# Presentation: 351257

## NIH-Primate Concentrate Supplement, A Novel  Non-Human Primate Diet Supplement

- Dennis Barnard1 and Carol Clarke2
**1 ****Veterinary Resources Program, Division of  Intramural Research Services, Office of Resource**** ****Services**
**2 ****Animal Care Branch, Division of Intramural Research, National Institute of Allergy and Infectious Diseases**

## Abstract

## Introduction

-        The nutritional status of monkeys involved in biomedical research can have a significant influence on experimental results.  Laboratory monkeys involved in research protocols may become anorexic and require dietary supplementation. Along with the anorexia related to research, New World primates such as Aotus and Callitrichidae are fastidious eaters that do not typically consume the nutritionally balanced laboratory primate diets available, therefore require dietary supplementation.   A common practice in primate research facilities is to provide dietary supplements produced in-house to monkeys that are anorexic.  The typical in-house non-human primate diet supplement is a combination of laboratory monkey diet, fruit, juice, water, and vitamin and mineral supplements.   The ingredients are mixed into a mash consistency that can be formed into meat balls or served in a cup.   To get the mash consistency, the major ingredient is water or juice, which is 60% or more of the total weight.  On an as-is basis, this results in a dilution of caloric density and nutrient concentration of the supplement by 60% or more.   Furthermore, the commercially available vitamin and mineral supplements that are used are not concentrated enough to overcome this dilution factor.    A very palatable supplement that does not provide the nutritional requirements, and may be functioning as a sole-source diet, can be detrimental to the monkeys’ health and experimental results.  The diet supplement, NIH Primate Concentrate (NIH-PC), has been developed to provide nutritional requirements to non-human primates that are anorexic.  The NIH-PC was formulated so that it can be diluted up to 60%, with whatever ingredients are required to make it  palatable, and still meet the monkey’s nutritional requirements.  The concentrate is composed of highly digestible ingredients.  Four sources of high quality protein are used to provide the complete amino acid requirements. The primary fat source is soy oil, the single source of dietary fat that can provide close to optimal essential fatty acid concentrations as well as fatty acid proportions.  A series of palatability studies utilizing three flavors (cherry, raspberry, and cheese) of the NIH-PCS were performed by feeding them to healthy _Aotus_ _spp_ (owl) and _Macaca_  _mulatta_ (rhesus) monkeys.

## NIH- Primate Concentrate Formulation

- 	**	**_**Ingredients**_**	****	**_**Percentage by Weight**_**	****	****	****Isolated Soy Protein****	****	****	****24.000**
**	****	****Casein Dried****	****	****	****20.000**
**	****	****Rice Protein Concentrate****	****	****16.000**
**	****	****Soy Oil****	****	****	****	****14.000**
**	****	****Calcium Phosphate Tri.****	****	****  8.500**
**	****	****Dryfat Plus****	****	****	****  7.972**
**	****	****Mineral Mix****	****	****	****  3.000**
**	****	****Sugar Food Product****	****	****	****  3.000**
**	****	****Vitamin Mix****	****	****	****  2.000**
**	****	****Sodium Chloride****	****	****	****  1.500**
**	****	****Tert-butylhydroquinone****	****	****  **_**0.028**_
**	****	****	****	****	****                ****100.000****	**
**	****	**

## NIH-Primate Concentrate Proximate Analysis

_**Nutrient**_**                    **_**Concentration**_
**Crude Protein      %****	****       44.0**
**Crude Fat             %        25.0**
**Crude Fiber          %****	****         0.3 **
**Ash                        %        11.9**
**   **

## NIH Primate Concentrate Supplement Formulations(Ingredients measured by weight)

- 		**	****	**_**Raspberry Flavor**_
**	****	****	****	****Primate Concentrate 40%**
**	****	****	****	****Monkey Diet 15%**
**	****	****	****	****Apple juice 24.995%**
**	****	****	****	****Bananas 20%**
**	****	****	****	****Raspberry flavor 0.5%**
**	****	****	****	**_**Cherry Flavor**_
**	****	****	****	****Primate Concentrate 40%**
**	****	****	****	****Monkey Diet 15%**
**	****	****	****	****Apple juice 24.995%**
**	****	****	****	****Bananas 20%**
**	****	****	****	****Cherry flavor 0.5%**
**	****	****	****	**_**Cheese Flavor**_
**	****	****	****	****Primate Concentrate 40%**
**	****	****	****	****Monkey Diet 10%**
**	****	****	****	****Apple juice 25%**
**	****	****	****	****Bananas 15%**
**	****	****	****	****Cheese flavor 10%**

## Proximate Analysis for NIH- Primate Concentrate Supplement

- 	  	Crude protein		%	Minimum  20.10
- 		Crude fat		%	Minimum  10.75
- 		Crude fiber		%	Maximum   1.90
- 		Gross Energy	         Kcal/g	Minimum    2.80

## Vitamin Concentrations for NIH- Primate Concentrate Supplement

- 		Vitamin A		IU/g		 23.00
- 		Vitamin D		IU/g		   6.90
- Alpha-tocopherol		PPM		 99.00
- 		Thiamin			PPM		 29.40
- Riboflavin		PPM		 21.80
- Niacin			PPM	             120.00
- Pantothenic Acid		PPM		 90.00
- Choline			PPM	            1637.00
- Pyridoxine		PPM	 	  19.40
- Folic acid		PPM		  33.00
- Biotin			PPM		    0.40
- Vitamin B12		Mcg/kg	    	  57.50
- Vitamin K		PPM	 	    9.50
- Vitamin C		PPM	             1920.00

## Mineral Concentrations for NIH- Primate Concentrate Supplement

- 		Calcium		%	   1.50
- Phosphorous	%	   0.74
- 		Potassium	%	   0.80
- 		Sodium		%	   0.27
- 		Magnesium	%	 ** ** 0.20
- 		 Iron		PPM 	203.00
- 		Zinc		PPM	 72.00
- 		Manganese	PPM	 58.00
- 		Copper		PPM	 10.30
- Cobalt		PPM	   0.92
- Iodine		PPM	 ** ** 2.70

## Amino Acid Concentrations for NIH- Primate Concentrate Supplement

- 		Arginine		% of total diet	   1.25
- Lysine  				   1.33
- Methionine 	    	   	   0.68
- 		Cystine				   0.24
- 		Tryptophan			  0.24
- 		Glycine				   0.35
- 		Histidine			   0.53
- 		Leucine				   1.73
- 		Isoleucine			   1.11
- 		Phenylalanine			   1.02
- 		Threonine			   0.79
- 		Valine				   1.25

## Equipment and Ingredients

- Batch Processor
- Balance
- Syringe
- NIH-Primate Concentrate
- Apple Juice
- Monkey Diet
- Bananas
- Fruit Flavor

## NIH-PCS Preparation Process

- Preparing to make the NIH-PCS
- Monkey biscuits after grinding in the batch processor
- NIH- Primate concentrate before and after being mixed with monkey biscuits
- NIH-PCS raspberry flavored meat balls

## Palatability Study Procedure

**Prepared NIH-PCS biweekly**
**25 Aotus monkeys were fed a 10 g NIH-PCS meat ball daily along with their normal diet.  Uneaten NIH-PCS was removed from the pans and cages and weighed 24 hours after feeding to determine how much of the NIH-PCS was eaten**
**21 rhesus monkeys were fed a 25 g NIH-PCS meat ball daily along with their normal diet.  Uneaten NIH-PCS was removed from the pans and cages and weighed 15 minutes after feeding to determine how much of the NIH-PCS was eaten**
**The raspberry and cherry flavors were increased from 0.05%  to 1.5% over time to determine the affect on palatability**
**Cheese flavor was increased from 10% to 12% over time to determine the affect on palatability**
**Daily observations were made to detect diarrhea and inappetence **
**Monkeys were weighed regularly**

## Raspberry NIH-PCS

- Aotus Monkeys

- Cherry NIH-PCS

- Cherry NIH-PCS

- Cheese NIH-PCS

- Cheese NIH-PCS

- Rhesus Monkeys

-  Fed each flavor for 20 to 40 days
-  Recorded gastrointestinal disorders ( ie diarrhea)
-  Recorded inappetence
-  Recorded body weights

## Results of the Palatability Study

- The NIH-Primate Concentrate Supplement is highly palatable
- The fruit flavors were more palatable than cheese
- The monkeys did not experience diarrhea or constipation
- The aotus and rhesus monkeys slightly increased their weight during the palatability study

## Slide 16

## Slide 17

## PF= (number of days monkeys ate more than 75% of supplement)/ (number of days monkeys were fed supplement)

## Discussion

-         In non-human primate research facilities it is a common practice to feed dietary supplements along with the commercial primate diet to monkeys showing signs of inappetence or anorexia.   There are factors that can affect research that must be considered when feeding diet supplements.  Feeding diet supplements to monkeys on experimental protocols will introduce a variable into the research.  To prevent introducing a dietary variable into the research it is important that there is a documented formulation for the supplement and that all modifications of the formulation are documented.   The preparation of the diet supplement can also introduce variables if the ingredients are not measured accurately and the finished supplement is not a homogeneous mixture of the ingredients.  Manual preparation is very labor intensive and inconsistencies in the homogeneity are probable, which will affect palatability and nutrient content.   To avoid these problems the NIH-Primate Concentrate Supplement (NIH-PCS) was prepared using a syringe to measure small volumes, batch processor, and balance.  The batch processor is highly recommended for primate research facilities because it is a labor saving device that can improve the ingredient homogeneity and nutrient consistency of the supplement.
-         Typically, the diet supplements are developed in-house, from a combination of laboratory monkey diet, fruit, water, juice, vitamin and mineral supplements, and anything else to make it palatable.   These in-house supplements are usually very palatable, but do not meet the nutritional requirements of the monkeys.  If the diet supplement becomes the primary source of nutrition it can be detrimental to the monkeys’ health and affect experimental results.  This problem is particularly true for New World monkeys such as the Aotus and callitrichids which are fastidious eaters1.    The open formula NIH Primate Concentrate (NIH-PC) has been developed to provide nutritional requirements to non-human primates that are anorexic.  The NIH-PC was formulated so that it can be diluted up to 60%, with whatever ingredients are required to make it  palatable, and still meet the monkey’s nutritional requirements.    The protein, fat, and gross energy for the NIH-Primate Concentrate Supplement formulations used in this study were 20.1%, 10.8%, and 2.8 kcal/g, respectively.  The vitamin, mineral, and amino acid concentrations met NRC requirements2.  The results show that the formulations of the NIH-PCS used in the feeding trial are very palatable to _Aotus_ _spp_ and _Macaca_ _mulatta_ monkeys.   The fruit flavors appeared to be slightly preferred over the cheese flavor.   The aotus and rhesus monkeys weights increased slightly from the start of the palatability study to the end, and no gastrointestinal problems were observed.  These results indicate that the NIH-PCS can be used as a dietary supplement for non-human primates.   Further evaluation will determine if it can be fed as a sole source diet.

## AcknowledgementThe authors acknowledge the contributions of Drs. Malcom Martin and Carol Long and the VRP Primate Unit technical staff to the concept of this project.

- References
- Barnard, D.E. and J.J. Knapka. “Callitrichidae Nutrition”, in _*The Cotton-Top Tamarin: A Primate Model for the Study of Colon Carcinogenesis*_, (N. Clapp, ed.), CRC Press, 1993, Chapter 3.
- National Research Council. “Nutrient Requirements of Nonhuman Primates”, National Academy of Science, Washington, D. C., 1978.