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CHAPTER3/ Hamster / Materials and Methods 117

3.1 a) Introduction to Hamster

Hamsters are rodents belonging to the subfamily Cricetinae. The subfamily contains about

25 species, classified into six or seven genera. 577

Hamsters are crepuscular animals that burrow underground in the daylight to avoid caught

by predators. Their diets include a variety of foods, including dried food, berries, nuts, fresh

fruits and vegetables. In the wild, they feed primarily on seeds, fruits and greens, and will

occasionally eat burrowing insects.2 They have an elongated pouch on each side of their heads

that extends to their shoulders, which they stuff full of food to be stored, brought back to the

colony or to be eaten later.578

Hamster behavior varies depending on their environment, genetics and interaction with

people. Hamsters often used as laboratory animals because they are easy to breed incaptivity.

Hamsters have also established as popular small house pets, and sometimes accepted even in

areas where other rodents disliked, and their typically solitary nature can reduce the risk of

excessive litters developing in households.579

Figure 3.1: Golden Syrian Hamster

CHAPTER3/ Hamster / Materials and Methods

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Table 3.1.1: Hamster Temporal Range

Middle Miocene–Recent

Scientific Classification

Kingdom Animalia

Phylum Chordata

Subphylum Vertebrata

Class Mammalia

Order Rodentia

Suborder Myomorpha

Super family Muroidea

Family Cricetidae

Subfamily Cricetinae

3.1.1 History

Although the Syrian hamster or golden hamster (Mesocricetus auratus) first described

scientifically in 1839, researchers were not able to successfully breed and domesticate hamsters

until 1939. The entire laboratory and pet populations of Syrian hamsters appear to be

descendants of a single brother-sister pairing. These littermates captured and imported in 1930

from Aleppo (Syria) by Israel Aharoni, a zoologist of the University of Jerusalem. In Jerusalem,

the hamsters bred very successfully. Years later, animals of this original breeding colony

exported to the USA, where Syrian hamsters became one of the most popular pets and laboratory

animals. Comparative studies of domestic and wild Syrian hamsters have shown reduced genetic

variability in the domestic strain. However, the differences in behavioral, chronobiological,

morphometrical, hematological and biochemical parameters are relatively small and fall into the

expected range of interstrain variations in other laboratory animals.579-381

http://en.wikipedia.org/wiki/Animal

http://en.wikipedia.org/wiki/Chordate

http://en.wikipedia.org/wiki/Vertebrata

http://en.wikipedia.org/wiki/Mammal

http://en.wikipedia.org/wiki/Myomorpha

http://en.wikipedia.org/wiki/Muroidea

http://en.wikipedia.org/wiki/Cricetidae


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3.1.2 Early Literature

In 1774, Friedrich Gabriel Sulzer, a companion of Johann-Wolfgang von Goethe, devoted a

whole academic monography in the domain of social sciences and natural history to hamsters,

entitled "An approach to a natural history of the hamster" ("Versuch einer Naturgeschichte des

Hamsters"). In several instances, he used the hamster to document the equal rights of all beings,

including Homo sapiens.582

3.1.3 Etymology

The name "hamster" is a loanword from the German, which itself derives from earlier Old

High German hamustro. It is possibly related to Old Russian choměstrǔ, which is both a blend of

the root of Russian khomiak ”hamster” and a Baltic word cf. Lithuanian staras "hamster" or of

Persian origin.583-584

3.1.4 Description

3.1.2 Roborovski Hamster

Hamsters are typically stout-bodied, with tails shorter than body length, and have small,

furry ears, short, stocky legs and wide feet. They have thick, silky fur, which can be long or

short, colored black, grey, honey, white, brown, yellow, red or a mix, depending on the species.

http://en.wikipedia.org/wiki/File:Roborofskiohamster.jpg


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Two species of hamster belonging to the genus Phodopus, Campbell's dwarf hamster and

the Djungarian, and two of the genus Cricetulus, the Chinese striped hamster and the Chinese

hamster have a dark stripe down their heads to their tails. The species of genus Phodopus are the

smallest, with bodies 5.5 to 10.5 cm long; the largest is the European hamster, measuring up to

34 cm, long, not including a short tail of up to 6 cm. The Angora hamster, also known as the

long-haired or teddy bear hamster, which is a type of the golden hamster is the second- largest

hamster breed, measuring up to 18 cm long.579

Figure 3.1.3: Skeleton of European Hamster


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Figure 3.1.4: Yawning white Syrian Hamster Showing Large Incisors

The hamster tail can be difficult to see, as it is usually not very long about 1/6 the length of

the body, with the exception of the Chinese dwarf hamster, which has a tail the same length as

the body. One rodent characteristic that can be highly visible in hamsters is their sharp incisors;

they have an upper pair and lower pair that grow continuously throughout life, so must regularly

wear down. Hamsters are very flexible, but their bones are somewhat fragile. They are extremely

susceptible to rapid temperature changes and drafts, as well as extreme heat or cold.

3.1.5 Senses

Hamsters have poor eyesight; they are nearsighted and colorblind. to compensate for their

poor sight when in unfamiliar territory, hamsters have scent glands on their flanks. A hamster

rubs these areas of its body against various objects, and leaves a trail of smells to follow to return

to its home den Hamsters also use their sense of smell to identify pheromones and gender, and to

locate food. They are also particularly sensitive to high-pitched noises and can hear and

communicate in the ultrasonic range.580, 585-586

http://en.wikipedia.org/wiki/File:Yawning_white_syrian_hamster.jpeg


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3.1.6 Diet

Hamsters are omnivores. Although they can survive on a diet of exclusively commercial

hamster food, other items, such as vegetables, fruits, seeds and nuts, can given, but these should

be removed before they become rotten. Hamsters in the Middle East have known to hunt in

packs to find insects for food. Hamsters are hindgut fermenters and must eat their own feces to

recover nutrients digested in the hindgut, but not absorbed.577, 590

3.1.7 Behavior

A behavioral characteristic of hamsters is food hoarding. They carry food in their spacious

cheek pouches to their underground storage chambers. When full, the cheeks can make their

heads double, or even triple in size.577

Social Behavior

Figure 3.1.5: Hamsters Fighting

Most hamsters are strictly solitary. If housed together, acute and chronic stress may

occur, and they may fight fiercely, sometimes fatally. Some dwarf hamster species may

tolerate conspecifics. Russian hamsters form close, monogamous bonds with their mates, and if

separated, they may become very depressed. This happens especially in males. Males will

http://en.wikipedia.org/wiki/File:Hamster_fight.jpg


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become inactive, eat more and even show some behavioral changes similar to some types of

depression in humans. This can even cause obesity in the hamster.581

3.1.9 Chronobiology

Evidence conflicts as to whether hamsters are crepuscular or nocturnal. Khunen writes,

"Hamsters are nocturnal rodents who are active during the night...” but others have written that

because hamsters live underground during most of the day, only leaving their burrows about an

hour before sundown and then returning when it gets dark, their behavior is primarily

crepuscular. Fritzsche indicated although some species have been observed to show more

nocturnal activity than others do, they are all primarily crepuscular.580-581

Wild Syrian hamsters are true hibernators and allow their body temperature to fall close to

ambient temperature but not below 20°C. This kind of thermoregulation diminishes

the metabolic rate to about 5% and helps the animal to reduce the need for food during the

winter. Hamsters may not hibernate per se, but instead reduce the rate of a number of

physiological systems, such as breathing and heart rate, for short periods. These periods

of torpor defined as "a state of mental or physical inactivity or insensibility" can last up 10 days.

588

3.1.10 Burrowing Behavior

All hamsters are excellent diggers, constructing burrows with one or more entrances, with

galleries connected to chambers for nesting, food storage, and other activities.1 they use their

fore and hindlegs, as well as their snouts and teeth, for digging. In the wild, the burrow buffers

extreme ambient temperatures, offers relatively stable climatic conditions and protects against

predators. Syrian hamsters dig their burrows generally at a depth of 0.7 m. A burrow includes a

steep entrance pipe 4–5 cm in diameter, a nesting, a hoarding chamber and a blind-ending branch

for urination. Laboratory hamsters have not lost their ability to dig burrows; in fact, they will do

this with great vigor and skill if they provided with the appropriate substrate. Wild hamsters will

also appropriate tunnels made by other mammals; the Djungarian hamster, for instance, uses

paths and burrows of the pika. 581, 589


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3.1.11 Reproduction

Figure 3.1.6 A Mother Syrian Hamster with Pups Under One Week Old

3.1.11.1 Fertility

Hamsters become fertile at different ages depending on their species. Both Syrian and

Russian hamsters mature quickly and can begin reproducing at a young age about 4–5 week,

whereas Chinese hamsters will usually begin reproducing at two to three months of age, and

Roborovskis at three to four months of age. The female’s reproductive life lasts about 18 months,

but male hamsters remain fertile much longer. Females are in estrus about every four days,

which indicated by a reddening of genital areas, a musky smell and a hissing, squeaking

vocalisation she will emit if she believes a male is nearby.

When seen from above, a sexually mature female hamster has a trim tail line; a male's tail

line bulges on both sides. This might not be very visible in all species. Male hamsters typically

have very large testes in relation to their body size. Before sexual maturity occurs, it is more

difficult to determine a young hamster's sex. When examined, female hamsters have their anal

and genital openings close together, whereas males have these two holes farther apart.579

3.1.11.2 Gestation and Fecundity

Hamsters are seasonal breeders and several litters a year with several pups in each litter. The

breeding season is from April to October in the Northern Hemisphere, with two to five litters of

one to 13 young being born after a gestation period of 16 to 23 days. Gestation lasts 16 to 18

days for Syrian hamsters, 18 to 21 days for Russian hamsters, 21 to 23 days for Chinese hamsters


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and 23 to 30 for Roborovski hamsters. The average litter size for Syrian hamsters is about seven

pups, but can be as great as 24, which is the maximum number of pups that can be contained in

the uterus. Campbell's dwarf hamsters tend to have four to eight pups in a litter, but can have up

to 13. Djungarian hamsters tend to have slightly smaller litters, as do Chinese and Roborovski

hamsters.587

3.1.11.3 Intersexual Aggression and Cannibalism

Female Chinese and Syrian hamsters knew for being aggressive toward the male if kept

together for too long after mating. In some cases, male hamsters can die after attacked by the

female. If breeding hamsters, separation of the pair after mating recommended or they will attack

each other.

Female hamsters are also particularly sensitive to disturbances while giving birth, and may

even eat their own young if they think they are in danger, although sometimes they are just

carrying the pups in their cheek pouches. If captive female hamsters left for extended periods

about three weeks or more with their litter, they may cannibalize the litter, so the litter must

removed by the time the young can feed and drink independently. 580

3.1.11.4 Weaning

Figure 3.1.7 An Adult Female with Pups Feeding


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Hamsters are born hairless and blind in a nest the mother will have prepared in advance

after one week, they begin to explore outside the nest. They completely weaned after three

weeks, or four for Roborovski hamsters. Most breeders will sell the hamsters to shops when they

are three to nine weeks old.579

3.1.12 Longevity

Syrian hamsters typically live no more than two to three years in captivity, and less in the

wild. Russian hamsters live about two to four years in captivity, and Chinese hamsters 2.5-3.0 yr.

The smaller Roborovski hamster often lives to three years in captivity.577

3.1.13 Hamsters as Pets

Figure 3.1.8 Golden Hamster

The best-known species of hamster is the golden or Syrian hamster (Mesocricetus auratus),

which the type most commonly kept as pets. It also sometimes called a "fancy" hamster. Pet

stores also have taken to calling them "honey bears,” "panda bears", "black bears", "European

black bears", "polar bears", "teddy bears" and "Dalmatian", depending on their

coloration. Several variations, including longhaired varieties, grow hair several centimeters long

and often require special care. British zoologist Leonard Goodwin claimed most hamsters kept in

the United Kingdom were descended from the colony he introduced for medical research

purposes during the Second World War.590-591


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3.1.9: A Russian Dwarf hamster

Other hamsters kept as pets are the various species of "dwarf hamster.” Campbell's dwarf

hamster is the most common, they also sometimes called "Russian dwarfs"; however, many

hamsters are from Russia, so this ambiguous name does not distinguish them from other species

appropriately. The coat of the Djungarian or winter-white Russian dwarf hamster turns almost

white during winter. The Roborovski hamster is extremely small and fast, making it difficult to

keep as a pet. The Chinese hamster, although not technically a true "dwarf hamster", is the only

hamster with a prehensile tail about 4 cm long most hamsters have very short, nonprehensile

tails. Many breeders also show their hamsters, so breed towards producing a good, healthy, show

hamster with a view to keeping one or two themselves. Therefore, quality and temperament are

of vital importance when planning the breeding.577, 579

3.1.14 Classification

Taxonomists generally disagree about the most appropriate placement of the

subfamily Cricetinae within the super family Muroidea. Some place it in a family Cricetidae that

also includes voles, lemmings, and New World rats and mice; others group all these into a large

family called Muridae. Their evolutionary history recorded by 15 extinct fossil genera and

extends back 11.2 million to 16.4 million years to the Middle Miocene Epoch in Europe and

North Africa; in Asia, it extends 6 million to 11 million years. Four of the seven living genera


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include extinct species. One extinct hamster of Cricetus, for example, lived in North Africa

during the Middle Miocene, but the only extant member of that genus is the European or

common hamster of Eurasia.

Subfamily Cricetinae

Genus Allocricetulus

A. Species A. curtatus—Mongolian hamster

B. Species A. eversmanni—Eversmann's or Kazakh hamster

Genus Cansumys

A. Species C. canus—Gansu hamster

Genus Cricetulus

A. Species C. alticola—Tibetan dwarf or Ladak hamster

B. Species C. barabensis, including "C. pseudogriseus" and "C. obscurus"—Chinese

striped hamster, also called Chinese hamster; striped dwarf hamster

C. Species C. griseus—Chinese (dwarf) hamster, also called rat hamster

D. Species C. kamensis—Kam dwarf hamster or Tibetan hamster

E. Species C. longicaudatus—long-tailed dwarf hamster

F. Species C. migratorius—gray dwarf hamster, Armenian hamster, migratory grey

hamster; grey hamster; migratory hamster

G. Species C. sokolovi—Sokolov's dwarf hamster

Genus Cricetus

A. Species C. cricetus—European hamster, also called common hamster or black-

bellied field hamster

Genus Mesocricetus—golden hamsters

A. Species M. auratus—golden or Syrian hamster

B. Species M. brandti—Turkish hamster, also called Brandt's hamster; Azerbaijani

hamster

C. Species M. newtoni, Romanian hamster

D. Species M. raddei, Ciscaucasia hamster

Genus Phodopus, dwarf hamsters

A. Species P. campbelli, Campbell's dwarf hamster


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B. Species P. roborovskii, Roborovski hamster

C. Species P. sungorus, Djungarian hamster or winter-white Russian dwarf hamster

Genus Tscherskia

A. Species T. triton, greater long-tailed hamster, also called Korean hamster

3.1.15 Relationships among Hamster Species

Neumann et al. (2006) conducted a molecular phylogenetic analysis of 12 of the above 17

species using DNA sequence from three genes: 12S rRNA, cytochrome b and von Willebrand

factor. They uncovered the following relationships: 592-593

Phodopus Group: The genus Phodopus found to represent the earliest split among hamsters.

Their analysis included both species. The results of another study suggest Cricetulus

kamensis and presumably, the related C. alticola might belong to either this Phodopus group

or hold a similar basal position.

Mesocricetus Group: The genus Mesocricetus also forms a clade. Their analysis included

all four species, with M. auratus and M. raddei forming one sub clade and M. brandti and M.

newtoni another.

Remaining Genera: The remaining genera of hamsters formed a third major clade. Two of

the three sampled species within Cricetulus represent the earliest split. This clade contains C.

barabensis, presumably the related C. sokolovi and C. longicaudatus.

3.1.16 Similar Animals

Some similar rodents sometimes called "hamsters" did not currently classify in the hamster

subfamily Cricetinae. These include the maned hamster, or crested hamster, which is really

the maned rat. Others are the mouse-like hamsters, and the white-tailed .594

C CHAPTER3/ Rat/ Materials and Methods 130

3.2 b) Introduction to Rat

The brown rat, common rat, street rat, sewer rat, Hanover rat, Norway rat, brown Norway

rat, Norwegian rat, wharf rat or hood rat is one of the best-known and most common rats.

One of the largest muroids, it is a brown or grey rodent with a body up to 25 cm long. In

addition, a similar tail length, the male weighs on average 350 g and the female 250 g.

Thought to have originated in northern China, this rodent has now spread to all continents

except Antarctica, and is the dominant rat in Europe and much of North America, making it by at

least this particular definition the most "successful" mammal on the planet after humans. Indeed,

with rare exceptions, the brown rat lives wherever humans live, particularly in urban areas.

Selective breeding of Rattus norvegicus has produced the laboratory rat, a model organism in

biological research, as well as pet rats. 595

3.2.1 Naming and Etymology

Originally called the "Hanover rat" by people wishing to link problems in 18th century

England with the House of Hanover, it is not known for certain why the brown rat is

named Rattus norvegicus, as it did not originate from Norway. However, the English

naturalist John Berkenhout, author of the 1769 book Outlines of the Natural History of Great

Britain, is most likely responsible for popularizing the misnomer. Berkenhout gave the brown rat

the binomial name Rattus norvegicus, believing it had migrated to England from Norwegian

ships in 1728, although no brown rat had entered Norway at that time. 596

By the early to middle part of the 19th century, British academics were aware that the

brown rat was not native to Norway, hypothesizing that it may have come from Ireland, Gibraltar

or across the English Channel with William the Conqueror. As early as 1850, however, a more

correct understanding of the rat's origins was beginning to develop.597-598

The British novelist Charles Dickens acknowledged the misnomer in his weekly journal, All

the Year Round, writing599

"Now there is a mystery about the native country of the best known species of rat, the

common brown rat. It frequently called, in books and otherwise, the 'Norway rat', and it said to


have imported into this country in a shipload of timber from Norway. Against this hypothesis

stands the fact that when the brown rat had become common in this country, it was unknown in

Norway, although there was a small animal like a rat, but really a lemming, which made its home

there."

Academics began to understand the origins and corrected etymology of the brown rat

towards the end of the 19th century, as seen in the 1895 text Natural History by American

scholar Alfred Henry Miles:"The brown rat is the species common in England, and best known

throughout the world. It is said to have travelled from Persia to England less than two hundred

years ago and to have spread from thence to other countries visited by English ships.” 600

Though the assumptions surrounding this species' origins were not yet entirely accurate, by

the 20th century, it established among naturalists that the brown rat did not originate in Norway;

rather the species came from central Asia and likely China. Despite this, this species' common

name of "Norway rat" is still in use today. 601

3.2.2 Description

Figure 3.2.1 Comparison of the Physique of a Black rat (Rattus Rattus) with a Brown Rat (Rattus Norvegicus)

http://en.wikipedia.org/wiki/File:Comparison_Black_Rat_Brown_Rat_EN.svg


The fur is coarse and usually brown or dark grey, while the under parts are lighter grey or

brown. The length can be up to 25 cm, with the tail a further 25 cm, the same length as the body.

Adult body weight averages 550 g in males and about 350 g in females, but a very large

individual can reach 900 g. Rats weighing over 1 kg are exceptional, and stories of rats as big as

cats are exaggerations, or misidentifications of other rodents, such as the coypu and muskrat.

Brown rats have acute hearing, are sensitive to ultrasound and possess a very highly

developed olfactory sense. Their average heart rate is 300 to 400 beats per minute, with a

respiratory rate of around 100 per minute. The vision of a pigmented rat is poor, around 20/600,

while a non-pigmented (albino) with no melanin in its eyes has both around 20/1200 vision and a

terrible scattering of light within its vision. Brown rats are dichromates that perceive colors

rather like a human with red-green colorblindness, and their colour saturation may be quite faint.

Their blue perception, however, also has UV receptors, allowing them to see ultraviolet lights

that some species cannot. 602

3.2.3 Biology and Behavior

Figure 3.2.2: Brown Rat Skull

The brown rat is nocturnal and is a good swimmer, both on the surface and underwater, but

unlike the related black rat, it is a poor climber. Brown rats dig well, and often excavate

http://en.wikipedia.org/wiki/File:Rattusnorvegicusskull.png


extensive burrow systems. A 2007 study found brown rats to possess metacognition, a mental

ability previously only found in humans and some primates, but further analysis suggested they

might have been following simple operant conditioning principles.603-604

3.2.4 Communication

Brown rats are capable of producing ultrasonic vocalizations. As pups, young rats use

different types of ultrasonic cries to elicit and direct maternal search behavior, as well as to

regulate their mother's movements in the nest.

Although pups will produce ultrasounds around any other rats at 7 days old, by 14 days old,

they significantly reduce ultrasound production around male rats as a defensive response. Adult

rats will emit ultrasonic vocalizations in response to predators or perceived danger; the frequency

and duration of such cries depends on the sex and reproductive status of the rat.16,17

The female

rat will also emit ultrasonic vocalizations during mating.605-611

3.2.5 Chirping

Rats may also emit short, high frequency, ultrasonic, socially induced vocalization during

rough and tumble play, before receiving morphine, or mating, and when tickled. The

vocalization, described as a distinct "chirping,” has been likened to laughter, and is interpreted as

an expectation of something rewarding. Like most rat vocalizations, the chirping is too high in

pitch for humans to hear without special equipment. Pet owners often use bat detectors for this

purpose. 612

In clinical studies, the chirping is associated with positive emotions, and social bonding

occurs with the tickler, resulting in the rats conditioned to seek the tickling. However, as the rats

age, the tendency to chirp appears to decline. Rat chirp also can use for mosquito control. Other

ultrasonic vocalizations, including a lower-frequency 'boom' or 'whoom' noise can be produced

by bucks in a calm state, when grooming or settling down to sleep. 613


3.2.6 Diet

The brown rat is a true omnivore and consumes almost anything, but cereals form a

substantial part of its diet.

Figure 3.2.3: Brown Rat Eating Sunflower Seeds

Martin Schein, founder of the Animal Behavior Society in 1964, studied the diet of brown

rats and concluded that the most-liked foods of brown rats include scrambled eggs, macaroni and

cheese, and cooked corn kernels. According to Schein, the least-liked foods were raw beets,

peaches and raw celery.

Foraging behavior is often population-specific, and varies by environment and food

source. Brown rats living near a hatchery in West Virginia catch fingerling fish. Some colonies

along the banks of the Po River in Italy will dive for mollusks, a practice demonstrating social

learning among members of this species. Rats on the island of Norderoog in the North Sea stalk

and kill sparrows and ducks. 614-619

3.2.7 Reproduction and Life Cycle

The brown rat can breed throughout the year if conditions are suitable, with a female

producing up to five litters a year. The gestation period is only 21 days, and litters can number up


to 14, although seven is common. They reach sexual maturity in about five weeks.

The maximum life span is up to three years, although most barely manage one. A yearly

mortality rate of 95% estimated, with predators and interspecies conflict as major causes. When

lactating,female rats display a 24-hour rhythm of maternal behavior, and will usually spend more

time attending to smaller litters than large ones. Brown rats live in large, hierarchical groups,

either in burrows or in subsurface places, such as sewers and cellars. When food is in short

supply, the rats lower in social order are the first to die. If a large fraction of a rat population

exterminated, the remaining rats will increase their reproductive rate, and quickly restore the old

population level. 620

3.2.8Social Behavior

Rats commonly groom each other and sleep together. As with dogs, rats create a

social hierarchy, and each rat has its own place in the pack. Rats said to establish an order of

hierarchy, so one rat will be dominant over another one. Groups of rats tend to "play fight",

which can involve any combination of jumping, chasing, tumbling, and "boxing". Play fighting

involves rats going for each other's necks, while serious fighting involves strikes at the others'

back ends. If living space becomes limited, rats may turn to aggressive behavior, which may

result in the death of some animals, reducing the burden over the living space. Rats like most

mammals also form family groups, a mother and her young. this applies to both groups of males

and females. However, rats are territorial animals, meaning that they usually act aggressively or

scared of strange rats. Rats will fluff up their hair, hiss, squeal, and move their tails around when

defending their territory. Rats will chase each other, groom each other, sleep in group nests,

wrestle with each other, have dominance squabbles, communicate, and play in various other

ways with each other.621-624

Huddling is an additional important part of rat socialization. Huddling is often supposed to

have a heat-conserving function. Nestling rats especially depend on heat from their mother, since

they cannot regulate their own temperature. Huddling is an extreme form of herding. Other forms

of interaction include, crawling under, which is literally the act of crawling underneath one

another, walking over, also explained in the name, then there is allo-grooming, so-called to

distinguish it from self-grooming. Lastly, there is another type of contact called nosing, where a

rat gently pushes with its nose at another rat near the neck.625


3.2.9 Burrowing

Rats knew to burrow extensively, both in the wild and in captivity, if given access to a

suitable substrate. Rats generally begin a new burrow adjacent to an object or structure, as this

provides a sturdy "roof" for the section of the burrow nearest to the ground's surface. Burrows

usually develop to include multiple levels of tunnels, as well as a secondary entrance. Older male

rats will generally not burrow, while young males and females will burrow vigorously. Burrows

provide rats with shelter and food storage, as well as safe, thermo-regulated nest sites. Rats use

their burrows to escape from perceived threats in the surrounding environment; for example, rats

will retreat to their burrows following a sudden, loud noise or while fleeing an

intruder. Burrowing can therefore describe as a "pre-encounter defensive behavior,” as opposed

to a "post-encounter defensive behavior,” such as flight, freezing or avoidance of a threatening

stimulus. 626-629

3.2.10 Distribution and Habitat

Likely originating from the plains of Asia, northern China and Mongolia, the brown rat

spread to other parts of the world sometime in the middle Ages. The question of when brown rats

became commensally with humans remains unsettled, but as a species, they have spread and

established themselves along routes of human migration and now live almost everywhere

humans are. 630-633

The brown rat may have been present in Europe as early as 1553, a conclusion drawn from

an illustration and description by Swiss naturalist Conrad Gesner in his book Historiae ani

malium, published 1551–1558. Though Gesner's description could apply to the black rat, his

mention of a large percentage of albino specimens, not common among wild populations of

brown rats, adds credibility to this conclusion. Reliable reports dating to the 18th century

document the presence of the brown rat in Ireland in 1722, England in 1730, France in 1735,

Germany in 1750 and Spain in 1800, becoming widespread during the Industrial Revolution. It

did not reach North America until around 1750–1755.634-637

As it spread from Asia, the brown rat generally displaced the black rat in areas where

humans lived. In addition to being larger and more aggressive, the change from wooden

structures and thatched roofs to bricked and tiled buildings favored the burrowing brown rats


over the arboreal black rats. In addition, brown rats eat a wider variety of foods, and are more

resistant to weather extremes. In the absence of humans, brown rats prefer damp environments,

such as riverbanks. However, the great majority now linked to manmade environments, such as

sewage systems.636, 638

Figure 3.2.4: Brown Rat

It often said that there are as many rats in cities as people, but this varies from area to area

depending on climate, living conditions, etc. Brown rats in cities tend not to wander extensively,

often staying within 20 m of their nest if a suitable concentrated food supply is available, but

they will range more widely, where food availability is lower. In New York City, there is great

debate over the size of the rat population, with estimates from almost 100 million rats to as few

as 250, 000. Experts suggest New York is a particularly attractive place for rats because of its

aging infrastructure, high moisture and poverty rates. In addition to sewers, rats are very

comfortable living in alleyways and residential buildings, as there is usually a large and

continuous food source in those areas. 639-640

In the United Kingdom, some figures show the rat population has been rising, with

estimations that 81 million rats reside in the UK. Those figures would mean there are 1.3 rats per

person in the country. High rat populations in the UK often attributed to the mild climate, which

allow them higher survival rates during the winter months. The only brown rat-free zones in the

world are the Arctic, the Antarctic, some especially isolated islands, such as Iceland, the


province of Alberta in Canada and certain conservation areas in New Zealand. Antarctica almost

completely covered by ice and has no permanent human inhabitants, making it uninhabitable by

rats. The Arctic has extremely cold winters that rats cannot survive outdoors, and the human

population density is extremely low, making it difficult for rats to travel from one habitation to

another. When the occasional rat infestation noticed and eliminated, the rats are unable to

reinfest it from an adjacent one. Isolated islands are also able to eliminate rat populations

because of low human population density and geographic distance from other rat populations.641-

644

3.2.10.1 Alaska

Rat Island in Alaska infested with brown rats after a Japanese shipwreck in 1780. They had

a devastating effect on the native bird life. An eradication program started in 2007 and the island

declared rat free in June 2009.

3.2.10.2 Alberta, Canada

Alberta, Canada, is unusual in that rat infestation eliminated by aggressive government

action. Although it is a major agricultural area, it is far from any seaport and only a portion of its

eastern boundary with Saskatchewan provides a favorable entry route for rats. They cannot

survive in the boreal forest to the north, the Rocky Mountains to the west, or the semiarid High

Plains of Montana to the south. The first brown rat did not reach Alberta until 1950, and in 1951,

the province launched a rat-control program that included shooting and poisoning rats, and

bulldozing, burning down, and blowing up rat-infested buildings. The effort backed by

legislation that required every person and every municipality to destroy and prevent the

establishment of designated pests. If they failed, the provincial government could carry out the

necessary measures and charge the costs to the landowner or municipality. 645

In the first year of the program, 64 tonnes of arsenic trioxide spread throughout 8,000

buildings on farms along the Saskatchewan border. In 1953, the much less toxic and more

effective poison, warfarin, introduced. By 1960, the number of rat infestations in Alberta

dropped to below 200 per year. Currently, only zoos, universities and research institutes allowed

owning caged rats in Alberta, and possession of an unlicensed rat including pet rats is punishable

by a $5,000 fine or 60 days in jail. The adjacent and similarly landlocked province of


Saskatchewan initiated a rat control program in 1972. Moreover, has managed to reduce the

number of rats in the province substantially, although they have not been eliminated.646-647

3.2.11 Diseases

Similar to other rodents, brown rats may carry a number of pathogens, which can result in

disease, including Weil's disease, rat bite fever, cryptosporidiosis, viral hemorrhagic fever, Q

fever and Hantavirus pulmonary syndrome. In the United Kingdom, brown rats are an important

reservoir for Coxiella burnetii, the bacterium that causes Q fever, with sero prevalence for the

bacteria found to be as high as 53% in some wild populations.648-649

This species can also serve as a reservoir for Toxoplasma gondii, the parasite that

causes toxoplasmosis, though the disease usually spreads from rats to humans when domestic

cats feed on infected brown rats. The parasite has a long history with the brown rat, and there are

indications that the parasite has evolved to alter an infected rat's perception to cat predation,

making it more susceptible to predation and increasing the likelihood of transmission.650-651

Surveys and specimens of brown rat populations throughout the world have shown this

species is often associated with outbreaks of trichinosis, but the extent to which the brown rat is

responsible in transmitting Trichinella larvae to humans and other synanthropic animals is at

least somewhat debatable. Trichinella pseudospiralis, a parasite previously did not consider to be

a potential pathogen in humans or domestic animals, has found to be pathogenic in humans and

carried by brown rats.652-655

Brown rats sometimes mistakenly thought to be a major reservoir of bubonic plague, a

possible cause of the Black Death. However, the bacterium responsible, Yersinia pestis, is

commonly endemic in only a few rodent species and usually transmitted zoonotically by rat

fleas; common carrier rodents today include ground squirrels and wood rats. However, brown

rats may suffer from plague, as can many nonrodent species, including dogs, cats, and humans.

The original carrier for the plague-infected fleas thought to be responsible for the Black Death

was the black rat, and it has hypothesized that the displacement of black rats by brown rats led to

the decline of bubonic plague. This theory has, however, been deprecated, as the dates of these

displacements do not match the increases and decreases in plague outbreaks.626-628


3.2.12 Control

Some of the common methods used to control the number of Brown rats include

Trapping: Using traditional break-back traps, glue traps, live cage traps and other

humane traps, such as guns and gas

Poisoning: There are many types of poison available for controlling the Brown rat. The

use of poison controlled in most of the countries in the world so it is important to check

state legislations.

Proofing: Prevention is the best cure. There are ways to stop the infestation occurring at

the first place, such as blocking access points, better waste management and better

sewage design.

3.2.13 Uses in Science

Selective breeding of albino brown rats rescued from killed in a now-outlawed sport

called rat baiting has produced the albino laboratory rat. Like mice, these rats are frequently

subjects of medical, psychological and other biological experiments, and constitute an

important model organism. This is because they grow quickly to sexual maturity and are easy to

keep and to breed in captivity. When modern biologists refer to "rats", they almost always

mean Rattus norvegicus.659

3.2.14 As Pets

The brown rat kept as a pet in many parts of the world. Australia, the United Kingdom, and

the United States are just a few of the countries that have formed fancy rat associations similar in

nature to the American Kennel Club, establishing standards, orchestrating events, and promoting

responsible pet ownership. The many different types of domesticated brown rats include

variations in coat patterns, as well as the style of the coat, such as Hairless or Rex, and more

recently developed variations in body size and structure, including dwarf and tailless fancy rats.

3.2.15 b.1) Introduction to Wistar Rat


Figure 3.2.5: A Wistar Rat

Wistar rats are an outbred strain of albino rats belonging to the species Rattus norvegicus.

This strain developed at the Wistar Institute in 1906 for use in biological and medical research.

and is notably the first rat strain developed to serve as a model organism at a time when

laboratories primarily used common house mice. More than half of all laboratory rat strains are

descended from the original colony established by physiologist Henry Donaldson, scientific

administrator Milton J. Greenman and genetic research/embryologist Helen Dean King. The

Wistar rat is currently one of the most popular rat strains used for laboratory research. It

characterized by its wide head, long ears, and having a tail length that is always less than its body

length. The Sprague Dawley rat and Long-Evans rat strains developed from Wistar rats. Wistar

rats are more active than other strains like Sprague Dawley rats. The spontaneously hypertensive

rat and the Lewis rat are other well-known strains developed from Wistar rats. 660.661

CHAPTER 3/ Rabbit/Materials and Methods

142

3.3 C) Introduction to Rabbit

Rabbits are small mammals in the family Leporidae of the order Lagomorpha, found in

several parts of the world. There are eight different genera in the family classified as rabbits,

including the European rabbit, cottontail rabbits, and the Amami rabbit. There are many other

species of rabbit, and these, along with pikas and hares, make up the order Lagomorpha. The

male called a buck and the female is a doe; a young rabbit is a kitten or kit.662

3.3.1 Habitat and Range

Figure3.3.1 Outdoor Entrance to a Rabbit Burrow

Rabbit habitats include meadows, woods, forests, grasslands, deserts and wetlands. Rabbits

live in groups, and the best-known species, the European rabbit, lives in underground burrows, or

rabbit holes. A group of burrows called a warren. More than half the world's rabbit population

resides in North America. They are also native to southwestern Europe, Southeast Asia, Sumatra,


143

some islands of Japan, and in parts of Africa and South America. They did not naturally find in

most of Eurasia that a number of species of hares are present. Rabbits first entered South

America relatively recently, as part of the Great American Interchange. Much of the continent

has just one species of rabbit, the tapeti, while most of South America's southern cone is without

rabbits. The European rabbit has introduced to many places around the world. 663

3.3.2 Biology

3.3.2.1 Evolution

Because the rabbit's epiglottis engaged over the soft palate except when swallowing, the

rabbit is an obligate nasal breather. Rabbits have two sets of incisor teeth, one behind the other.

This way they can be distinguished from rodents, with which they are often confused. Carl

Linnaeus originally grouped rabbits and rodents under the class Glires; later, they separated, as

the scientific consensus is that many of their similarities were a result of convergent evolution.

However, recent DNA analysis and the discovery of a common ancestor has supported the view

that they share a common lineage, and thus rabbits and rodents are now often referred to together

as members of the super class Glires.665

3.3.2.2 Morphology

Figure 3.3.2: A White Fur Rabbit


144

The rabbit's long ears, which can be more than 10 cm long, are probably an adaptation for

detecting predators. They have large, powerful hind legs. The two front paws have 5 toes, the

extra called the dewclaw. The hind feet have four toes. They are plantigrade animals while at

rest; however, they move around on their toes while running, assuming a more digitigrade form.

Wild rabbits do not differ much in their body proportions or stance, with full, egg-shaped bodies.

Their size can range anywhere from 20 cm in length and 0.4 kg in weight to 50 cm and more

than 2 kg. The fur is most commonly long and soft, with colors such as shades of brown, gray

and buff. The tail is a little plume of brownish fur, white on top for cottontails.663, 666

3.3.2.3 Ecology

Rabbits are hindgut digesters. This means that most of their digestion takes place in

their large intestine and cecum. In rabbits, the cecum is about 10 times bigger than the stomach

and it along with the large intestine makes up roughly 40% of the rabbit's digestive tract. The

unique musculature of the cecum allows the intestinal tract of the rabbit to separate fibrous

material from more digestible material; the fibrous material passed as feces, while the more

nutritious material encased in a mucous lining as a cecotrope. Cecotropes sometimes called

"night feces,” are high in minerals, vitamins and proteins that are necessary to the rabbit's health.

Rabbits eat these to meet their nutritional requirements; the mucous coating allows the nutrients

to pass through the acidic stomach for digestion in the intestines. This process allows rabbits to

extract the necessary nutrients from their food.667-668

Rabbits are prey animals and are therefore constantly aware of their surroundings. For

instances, in Mediterranean Europe, rabbits are the main prey of red foxes, badgers, and Iberian

lynxes. If confronted by a potential threat, a rabbit may freeze and observe then warn others in

the warren with powerful thumps on the ground. Rabbits have a remarkably wide field of vision,

and a good deal of it is devoted to overhead scanning. They survive predation by burrowing,

hopping away in a zigzag motion, and, if captured, delivering powerful kicks with their hind

legs. Their strong teeth allow them to eat and to bite in order to escape a struggle.669-671


145

3.3.3 Sleep

Rabbits are crepuscular, most active at dawn and dusk. The average sleep time of a captive

rabbit said to be four to eight hours.672

3.3.4 Lifespan

Figure3.3.3: A Litter of Rabbit Kits Figure3.3.4: A Nest Containing Baby Rabbits

The expected rabbit lifespan is about 9–12 years; the world's longest- lived was 18 years.673-675

3.3.5 Diet and Eating Habits

Figure3.3.5: Young Rabbits in the Grass


146

Rabbits are herbivores that feed by grazing on grass, forbs and leafy weeds. In consequence,

their diet contains large amounts of cellulose, which is hard to digest. Rabbits solve this problem

by passing two distinct types of feces: hard droppings and soft black viscous pellets, the latter of

which known as caecotrophs and are immediately. Rabbits reingest their own droppings to digest

their food further and extract sufficient nutrients.676

Rabbits graze heavily and rapidly for roughly the first half hour of a grazing period usually

in the late afternoon, followed by about half an hour of more selective feeding. In this time, the

rabbit will also excrete many hard fecal pellets, being waste pellets that will not reingested. If the

environment is relatively non-threatening, the rabbit will remain outdoors for many hours,

grazing at intervals. While out of the burrow, the rabbit will occasionally reingest its soft,

partially digested pellets; this is rarely observed, since the pellets are reingested as they are

produced. Reingestion is most common within the burrow between 8 o'clock in the morning and

5 o'clock in the evening, carried out intermittently within that period. 663

Hard pellets made up of hay- like fragments of plant cuticle and stalk, being the final waste

product after redigestion of soft pellets. These only released outside the burrow and did not

reinvest. Soft pellets usually produced several hours after grazing, after the hard pellets have all

excreted. They made up of microorganisms and undigested plant cell walls. The chewed plant

material collects in the large cecum, a secondary chamber between the large and small intestine

containing large quantities of symbiotic bacteria that help with the digestion of cellulose and

produce certain B vitamins. The pellets are about 56% bacteria by dry weight, largely accounting

for the pellets being 24.4% protein on average. These pellets remain intact for up to six hours in

the stomach; the bacteria within continue to digest the plant carbohydrates. The soft feces form

here and contain up to five times the vitamins of hard feces. After excreted, they ate completely

by the rabbit and redigested in a special part of the stomach. This double-digestion process

enables rabbits to use nutrients that they may have missed during the first passage through the

gut, as well as the nutrients formed by the microbial activity and thus ensures that maximum

nutrition derived from the food they eat. This process serves the same purpose within the rabbit

as rumination does in cattle and sheep. Rabbits are incapable of vomiting. 677-668


147

3.3.6 Rabbit Diseases

Rabbits may affected by a number of diseases. These include pathogens that also affect

other animals and/or humans, such as Bordetella bronchiseptica and Escherichia coli', as well as

diseases unique to rabbits such as rabbit haemorrhagic disease and myxomatosis. Rabbits and

hares are almost never found to infect with rabies and have not known to transmit rabies to

humans. Among the parasites that infect rabbits are tapeworms such as Taenia serialis, external

parasites like fleas and mites, coccidia species, and Toxoplasma gondii. 679-680

3.3.7 As Pets

Figure 3.3.6: A Rabbit as a Pet

Domestic rabbits can kept as pets in a back yard hutch or indoors in a cage or house trained

to have free roam. Rabbits kept indoors often referred to as house rabbits. House rabbits typically

have an indoor pen or cage and a rabbit-safe place to run and exercise, such as an exercise pen,

living room or family room. Rabbits can trained to use a litter box and some can learn to come

when called. Domestic rabbits that do not live indoors can also serve as companions for their


148

owners, typically living in a protected hutch outdoors. Some pet rabbits live in outside hutches

during the day for the benefit of fresh air and natural daylight and brought inside at night.

Whether indoor or outdoor, pet rabbits' pens are often equipped with enrichment activities such

as shelves, tunnels, balls, and other toys. Pet rabbits often provided additional space in which to

get exercise, simulating the open space a rabbit would traverse in the wild. Exercise pens or lawn

pens often used to provide a safe place for rabbits to run. A pet rabbit's diet typically consists of

unlimited timothy-grass or other hay, a small amount of pellets, and a fair quantity of fresh

vegetables and need unrestricted access to fresh clean water. Rabbits are social animals. Rabbits

as pets can find their companionship with a variety of creatures, including humans, other rabbits,

birds, chinchillas, guinea pigs, and sometimes even cats and dogs. Rabbits can make good pets

for younger children when proper parental supervision provided. As prey animals, rabbits are

alert, timid creatures that startle easily. They have fragile bones, especially in their backs that

require support on the belly and bottom when picked up. Older children and teenagers usually

have the maturity required to care for a rabbit.682

3.3.8 Aggression in Rabbits

Rabbits may grunt, lunge and even bite. Usually they do not bite hard enough to break skin.

Rabbits become aggressive when they feel threatened. This behavior can corrected with the

proper tools. House Rabbit Society says that the owner of the pet needs to win its trust, with

certain behavioral tools. 683

3.3.9 As Food and Clothing

Figure 3.3.7: Rabbit Meat Sold Commercially Figure 3.3.8: Rabbit Pelt; Rabbit Pelt is Prized for its Softness

http://en.wikipedia.org/wiki/File:Rabbit_meat.jpg


149

Leporids such as European rabbits and hares are a food meat in Europe, South America,

North America, some parts of the Middle East. Rabbit still sold in UK butchers and markets, and

some supermarkets sell frozen rabbit meat. Additionally, some have begun selling fresh rabbit

meat alongside other types of game. At farmers markets and the famous Borough Market in

London, rabbits will display dead and hanging unbutchered in the traditional style next to braces

of pheasant and other small game. Rabbit meat once commonly sold in Sydney, Australia, the

sellers of which giving the name to the rugby league team the South Sydney Rabbitohs, but

quickly became unpopular after the disease myxomatosis introduced in an attempt to wipe out

the feral rabbit population. 684

When used for food, rabbits both hunted and bred for meat. Snares or guns usually

employed when catching wild rabbits for food. In many regions, rabbits also bred for meat, a

practice called cuniculture. Rabbits can then killed by hitting the back of their heads, a practice

from which the term rabbit punch derived. Rabbit meat is a source of high quality protein. it can

use in most ways chicken meat is used. In fact, well-known chef Mark Bittman says that

domesticated rabbit tastes like chicken because both are blank palettes upon which any desired

flavors can layer. Rabbit meat is leaner than beef, pork and chicken meat. Rabbit products are

generally labeled in three ways, the first being Fryer. This is a young rabbit between 4.5 and 5

pounds and up to 9 weeks in age. This type of meat is tender and fine-grained. The next product

is a Roaster; they are usually over 5 pounds and up to 8 months in age. The flesh is firm and

coarse grained and less tender than a fryer. Then there are giblets that include the liver and heart.

One of the most common types of rabbit to breed for meat is New Zealand white rabbit. 685-687

Issues associated several healths with the use of rabbits for meat. One of which

is tularemia or rabbit fever. Another is so-called rabbit starvation, due most likely to deficiency

of essential fatty acids in rabbit meat. Rabbits are a common food item of large snakes, such as

Burmese pythons and reticulated pythons, both in the wild and in captivity. 688

Rabbit pelts sometimes used for clothing and accessories, such as scarves or hats. Angora

rabbits bred for their long, fine hair, which can shear and harvested like sheep wool. Rabbits are

very good producers of manure; additionally, their urine, being high in nitrogen, makes lemon


150

trees very productive. Their milk may also be of great medicinal or nutritional benefit due to its

high protein content.689

3.3.10 Environmental Problems

Rabbits have been a source of environmental problems when introduced into the wild by

humans. Because of their appetites, and the rate at which they breed, feral rabbit depredation can

be problematic for agriculture. Gassing, barriers, shooting, snaring and ferreting have used to

control rabbit populations, but the most effective measures are diseases such

as myxomatosis and calicivirus. In Europe, where rabbits farmed on a large scale, they protected

against myxomatosis and calicivirus with a genetically modified virus. The virus developed in

Spain, and is beneficial to rabbit farmers. If it were to make its way into wild populations in

areas such as Australia, it could create a population boom, as those diseases are the most serious

threats to rabbit survival. Rabbits in Australia and New Zealand considered such a pest that

landowners are legally obliged to control them.690-691

3.3.11 Urban Legends

It commonly believed that pregnancy tests based on the idea that a rabbit would die if

injected with a pregnant woman's urine. This is not true. However, in the 1920s it discovered

that if the urine contained the HCG, a hormone found in the bodies of pregnant women, the

rabbit would display ovarian changes. The rabbit would then kill to have its ovaries inspected,

but the death of the rabbit was not the indicator of the results. Later revisions of the test allowed

technicians to inspect the ovaries without killing the animal. A similar test involved

injecting Xenopus frogs to make them lay eggs, but animal tests for pregnancy have been made

obsolete by faster, cheaper, and simpler modern methods.691

CHAPTER 3 / Materials and Methods 151

3.4 STAGE 1

3.4.1 Induction of Diabetes

In this study, we induce experimental diabetes with Streptozotocin to study the effects of

adding bivalent ions (calcium and magnesium) and mono-valent ions (sodium and potassium) to

the drinking water of hamster, offspring sexes was investigated. In addition, we study the effects

of adding bivalent ions (calcium and magnesium) and mono-valent ions (sodium and potassium)

to the drinking water of the first and second generation of hamsters, rats and rabbits offspring

sexes investigated. Streptozotocin, Streptozocin, Izostazin or Zanosar (STZ) is a synthetic

antineoplastic agent. An anti-tumor antibiotic and chemically related to other nitrosureas used

that classifically in cancer chemotherapy. Streptozotocin sterile powders provided and prepared

as a chemotherapy agent. Each vial of sterilized Streptozotocin powder contains 1 gr. of

Streptozotocin active ingredient with the chemical name, 2-Deoxy-2- [(methylnitrosoamino) -

carbonyl] amino]-D-glucopyranose and 200 mg citric acid. Pharmacia Company supplied

streptozotocin. Streptozotocin is available for intravenous use as a dry-frozen, pale yellow,

sterilized product. Pure Streptozotocin has alkaline pH. When it dissolved inside the vial in

distilled water as instructed, the pH in the solution inside the vial will be 3.5-4.5 because of the

presence of citric acid. This material is prepared in 1-gr vials and kept in cold store and

refrigerator temperature (2-8 °C) away from light. Control animals give an equivalent volume of

citrate buffer solution.

In this study, for accuracy of the experimental, we experimented on the 10 hamsters, rats,

rabbits and also first generation and second generation of them.

3.4.1.1 a) Induction of Diabetes on Hamsters

To Induction of diabetes, hamsters were fasted for 12 h before inducing diabetes, 20 adult

hamsters weighting 150-180 g (42-56 days old) used for inducing diabetes. The hamsters

injected by a single intra-peritoneal streptozotocin at the dose of 40 mg/kg of the body weight.

STZ freshly dissolved in 0.05 M citrate buffer, pH 4.5. For the intraperitoneal injection of STZ,


the hamsters was held in one hand in dorsal position, the injection site was swabbed using

povidone- iodine solution and the designated amount of STZ was injected within 10mn after

preparation in the caudal abdominal cavity using sterile 25g insulin needle. Streptozotocine

induces diabetes within 3 days by destroying the beta cells. Tail blood collected for glucose

determination using a glucometer (Accutrend Glucose, Roche Diagnostics and Mannheim,

Germany). Blood glucose levels measured on the third day, STZ injected hamsters with blood

glucose levels 15 m mol/l (270mg/dl) as well as polydipsia, polyuria and polyphagia for at least

one week considered diabetes.

40 control hamsters [10non diabetic (Ca, Mg), 10 neither diabetic nor (Ca, Mg) and 10 non-

diabetic (Na, K) and 10 neither diabetic nor (Na, K)] were injected with an equal volume of


3.4.1.2 b) Induction of Diabetes on Rats

To Induction of diabetes, rats were fasted for 12 h before inducing diabetes, 20 adult rats

weighting 100-130 g (70-80 days old) used for inducing diabetes. The rats injected by a single

intraperitoneal streptozotocin at the dose of 40 mg/kg of the body weight. STZ freshly dissolved

in 0.05 M citrate buffer, pH 4.5. For the intraperitoneal injection of STZ, the rats was held in one

hand in dorsal position, the injection site was swabbed using povidone- iodine solution and the

designated amount of STZ was injected within 10mn after preparation in the caudal abdominal

cavity using sterile 25g insulin needle. Streptozotocine induces diabetes within 3 days by

destroying the beta cells. Tail blood collected for glucose determination using a glucometer

(Accutrend Glucose, Roche Diagnostics and Mannheim, Germany). Blood glucose levels

measured on the third day, STZ injected rats with blood glucose levels 15 m mol/l (270mg/dl) as

well as polydipsia, polyuria and polyphagia for at least one week considered diabetes.

40 control Wistar rats [10non diabetic (Ca, Mg), 10 neither diabetic nor (Ca, Mg) and 10

non-diabetic (Na, K) and 10 neither diabetic nor (Na, K)] were injected with an equal volume of



3.4.1.3 c) Induction of Diabetes on Rabbits

To Induction of diabetes, rabbits were fasted for 12 h before inducing diabetes, 14 adult

rabbits weighting 1100-1300 g (9 months old) used for inducing diabetes. The rabbits injected by

a single intraperitoneal streptozotocin at the dose of 40 mg/kg of the body weight. STZ freshly

dissolved in 0.05 M citrate buffer, pH 4.5. For the intraperitoneal injection of STZ, the hamsters

was held in one hand in dorsal position, the injection site was swabbed using povidone- iodine

solution and the designated amount of STZ was injected within 10mn after preparation in the

caudal abdominal cavity using sterile 25g insulin needle. Streptozotocine induces diabetes within

3 days by destroying the beta cells. Tail blood collected for glucose determination using a

glucometer (Accutrend Glucose, Roche Diagnostics and Mannheim, Germany). Blood glucose

levels measured on the third day, STZ injected hamsters with blood glucose levels 15 m mol/l

(270mg/dl) as well as polydipsia, polyuria and polyphagia for at least one week considered

diabetes.

28 control rabbits [10non diabetic (Ca, Mg), 10 neither diabetic nor (Ca, Mg) and 10 non-

diabetic (Na, K) and 10 neither diabetic nor (Na, K)] were injected with an equal volume of



Table 3.4.1: Es timated Minerals Requirements of Adult Mice and Human

Mouse** (g/Kg)

Minerals

Amount

Per Kg diet

Human *(mg -ug/day)

Calcium 5.0 1000

Chloride 0.5 750

Magnesium 0.5 2-5

Phos phorus 3.0 700

Sodium 0.5 500

Potassium 2.0 2000

iron 35.0 8

Manganese 10.0 2-5

Zinc 10.0 10-12

Iodine 150.0 150

Molybdenum 150.0 75-250(ug)

**adapted from Nutrient Requirements of Nonhuman Primates

* Adapted from Lanus Micronutrient informat ion Center, Oregon State Unit


3.5 STAGE 2

3.5.1 Housing of Female and Male Hamsters

60 Adult female hamster weighting 150-180g (42-56 days old) and still in their reproductive

phase , were kept under constant conditions of light (12 h light-dark cycle) and humidity, fed

with standard laboratory chow ad libitum (Trouw, Gent, Belgium), and had free access to tap

water. Before initiation, the hamsters allowed to adapt for one week. The hamsters then weighed,

and blood sample tested for glucose and insulin levels. Vaginal wet smears made to determine

the estrous cycle of the hamsters. On the evening before estrus, female hams ters housed

overnight with male hamster; the presence of spermatozoa in a vaginal smear the next morning

defined as day one of pregnancy.

Diabetic hamsters and non-diabetic control group were kept in metabolic cages individually

and separately and within 16-21 days, on the specified diets (Ca, Mg , non Ca, Mg and Na, K and

non Na, K ) feeding and metabolism control (15g Ca /kg , 1.5 g Mg /kg and1% sodium

,potassium ) (Table 3.4.1). The first group diabetics (Ca, Mg)and second group non diabetics

(Ca, Mg) was supplied with drinking water mixed with 1% calcium and magnesium, the third

group was chosen as a control group neither diabetics nor Ca, Mg, pure drinking water was

supplied . The first group diabetics (Na, K) and second group non diabetics (Na, K) was supplied

with drinking water mixed with 1% sodium and potassium, the third group was chosen as a

control group neither diabetics nor Na& K, pure drinking water was supplied. After 16-21 days,

on the specified diets, the hamsters at the oestrus stage of the reproductive cycle caged with male

hamsters for mating and gestational day 1, confirmed on the observation of a vaginal plug. At

postnatal day two, the number of litters and the gender of pups recorded. Pups sexed by means of

the ano-genital distance, which is longer in males 692; this confirmed in later examinations during

pre weaning development. The data entered and analyses by SPSS software using t.test and the

p-value less than 0.05 considered as significant.


3.5.2 b) Housing of Female and Male Wistar Rats

60 Adult female Wistar rat weighting 100-130 g (70-80 days old) and still in their

reproductive phase , were kept under constant conditions of light (12 h light-dark cycle) and

humidity, fed with standard laboratory chow ad libitum (Trouw, Gent, Belgium), and had free

access to tap water. Before initiation, the rats allowed to adapt for one week. The rats then

weighed, and blood sample tested for glucose and insulin levels. Vaginal wet smears made to

determine the estrous cycle of the rats. On the evening before estrus, female rats housed

overnight with male rat; the presence of spermatozoa in a vaginal smear the next morning


Diabetic rats and non-diabetic control group were kept in metabolic cages individually and

separately and within 16-21 days, on the specified diets (Ca, Mg , non Ca, Mg and Na, K and


,potassium ) (Table 3.4.1). The first group diabetics (Ca, Mg)and second group non diabetics

(Ca, Mg) supplied with drinking water mixed with 1% calcium and magnesium, the third group

was chosen as a control group neither diabetics nor Ca, Mg, pure drinking water was supplied.

and also The first group diabetics (Na, K)and second group non diabetics (Na, K) was supplied

with drinking water mixed with 1% sodium and potassium, the third group was chosen as a

control group neither diabetics nor Na& K, pure drinking water was supplied. After 16-21 days,

on the specified diets, the rats at the oestrus stage of the reproductive cycle caged with male rats

for mating and gestational day 1, confirmed on the observation of a vaginal plug. At postnatal

day two, the number of litters and the gender of pups recorded. Pups sexed by means of the ano-

genital distance, which is longer in males 692; this confirmed in later examinations during pre

weaning development. The data entered and analyses by SPSS software using t.test and the p-

value less than 0.05 considered as significant.

3.5.3 C) Housing of Female and Male Rabbits

42 Adult female rabbits weighting 1100-1300 g (9 months old) and still in their

reproductive phase , were kept under constant conditions of light (12 h light-dark cycle) and

humidity, fed with standard laboratory chow ad libitum (Trouw, Gent, Belgium), and had free


access to tap water. Before initiation, the rabbits allowed to adapt for one week. The rabbits then

weighed, and blood sample tested for glucose and insulin levels. Vaginal wet smears made to

determine the estrous cycle of the rabbits. On the evening before estrus, female rabbits housed

overnight with male rabbits; the presence of spermatozoa in a vaginal smear the next morning


Diabetic rabbits and non-diabetic control group were kept in metabolic cages individually

and separately and within 16-21 days, on the specified diets (Ca, Mg , non Ca, Mg and Na, K and


,potassium ) (Table 3.4.1). The first group diabetics (Ca, Mg) and second group non diabetics

(Ca, Mg) was supplied with drinking water mixed with 1% calcium and magnesium, the third

group was chosen as a control group neither diabetics nor Ca, Mg, pure drinking water was

supplied. and also The first group diabetics (Na, K)and second group non diabetics (Na, K)

supplied with drinking water mixed with 1% sodium and potassium, the third group was chosen

as a control group neither diabetics nor Na& K, pure drinking water was supplied. After 16-21

days, on the specified diets, the rabbits at the oestrus stage of the reproductive cycle caged with

male rabbits for mating and gestational day 1, confirmed on the observation of a vaginal plug. At

postnatal day two, the number of litters and the gender of pups recorded. Pups sexed by means of

the ano-genital distance, which is longer in males 692; this confirmed in later examinations during

pre weaning development. The data entered and analyses by SPSS software using t.test and the

p-value less than 0.05 considered as significant.

3.6 STAGE 3

3.6.1 a) Housing of First Generation of Female and Male Hamsters

20 hamsters have chosen from the first generation of the previous experimented [(Ca, Mg)

and none (Ca, Mg)] and divided into two groups in the ratio male to female 1:5. The first group

was Ca and Mg, the second group was control unit without Ca and Mg. and also 20 hamsters

chosen from the first generation of the previous experimented [(Na, K) and non (Na, K)] and

divided into two groups in the ratio male to female 1:5. The first group was Na, K; the second

group was control unit without Na, K.


3.6.2 b) Housing of First Generation of Female and Male Rats

20 Wistar rats have chosen from the first generation of the previous experimented [(Ca, Mg)


was Ca and Mg, the second group was control unit without Ca and Mg. and also 20 Wistar rats




3.6.3 C) Housing of First Generation of Female and Male Rabbits

20 rabbits have chosen from the first generation of the previous experimented [(Ca, Mg)


was Ca and Mg, the second group was control unit without Ca and Mg. and also 20 rabbits




3.7 STAGE 4

3.7.1 a) Housing of Second Generation of Female and Male Hamsters

20 hamsters have chosen from the second generation of the previous experimented [(Ca,

Mg) and none (Ca, Mg)] and divided into two groups in the ratio male to female 1:5. The first

group was Ca and Mg, the second group was control unit without Ca and Mg. and also20

hamsters have chosen from the second generation of the previous experimented [(Na, K) and

non (Na, K)] and divided into two groups in the ratio male to female 1:5. The first group was

Na, K; the second group was control unit without Na, K.

3. 7.2 C) Housing of Second Generation of Female and male Rats

20 Wistar rats have chosen from the second generation of the previous experimented [(Ca,

Mg) and none (Ca, Mg)]; and divided into two groups in the ratio male to female 1:5. The first


Group was Ca and Mg, the second group was control unit without Ca and Mg. and also20 Wistar

rats have chosen from the second generation of the previous experimented [(Na, K) and none

(Na, K)]; and divided into two groups in the ratio male to female 1:5. The first group was Na, K;

the second group was control unit without Na, K.

3. 7.3 C) Housing of Second Generation of Female and Male Rabbits

20 rabbits have chosen from the second generation of the previous experimented [(Ca, Mg)

and none (Ca, Mg)]; and divided into two groups in the ratio male to female 1:1. The first group

was Ca and Mg, the second group was control unit without Ca and Mg. and also 20 rabbits have

chosen from the second generation of the previous experimented [(Na, K) and none (Na, K)];

and divided into two groups in the ratio male to female 1:1. The first group was Na, K; the

second group was control unit without Na, K.

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