_74 133 _+42 932 18 77+_ 14 251 + 74 249 + 49 Ill + 17 51 +6 167 + 51 109+35 4695 100 389 2 80 973 + 234 902 _+201 1722 + 245 1722 _+245
Fat nutria, dry
Table 2. Increase
Non-fat nutria, dry
&M)
65 + 9 -20_+8 231 F 30 230 + 59 152+30 140+35 53+ 11 32+ 10 104+_24 77 + 24 74_t 15 74+ 15 172 +36 64+ 19 116+23 104_C32 23 + 5 4+_2 235k40 95+2S 174 +29 109+41 509+52 357 & 78
109+20 70+ 13 374 +_83 335+64 -
acid concentrations
123+35 90+ 17 491 t_ 18 356 + 27 95+ 16 7+_5 S6+ 13 -15+5 354 f 97 263 + 37 217rt27 135+_5 74+20 51* 19 180-124 138+24 62 + 8 41* 14 148 + 36 98 + 23 127&20 7.5+9 45+8 6+_5 346+55 346 f 55 402f59 275 It 76 8002 114 _._ .._ 562 F 13U
amino
325 145 695 321 267 115 210 90 423 196 267 84 188 102 244 166 189 118 312 197 161 58 203 94 674 402 1830 1190 2300 ._._ IYltJ
Non-fat
in plasma
’
254 rl: 95 191 * 74 962 ?r 97 473 i 184 433 Ifr 61 223 C 56 446+30 171&93 792 + I42 228 rf: 128 336 + 52 69rfi 16 170227 73+26 289 + 116 176+67 i44i22 93*31 332 & 53 158 rt 35 289f48 36 F 24 243 + 25 133+_50 1393 _+282 576 f 222 665 +_ 16 511+ 135 2463 + 671 ..__ _~_ 1 IKZ * 355
dry
Non-fat chicken. 315 230 1072 517 418 420 405 405 688 591 203 172 213 213 180 130 159 132 234 144 134 80 192 130 838 645 558 313 1602 1262
dry
Fat chicken, 171 154 740 740 113 113 227 134 47t 449 155 150 118 104 248 240 60 21 274 214 287 136 13s 95 571 463 86 73 997 892
Casein
at the peak and at 30 hr after feeding
3014 2214 2124
4400
69 26 296 152 4 -2 113 43 208 114 130 34 6 -12 89 107 241 145 239 186 55 17 74 66 221 121
Gelatin
various
130 49 550 550 77 17 83 183 294 294 147 147 55 23 143 143 265 265 110 110 197 197 so 50 351 351 397 397 586 496
Edestin
foods corn G&din gluten ~____ 48 91 -7 67 305 280 192 190 23 40 8 32 178 171 177 165 279 867 190 617 137 loo 70 94 21 i __6 -20 231 54 138 IS 326 32 238 32 49 95 34 27 171 104 72 a2 212 40 114 36 4459 309 2439 241 163l 5 I255 -8 1616 293 1451 164
~._~ SOY 163 & 48 163 k48 456 f 35 4S6*35 10+4 -4&l 211&40 204?,11 413 *66 413 +_66 184_t25 184 + 25 48k 15 401t: II 51 *4 47 & 27 177t_33 l46+27 250 +- 57 212 &42 155 &70 89t I1 91 If: 13 71 I5 452 f 58 452 + 58 283 rt 68 245 & 74 663 * 152 663 f 152
454
ROLAND
A. COWSON el ai.
control values) at the time each was at its peak. Where the number of alligators in an experiment were adequate, the standard error of the mean (SEM) is shown. Arbitrarily, the concentration at a second time (30 hr) appears beneath the peak value. This type of data presentation represents a compromise between including all the data for 4,7, 10, 14,24, 30, 37, 48, 54 and 72 hr, and only showing the peak concentration. Where the value at 30 hr was well below that at the peak, the concentration was declining rapidly, and where both values were about equal, removal was slow. Apparent biological value of the diets summarized
Table 3 summarizes the apparent deficiencies in the several diets based both on the peak concentrations in the plasma relative to those found after feeding the animal diets, and on the relative time at which the peaks were reached. Some comments on the diets other than nutria, fish and chicken appear below. Casein. This protein was slow to digest but nutritionally complete with the exception of arginine and glycine. It was well tolerated and little appeared in the feces. Gektin. Perhaps the poorest protein available, it proved quite easy to digest. Not only is it lacking several amino acids, but those present are in such an improper ratio as to render body protein synthesis from its component amino acids impossible. Failure to remove the absorbed amino acids by incorporating them into protein forces the alligators to resort to the slower catabolic routes for their disposal. Feeding large amounts (at 28°C) to alligators and turtles in previous experiments (Herbert and Coulson, 1976) caused a number of deaths. Edestin. Only a single alfigator was fed this protein. Aithou~h it digested slowly, and was deficient in several amino acids, none of the deficiencies were serious. Gliadin and corn gluten meal. Gliadin, an important protein of wheat flour, digested slowly at 31°C and much more slowly (if at all) at 28°C (Coulson and Hernandez, 1983). Although of limited nutritional value, its paste-like consistency when wet should make it an excellent binder for making diet pellets. In contrast, corn gluten meal, which contains a protein of the same class as gliadin, appeared to have less
satisfactory binding properties (if its consistency when wet is a valid measure). Isolated soybean protein. As far as the alligator is concerned, this vegetable protein, commonly used in animal and human foods, is not a satisfactory substitute for animal protein. It was deficient in some amino acids, slow to digest, and considerable amounts appeared in the feces. Staton mix. Its composition (see above) was designed to provide a mixture of acceptable feed grade proteins with high stability at room temperature in the dry state. The results reported here were derived from tests on only one such mix, and they are of value principally for illustrating the single feeding approach to determine biological value. Changes in individual amino acids following force-feeding dry nutria powder are compared with those following feeding the mix (Fig. 1). For some unknown reason, the curves are somewhat irregular with a notch or shoulder appearing a few hours after feeding. It is possible that this is an artifact caused by dilution of the plasma amino acids by absorption of a large amount of water from the stomach between the seventh and tenth hours after feeding. Numbers appearing inside the curves refer to peak values (PM). Where an amino acid was present in sub-optimal amounts in the protein, the peak concentration was low and the total area was also small compared with that for other amino acids in plentiful supply. To the right are bar graphs comparing the relative areas of the curves. When nutria was fed, the area of each amino acid curve was about the same (excluding the nitrogen carriers, glycine, alanine and glutamine). When the mix was fed, the amino acid areas varied, those that were deficient being smaller. Deficiencies in methionine, isoleucine and glutamate are apparent. e~~tarn~ne, glycine, alun~ne, and glutamate
The first three are the principal distributors of nitrogen, accounting for about half the plasma total. Glutamate (like aspartate) is never a major constituent in plasma (Coulson and Hernandez, 1983). Glutamine. That an alligator can digest a protein rich in glutamine and absorb this amino acid unchanged may be seen from the results of the gliadin experiment where peak glutamine exceeded 4 mM, or 46% of the total free amino acids. It may then be
Table 3. Apparent amino acid deficiencies in various foods as determined in the alligator -_.--..Mix Mix + Met Casein Gelatin Edestin Gliadin Corn gluten SOY
__ _
Thr ~~
~~~Val_
__Met._ x*
x
_tie _ x x
_Leu
Phe
x ix
x *x
x x x
l x = deficiency. ** t x = possible or marginal deficiency. Fresh nutria muscle 1 Non-fat. drv nutria 1 Fat. dry n&a No deficiencies evident Non-fat, dry fish Non-fat, dry chicken Fat, dry chicken
IX
His Lys + x ** *x x x x x x
Arg
Ser
x
GUY
x x
x
Glu x x
ix x
TY~
ix zkx x
x ,x
+x x x
x
Protein nutrition in the alligator
455
Rolatlvo
20
40
amar
60
Time
(hrl
Fig. 1. The effects of feeding 10 g of protein/kg in the form of dry, non-fat nutria powder compared with those seen after feeding an equal amount of protein in the mix. So that the relative changes could be compared more easily, in each case the peak concentration was given an arbitrary value of 100, and the amount of the changes before and after the peak are plotted as % of the peak value. Numbers inside the curves represent the increase in each amino acid in the plasma @M) above that of the non-fed controls. For SEM at the peak and at 30 hr, see Table 2. Bars to the right indicate the relative areas of the amino acid curves. Where the areas of the curves were nearly uniform in the nutria-fed group (glycine, alanine and glutamine excepted), they are quite irregular in those fed the mix. The reason for the consistent notching of the curves at about 10 hr after feeding is unknown, but it could be the result of dilution of the plasma from absorption of the large amount of water given with the food.
assumed that a low peak after feeding would indicate a low glutamine content in the protein fed. Glycine. Evidently, peak concentration of this amino acid also reflects its content in the protein fed.
It was very high in the gelatin experiment and almost absent after corn gluten meal feeding. In common with glutamine and alanine, synthesis from other amino acids was responsible for its more prolonged elevation (Table 1, Fig. 1). Alunine. The range in peaks was from 293 p M after corn gluten to 2463 PM in those fed chicken. Even where the rise was small, adequate amounts for protein synthesis seem to have been available during digestion of all 14 diets. Glutamate. Most of the glutamic acid in food protein is apparently degraded in the gut wall in the dog (Neame and Wiseman, 1957). However, our results seem to indicate that in the alligator at least, the plasma content after feeding was related to the content in the protein. For example, the Staton mix had only about 40% of the glutamine plus glutamate content of fish, an amount apparently below that needed to prevent a deficit in the plasma. One might expect glutamine to serve as an adequate substitute, but it did not seem to. There was some correlation between the plasma glutamine concentration and that of glutamate, but the significance is questionable.
Relationship between the amino acid content of proteins and peak values
The reported amino acid content of the various proteins was related in most cases to the height of the peak value, but there were some notable exceptions. Plasma alanine concentrations were usually considerably higher than would have been predicted from the protein alanine content, and in part, this was true for glycine. In all cases where the protein content of an essential amino acid was half or less of what would be considered ideal (using the composition of fish or nutria as an “ideal” standard), the peak plasma concentration was very low, and in the case of both methionine and lysine, a modest deficit in the fed protein always led to a major shortage in the plasma. Unfortunately, the exact content of glutamine and glutamate in most of the proteins tested was unknown. Hydrolysis in either acid or alkali converts glutamine to glutamate and therefore the combination in the protein can only be designated as Glx, the symbol for the combination of the two. Where the peak glutamine concentration was very high, it would be reasonable to assume that most of the Glx was glutamine, and where it was very low, it was probably principally in the form of glutamic acid (see above).
ROLAND A. COULSON
456
et al.
Effect of fat on the rate of digestion As noted above, fat delayed the digestion of nutria and chicken. The difference between the fat and non-fat experiments is shown in Fig. 2. Long-term growth rates of alligators fed non-fat nutria powder Two alligators fed 10 g of protein/kg per day in the form of nutria powder on 5 consecutive days each week grew rapidly (Fig. 3). They were weighed weekly at the conclusion of the Z-day fast for a total of 14 weeks. The plotted weights gave a remarkably straight line. The gain in per cent per week fell as the alligators grew, while the actual gain in grams remained constant. At the conclusion of the feeding experiment, the alligators appeared in good health. Considering the metabolic rate of the alligator, even 14 weeks should be judged a short-term experiment, and had the feeding been continued for several more months, deficiencies in vitamins, essential fatty acids, etc., might have been observed. NITROGEN EXCRETION PATTERNS
Since alligators raised in captivity sometimes develop gout (Coulson et al., 1973) nitrogen excretion patterns and plasma urate levels were monitored in groups of animals force-fed different diets or forcefed the same diet at different temperatures. In some experiments a single meal was fed to fasted animals; in others, the animals were fed 5 days a week for l-14 weeks. E#ect of iemperatare Groups of three alligators were force-fed lean nutria muscle, 2.5% of body weight, while being maintained at 25, 28 or 31”C, and nitrogen excretion, plasma urate, and plasma amino acid levels monitored daily for 7-8 days. Blood was obtained at 0 hr (just before feeding), and at 20, 44, 69, 92, 116, 140, and 165 hr after feeding. The animals were kept dry during collection of timed urine samples for 4-6 hr each day. At ail other times they were kept in water in covered containers.
Time
ihr)
Fig. 2. The effect of fat on the rate of digestion and assimilation of dry, powdered nutria. Both groups (six animals in each) were fed 10 g of nutria protein per kg. For SEM at the peaks and at 30 hr, see Table 2. The fat content was about 8%.
700:
I 20
I
I
40 60 Time (days)
I 80
I 100
Fig. 3. Increase in body weight in two alligators fed dry, powdered, defatted nutria for several weeks. No vitamin supplements were given, but they were exposed to sunlight for about 2 hr/day, starting at week 5. At 25°C a temperature at which captive animals ordinarily will not eat much voluntarily, digestion was incomplete, and some animals regurgitated portions of undigested meat 2-3 days after feeding. At this temperature (N = 6), plasma urate was high after feeding [1.02 & 0.19 mM (mean + SEM) at 20 hr], uric acid excretion was increased about six-fold (to 6.13 + 0.68 mmol nitrogen~kg per day at 92 hr), but ammonia excretion increased only a little (to 2.98 + 0.51 mmol/kg per day at 92 hr). Since feeding at th% temperature was obviously unphysiological, further experiments at this temperature were not conducted. Although digestion is somewhat slower at 28 than at 31°C (Coulson and Coulson, 1986), there was no apparent difference in nitrogen excretion patterns, changes in plasma urate, or uric acid clearance between the two groups (see Table 4).
Fish powder us nutria powder Table 2 indicates that feeding fish powder resulted in greater increases in plasma glycine and alanine than did feeding nutria powder. These amino acids are important precursors of uric acid in many animals, and gout was a perennial problem when our alligators were routinely fed a diet of ground marine fish (Coulson et al., 1973). Therefore, three alligators were force-fed a single meal of defatted fish powder, 10 g protein/kg body wt, and their nitrogen excretion patterns monitored for 1 week. When plasma and urine values had returned to control levels, the same three animals were fasted for five days, fed defatted nutria powder (10 g protein/kg) and nitrogen excretion and plasma monitored as before. Although these experiments confirmed that plasma glycine and alanine rose significantly higher in the fish-fed group than in those fed nutria (Fig. 4) there was no apparent difference in plasma urate levels or in nitrogen excretion patterns in the two groups, at least in this short-term, single-meal experiment. Effect of long-term feeding Nitrogen excretion patterns and plasma urate were monitored in two alligators force-fed defatted nutria
Protein
nutrition
in the alligator
457
Table 4. Changes in nitrogen excretion, plasma orate, and uric acid clearance in alligators fed lean nutria muscle at 28 and 31°C (mean + SEM) Hours after feeding 44
Temp. I”C)
N
0
20
28 3t
6 9
0 + 0.03 0 f 0.05
0.25 it 0.04 0.27 2 0.08
0.21 rf:0.06 0.22 * 0.06
0.14rto.04 0.15 iO.08
28 31
6 9
O&O.31 0 2 0.48
3.89 f 0.68 5.88 * 0.92
5.07 rf 1.44 6.97 & 1.67
4.03 * 1.52 6.18 f 2.19
3.34* 1.31 3.10 4 1.65
28 31
6 9
020.31 0 i: 0.45
5.83 & 1.87 5.36 rt 1.14
7.39+ 1.15 6.50 t 0.88
3.62 + 0.50 3.29 + 0.77
2.39 + 0.36 0.76 + 0.65
28 31
6 9
0+0.19 0 2 0.23
1.46$0.16 2.09 _+0.20
2.14_+0.31 2.54 & 0.26
1.80f 0.46 2.46 L-0.53
2.19 +0.74 2.44 + 1.05
Plasma urate (mM) Uric acid nitrogen excretion (mmolikg per day) Ammonia nitrogen excretion (mmol/kg per day) Uric acid clearance (l/kg per day)
92
69
0.06 & 0.01 -0.01 f 0.05
0.6
-ktl=; 1 E
Plosmo
0.4
02
/
I/
‘p-
urote
4-
R2;
_ _
a
I
Ez E
I
Uric acid nltroqut
1
: I
-~
20
acid
40 I7m6
amino ’
acid6
\ t \ \
0
nittqllt
Amonio
Uric
Ementlol
II’
3
0
0
T
c16oronce
60 (hr)
80
loo Time
lhrf
Fig. 4. Plasma mate, nitrogen excretion, urine volumes, uric acid clearance, and changes in plasma amino acid levels (Mean + SD) in three alligators force-fed a single meal of dried, defatted fish powder, 10 g protein/kg body wt, (-A--), or dried, defatted nutria powder, 10 g protein/kg (-0-J The “essential amino acid” segment represents the change (from fasting control levels) in the sum of six amino acids: 7’hr, Val, Met, He, L_euand Phe. Uric acid excretion is plotted as uric acid nitrogen (mmoi uric acid x 4) to facilitate comparison of the two routes of nitrogen excretion.
ROLANDA. COULSONet al.
458
Time (days)
Fig. 5. Mean nitrogen excretion, plasma urate, and urine volumes in two alligators force-fed dried, defatted nutria powder 5 days a week for 14 weeks. Blood and urine samples were collected 46 hours after the 5th feeding
each week. Individual
values varied
powder (10 g protein/kg body wt) 5 days a week for 14 weeks. Two-hour timed urine samples were collected 4-6 hr after feeding on day 5 of each week; blood was taken for uric acid analysis at the end of the urine collection period. Figure 5 shows ammonia and uric acid nitrogen excretion levels, plasma urate concentrations and urine volumes through 96 days on this diet. Although ammonia nitrogen excretion was only about twice the uric acid nitrogen excretion in the early stages, continued feeding resulted in an increase in ammonia and a decrease in uric acid nitrogen excretion until ammonia exceeded uric acid nitrogen by about six-fold. Plasma urate levels fell steadily from an initial value of 0.53 mM to about 0.2 mM after 82 days of feeding. Uric acid clearance ranged from about 8.5 l/kg per day in the first week to about 10.5 l/kg per day at the end of the experiment. Urine volumes are included to show the close correlation with ammonia excretion levels. The urine ammonia concentration was therefore nearly constant at 149 + 2 mM (N = 26). There was no obvious ill effect of feeding this diet on either growth rates (Fig. 3), or on nitrogen excretion rates or plasma urate levels (Fig. 5). At the termination of this experiment, the same animals were fed the Staton mix diet for 7 days, then the “Mix + Met” formulation for 5 days. Blood and urine were collected on the last day of feeding each diet and analyzed. The values obtained in these short-term, multiple-feeding experiments were as follows: plasma urate, 0.22 mM (Mix) and 0.23 mM (Mix + Met); ammonia nitrogen excretion, 42 and 44 mmol/kg per day; uric acid nitrogen excretion, 10 and 11 mmol/kg per day; urine volumes, 282 and 302 ml/kg per day; uric acid clearance, 11 and 12 l/kg per day. These values are comparable to those obtained after long-term nutria feeding (Fig. 5) and indicate no obvious problem for nitrogen excretion mechanisms in the short term at least. CONCLUSIONS
Although some progress has been made toward an understanding of nutrition in the alligator, much
less than
10% from the mean values
shown.
remains to be done. The amount of fat that should be included in the diet is unknown. Too much fat leads to steatitis, a condition in which the liver may be destroyed by a massive intrusion of fat. Too little fat might limit the maximum rate of growth by forcing the alligators to rely on protein for all of the energy needs, and for the gluconeogenesis so necessary to prevent hypoglycemia. Although fat is not usually considered to be “protein sparing”, in an animal with a small brain and a low metabolic rate, the glycerol in fat can provide the nervous system with an adequate amount of glucose without the need for glycogenic amino acids. Presumably, if the animals would use only fat for their energy needs, more protein would be available for growth. In the experiments reported here, 10 g of protein were fed per kg. That quantity was not adopted for any valid scientific reason, but rather as a convenient amount for the preliminary experiments. Should this amount have exceeded the ability of the alligator to synthesize body protein, the excess would be converted to fat, which would increase the weight of the animal but not affect its length. So long as the alligator lengthens, the animal may be considered to be growing, in contrast to just gaining weight through fat deposition. The amount of each of the probably large number of vitamins required by a growing alligator is also unknown. It may be assumed that the vitamin requirement is a function of the metabolic rate, as that rate will govern the rate of their excretion, metabolic degradation, etc. Certain vitamins given in excess are toxic to mammals, and this would probably be true in an alligator. The stability of vitamins in the dry state at room temperature, when blended with other components of the diet, may also be a problem. Deficiencies in the Staton mix should not be considered proof that it would not be of value as an alligator feed. If those amino acids present are in a combination that will lead to growth at a near maximal rate if eaten in sufficient amounts, then the problem would be less one of nutrition than one of economics. Improvements in the balance could then decrease the amount needed and lower the cost.
Protein nutrition in the alligator Results
of ongoing
feeding
trials
with formulated
feeds will be presented elsewhere. Acknowledgements-We thank the Louisiana Department of Wildlife and Fisheries for alligators and alligator food. The Louisiana Board of Regents Research and Development Program and the Louisiana Department of Wildlife and Fisheries provided financial support. REFERENCES
Archibald R. M. (1957) Calorimetric measurement of uric acid. Clin. Chem. 3, 102-105. Conway E. J. and Byrne A. (1933) An absorption apparatus for the micro-determination of certain volatile substances. 1, The micro-determination of ammonia. Biochem. J. 27, 419429.
Coulson R. A. and Coulson T. D. (1986) Effect of temperature on the rates of digestion, amino acid absorption and assimilation in the alligator. Camp. Biochem. Physiol. 83A, S-588.
Coulson T. D., Coulson R. A. and Hernandez T. (1973) Some observations on the growth of captive alligators. Zoologica 58, 47-52.
Coulson R. A. and Hernandez T. (1964) Biochemisrry of the Alligator, A Study of Metabolism in Slow Motion. Louisiana State University Press, Baton Rouge. Coulson R. A. and Hernandez T. (1968) Amino acid metabolism in chameleons. Comp. Biochem. Physiol. 25, 861-872.
459
Coulson R. A. and Hernandez T. (1970) Protein digestion and amino acid absorption in the caiman. J. Nurr. 100, 81&826. Co&on R. A. and Hernandez T. (1974) Intermediary metabolism of reptiles. In Chemical.Zooiogy (Edited by Florkin M. and Scheer B. T.). Vol. 9. DD. 217-247. AA Academic Press, New York. ” Coulson R. A. and Hernandez T. (1983) Alligator Merabolism. Studies on Chemical Reactions in vivo. Pergamon Press, Oxford. Herbert J. D. (1981) Nitrogen excretion in “maximally-fed” crocodilians. Comp. Biochem. Physiol. 69B, 499-504. Herbert J. D. and Coulson R. A. (1976) Plasma amino acids in reptiles after feeding protein or amino acids and after injecting amino acids. J. Nurr. 106, 1097-l 101. Joanen T. and McNease L. (1987) Alligator farminn research in Louisiana. Proceedings of- a Technic> Conference on Crocodile Conservation and Management,
January 1985, Darwin, Australia (in press). Lance V., Joanen T. and McNease L. (1983) Selenium, vitamin E, and trace elements in the plasma of wild and farm-reared alligators during the reproductive cycle. Can. J. Zool. 61, 17441751. Neame K. D. and Wiseman G. (1957) Transamination of glutamic and aspartic acids during absorption by the small intestine of the dog in uivo. J. Physiol., Land. 135, 442450.
Spackman D. N., Stein W. and Moore S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyr. Chem. 30, 119&1206.