CHAPTER IIICHAPTER IIICHAPTER IIICHAPTER III

REPRODUCTIVE REPRODUCTIVE REPRODUCTIVE REPRODUCTIVE

TOXICITY TOXICITY TOXICITY TOXICITY

OF OF OF OF OPUNTIA ELATIOROPUNTIA ELATIOROPUNTIA ELATIOROPUNTIA ELATIOR

FRUIT EXTRACT IN FRUIT EXTRACT IN FRUIT EXTRACT IN FRUIT EXTRACT IN

MALEMALEMALEMALE SWISS ALBINO SWISS ALBINO SWISS ALBINO SWISS ALBINO

MICEMICEMICEMICE

44

1.1.1.1. IntroductionIntroductionIntroductionIntroduction

The geometric growth of world population has brought a great pressure on

land, water, energy, and biological resources to provide adequate supply of food while

maintaining the integrity of our ecosystem. Expansion of world population results in

increasing food problem, with the increase in the number of malnourished children.

Though India has a landmass of 2.4% of the world, it has 16% of the global

population. Limited access to health care, contraception, and education in many of

these countries has resulted in national demographic trends that exhibit stark contrasts

with those of the industrialized world, leading to high population densities. Population

growth, therefore, poses a number of socioeconomic and geopolitical challenges.

There is a global need to support individuals in family planning due to the

increasing growth rate of the world’s population with its negative impact on the

environment, economic growth and poverty reduction in underdeveloped countries.

India is first among the countries which adopted an official family planning program,

as early as 1950, but has not prevented the population touching one billion mark

(Rao., 2001). In spite of a number of exciting developments in recent years, in which,

since 1971, the WHO special program of Research, Development and Research

Training in Human Reproduction has played a key role through its task force on

methods for the regulation of male fertility (De Kretser., 1978). Relatively few

realistic approaches towards male contraception include: a) the suppression of sperm

production b) disruption of sperm maturation and function c) interruption of sperm

transport (Thakur et al., 2010).

45

Despite the numerous areas of research currently being pursued, the prospect

of a marketable male contraceptive remains several years away. Two problems

common to all avenues of investigation for male fertility control remain to be resolved

– acceptability and methods of assessing efficacy. The question of which methods can

be used to test the efficacy of new male contraceptives remains unanswered (De

Kretser., 1978). Currently available methods of fertility regulation for men and

women do not adequately meet the varied and changing personal needs to couples in

their reproductive lives. While increasing the choice available to either partner will

ensure the widest availability of safe and effective means for fertility regulation, the

shortcomings of currently available male methods are a major barrier to the greater

involvement of men in family planning. Although increased understanding of gender

issues and roles are required for improved reproductive health, there is more

recognition for the need for shared contraceptive responsibility. The availability and

use of acceptable male contraceptive methods could reduce the burden traditionally

placed almost exclusively on the female partner (Vogelsong.,2005). Thus the present

study aimed for male contraception.

Acceptable antifertility drugs for men are proving difficult to produce. Such

drugs must aim to achieve complete azoospermia over a long period. This

requirement may be relaxed only if it can be shown that the residual sperms produced

by men whose spermatogenesis has been suppressed by antifertility drugs to

oligospermia are incapable of fertilizing ova (Waites., 1986).

A large number of chemical agents have been described under non –

hormonal male contraception but all tend to lead to total spermatogenic arrest and

46

ultimately to irreversible sterility (Thakur et al., 2010). The endocrine suppression of

sperm production is difficult to achieve without simultaneously suppressing the

production of androgens, which have an important role in erythropoiesis, protein

anabolism, bone metabolism and secondary sex and behavioral characteristics as well

as libido potency (De Kretser., 1978; Waites., 1986). Estrogens will suppress

spermatogenesis quite effectively, but even with the addition of androgens, feminizing

symptoms such as gynecomastia have remained as a risk (Waites., 1986).

Available scientific data indicate that the prospects of reversible male

contraception appear promising. Acceptable contraceptive methods for use by men

should focus on a) inhibition of sperm production b) interference with sperm function

and structure c) interruption of sperm transport d) interruption of sperm deposition e)

prevention of sperm – egg interaction (Vogelsong.,2005).

The interest in drugs of plant origin is mainly because it is conventional, with

no side effects. Ecological awareness suggests that the use of “natural” products as

drugs are harmless (Raghavendra et al., 2009). For centuries people have used plants

for healing. Plant products – as parts of foods or botanical portions and powders –

have been used with varying success to cure and prevent diseases throughout history.

Uses of indigenous drugs from plant origin are the major sources as an alternative

system of medicine or traditional system of medicine since ancient (Joshi et al.,

2004). WHO define the traditional system of medicine as “diverse health practice,

approach, knowledge and belief incorporating plant, animal and / or mineral based

medicine, spiritual therapy, manual technique and exercise applied singularly or in

combination to maintain well being as well as to treat, diagnose or prevent illness”.

47

This system aims to promote healthy and enhanced quality of life (Patwardhan et al.,

2006). Neutraceuticals and cosmoceuticals are of great importance as a reservoir of

chemical diversity aimed at new drug discovery and are explored for antimicrobial,

cardiovascular, immunosuppressant and anticancer drugs. Natural products including

plants, animal and minerals have been the basis of treatment of human diseases

(Patwardhan et al., 2004). It is suggested that traditional medicine may offer better

routes to the discovery, development and delivery of new drugs with enhanced

performance in terms of cost, safety and efficacy. To this end, it is also believed that

the basic principles, experiential wisdom, holistic approach and systematic database

of Ayurveda may offer useful bio prospecting tools and an efficient discovery engine

(Patwardhan et al., 2004). The mass screening of plants in the development of new

leads or drugs is tremendously expensive and inefficient. However, the traditional

knowledge based on bio prospecting offered better leads for the treatment of AIDS

and cancer. About 60% of anticancer and 75% of anti-infective drugs approved from

1981 to 2002 was developed from natural origins (Gupta et al., 2005).

Opuntia species has been used by humans for thousands of years. Besides

being consumed as food or beverages, most portions of the plants have been used as

medicine and in modern times have also been prepared as juice, jam, flour, frozen

fruit, juice concentrates, and spray-dried juice powder (Smith., 1967; Stintzing and

Carle., 2005; Stintzing and Carle., 2006; Feugang et al., 2006). A remarkable number

of cacti are used by indigenous people of the New World for healing. The plant is

bitter, laxative; stomachic, carminative, antipyretic. Cures biliousness, burning,

leucoderma, urinary complains, tumors, loss of consciousness, piles, inflammations,

48

anemia, ulcers, respiratory disorders like asthma and the enlargement of the spleen

(Parmar and Kaushal., 1982; Kirtikar and Basu., 1999; Patil et al., 2008).

The fruit of Opuntia is considered a refrigerant, and is said to be useful in

gonorrhea. The baked fruit is said to be given in whooping cough and the syrup of the

fruit is said to increase the secretion of bile and control spasmodic cough and

expectoration (Kirtikar and Basu, 1999; The Wealth of India, 2001). Both fruit and

stem have been regarded to be safe for food consumption. The constantly increasing

demand for nutraceuticals is paralleled by a more pronounced request for natural

ingredients and health-promoting foods. The multiple functional properties of cactus

pear fit well this trend. Recent data revealed the high content of some chemical

constituents, which can give added value to this fruit on a nutritional and

technological functionality basis. High levels of betalains, taurine, calcium,

magnesium, and antioxidants are noteworthy (Piga, 2004; Stintzing and Carle, 2005;

Feugang et al., 2006).

The phylloclade is also considered to be of medicinal values. The juice from

the phylloclade is being used to treat cuts, boils, wounds (Singh et al., 2011; Kumar

and Abbas., 2012), knee aches (Dushing and Patil, 2010), fever, indigestion, chest

complaint, urine stone, stomach ache (Rothe., 2011), muscle pain (Bhosle et al.,

2009) and swelling of nails (Patil and Biradar 2011). It is also used for the treatment

of anthrax (Singh et al., 2011), rheumatism (Patil and Biradar 2011) and asthma (Patil

et al., 2008). It also enhances the milk flow of a nursing mother, when the heated

plant is applied to the breasts (Sayed et al., 2007). The root bark is used to treat

menstrual problem (Patil et al., 2010a). The flowers are also not set apart from their

49

medicinal value. The dried flowers are used to treat against hiccough (Rothe., 2011),

and to cure hemorrhoid (Ghorband and Biradar., 2011).

Opuntia elatior belongs to the family Cactaceae and is usually grown in arid

and semiarid regions. The uses of O. elatior whole plant and other species of Opuntia

are enormous (Ramyashree et al., 2012). The fruit being considered to be edible

(Tiwari et al., 2010) has been documented as a medicinal plant in Vijayanagar forest

(Vegda et al., 2012). In addition fruits of O. elatior are used for hematinic, anti-

asthmatic and spasmolytic action by tribal people of Saurashtra region of Gujarat

state, and have been successfully controlled the disease as well (Chauhan., 2010). The

O. elatior fruits have been used for whooping cough, diabetes, high blood cholesterol,

obesity, as a blood purifier (Kshirsagar et al., 2012a., 2012b). The fruit pulp is also

fed for the infant’s stomach ache (Patil and Biradar 2011) and to cure asthma (Patil et

al., 2008), rheumatism (Patil and Ahirrao., 2011), burning sensation in the stomach

(Kumar et al., 2008) and diphtheria in livestock (Kumar and Bharathi., 2012).

Interestingly, O. elatior fruits have been used since date back as a source of

contraceptive medicine by tribal women mixing it with jaggery and taken orally for 2-

3 days for complete sterility (Jain et al., 2007). As there is no scientific data available

on the antifertility effect of this plant except tribal knowledge we have examined the

antifertility effect of O. elatior using male Swiss albino mice.

The synthetic and hormonal contraceptives side effects prompted scientists to

examine herbal contraception and identify suitable antifertility inducing biomolecules.

The major sources of the alternative medicine since ancient period are the potential

use of biologically active components from plant origin (Joshi et al., 2004). Scientific

50

studies using different plants on male contraception have shown promising results as

safe and effective contraceptive. The methonolic phylloclade extract of Opuntia

dillenii caused antispermatogenic effect in mice (Gupta et al., 2002; Bajaj and Gupta.,

2011), reduced sperm count and decreased sperm motility as has reported using seed

extract of Vitex negundo (Das et al., 2004), Albizzia lebbeck (Gupta et al., 2005),

Cestrum parqui (Souad et al., 2007), leaf extract of Aegel marmelos (Kumar et al.,

2011a), Azadirachta indica (Parshad et al., 1997; Kasturi et al., 2002; Ghosesawar et

al., 2003; Aladakatti and Ahamed., 2005), seed extract of Thespesia populnea

(Nagashree, 2010), Madhuca indica (Shivabasavaiah et al., 2011), and Cyamposis

psoralioides (Thejashwini et al., 2009a, b, 2012), but no work has been carried out

using O. elatior fruit extract.

Many plants and its parts have been tested for antifertility activity, but most of

them have its effect on libido thus affecting the sexual behavior of the treated animal,

this is another test to examine the antifertility activity of the plant extract without

disturbing the libido and the sexual behavior of the animal. As this fruit is used as an

antifertility agent by the tribal women we got an opportunity to test this fruit extract

scientifically for antifertility activity. The unavailability of scientific data about the

fruit paved a way for further scientific investigation about the phytochemical

screening and antifertility activity of the plant extract.

2.2.2.2. RRRReview of literatureeview of literatureeview of literatureeview of literature

India is known as the emporium of medicinal plants. It has a very long, safe and

continuous usage of many herbal drugs in the officially recognized alternative

systems of health viz. Ayurveda, Yoga, Unani, Siddha, Homeopathy and Naturopathy.

51

The World Health Organization (WHO) has recognized the traditional systems of

medicine as one of the tools to achieve its aim “Health for all”.

The ethanolic extract of Rauwolfia vomitoria root bark at a dosage of 600mg/ kg

bw caused a significant reduction in the spermatozoa count, motility, viability and

volume as well. The concentration of the serum testosterone level was also noticed.

There was also histopathological conditional observed which was irreversible

(Olanrewaju et al., 2012). The methanolic extract of Momordica dioica root at a dose

level of 20mg/ kg bw caused a significant reduction in the body weight, spermatozoa

count and motility. The concentration of the serum testosterone level was also

noticed. There was a significant reduction in the values of biochemical parameters

also. However, after cessation of the treatment, recovery in all the parameters were

observed (Kachchawa et al., 2010). The subchronic administration of U and Dee

sweet bitter herbal supplement posed a toxic effect on the accessory reproductive

organs of male Wistar rats. It also brought down the epididymal sperm count

(Ejeatuluchukwu et al., 2011). The alcoholic extract of Citrus limonum showed a

reversible antifertility activity. The plant extract was responsible for the reduction in

the weight of the epididymis, decreased sperm count, motility and viability (Kulkarni

et al., 2012). The aqueous and ethanolic extract of Adiatum lunulatum Burm has its

antiandrogenic effect on the testis and accessory reproductive organs of male albino

rats. Administration of the ethanolic extract of the fern resulted in the arrest of the

spermatogenesis. The extract has adverse effect on the histoarchitecture of the

reproductive organs. Even though no alteration in the fertility rate was observed in

low dose treated animals but at higher dosage the extract induced complete infertility

(Bhatia et al., 2010). Methanolic extract of Nyctanthes arbortristis caused a

52

significant reduction in the reproductive organ weight, sperm motility and density

which in turn is the major cause for reduced fertility. The extract was also responsible

for the reduced levels of biochemical parameters. Reduction in the germ cell

population is one of the main causes for the impairment of testicular function (Gupta

et al., 2006). The ethanolic seed extract of Abrus precatorius showed a permanent

antifertility activity at higher dosage showing highly significant decrease in the total

sperm count. However, at lower dosage the effect was temporary which could be

withdrawn to normal levels after cessation of the treatment (Abu et al., 2012). The

alcoholic extract of Ruta graveolens has its negative effect on sperm count, motility

and finally the fertilizing capacity of the sperms (Rahim et al., 2010). The antifertility

effect of an ethanolic fruit extract of Trachyspermum ammi (Linn) shows decreased

testis weight, sperm count, motility and increased abnormal sperms, but there was a

recovery in all the parameters related to sperm count, motility and abnormal

spermatozoa that were altered (Kumar et al., 2011b). The aqueous leaf extract of

Andrographis paniculata shows decreased weight of testis, epididymis and seminal

vesicle. A dose dependent reduction of testicular sperm count, epididymal sperm

count, motility and abnormal sperm count was observed (Sathiyaraj et al., 2011). The

effect of Corynanthe Yohimbe (Yohimbe) shows decreased sperm count, increased

sperm abnormality and reduced fertility rate, which proves it to be an antifertility

agent (Al-Majed et al., 2006). The ethanolic extract of Capparis aphylla (Roth)

caused antisteroidogenic activity where the total cholesterol and ascorbic level were

increased, which is the principal precursor of androgen in the biogenic pathway in the

testis. Thus the plant extract was responsible for the inhibition of testicular

steroidogenesis, which acts as an antifertility agent (Saratchandiran et al., 2007). 50%

ethanolic extract of Calendula officinalis in male rats brought about a significant

53

reduction in the reproductive organ weight, serum testosterone level and reduced the

levels of glycogen and protein. The plant extract was also responsible for reduced

fertility rate (Agarwal et al., 2011). The seed extract of Madhuca latifolia caused a

significant decrease in the weight of the reproductive organs, sperm count, and serum

testosterone level which in turn may be used as an effective antifertility agent

(Gopalakrishnan and Shimpi., 2011). The ethanolic leaf extract of Enicostemma

axillare and Urena lobata was responsible for reduced reproductive organs weight,

sperm count and there was a marked increase in the number of sperm abnormality,

testicular cholesterol and total ascorbic acid, which acts as an antifertility agent

(Dhanapal et al., 2012a). The aqueous ethanolic leaf extract of Lawsonia inermis Linn

acts as a spermicidal agent causing immobilization of the spermatozoa on the in vitro

application of the extract on the collected semen of male rats (Hyacinth et al., 2012).

The aqueous leaf extract of Oldenlandia affinis caused a significant decrease in the

testis weight and testosterone level but there was no such significant difference in the

sperm count, but has interfered in process of spermatogenesis in rats, thus it can be

considered as an antifertility agent (Sewani-Rusike and Gundidza., 2011). The

hydroalcoholic extract of Cissampelos pareira caused a significant reduction in the

weight of the reproductive organs, serum testosterone level, sperm count and motility,

thus affecting the fertilizing ability of the male rats (Harisha., 2012). The aqueous

extract of Phyllanthes niruri brought about a significant decrease in the sperm count

and motility and decreased testosterone level which has reduced the fertilizing ability

of the male rats. Hence the plant can be considered as an effective antifertility agent

(Ezeonwu., 2011). The Momordica charantia Linn seed extract caused an

antispermatogenic effect in male mice (Patil and Patil., 2011). The alcoholic extract of

Iranian neem seed extract caused a decreased sperm motility and increased sperm

54

abnormality thus reducing the fertility rate in male mice (Dehgan et al., 2005). The

aqueous, methanolic and saponins extract of Ziziphus maurotiana has a sperm

immobilization activity in human spermatozoa (Dubey et al., 2011). The ethanolic

leaf extract of Spondias mombin is responsible for reduced sperm count and decreased

serum testosterone level which in turn suppress the spermatogenesis, thus acting as an

antifertility agent in male rats (Asuquo et al., 2012). The ethanolic extract of

Cyamposis psoralioides caused a significant decrease in the weight of the

reproductive organs, sperm count, serum testosterone level, total cholesterol, protein,

glycogen and ascorbic acid level in mice, which was reversible after cessation of the

treatment, thus acting as fertility regulating agent in mice (Thejashwini et al., 2012).

The methanolic seed extract of Cuminum cyminumin caused a significant reduction in

the weight of the reproductive organs, sperm motility and the biochemical parameters,

which resulted in impairment in the testicular function and has affected the

spermatogenesis, thus affecting the fertility rate in male rats (Muthu and

Krishnamoorthy., 2011). The aqueous ethanolic stem bark extract of Hymenocardia

acida acts as an in vitro sperm immobilizing agent in male rats (Abu et al., 2011). The

ethanolic fruit pulp extract of Feronia limonia has reversible antispermatogenic and

antisteroidogenic effect in male rats (Dhanapal et al., 2012b). The ethanolic extract of

Lepidagathis alopecuroides caused a significant reduction in the weights of the testis,

a number of various spermatogenic elements, results in reduced fertility, thus acting

as an antifertility agent (Orly et al., 2012). The aqueous seed extract of Solanum

surattense decreased the oxidative potential of the cauda epididymal spermatozoa

which acts an antifertility effect (Thirumalai et al., 2012). The ethanolic root bark

extract of Chrysophyllum albidum caused a significant reduction in the sperm count,

serum testosterone, FSH and LH levels, thus acting as an antifertility agent (Onyeka

55

et al., 2012). The 50% ethanolic flower extract of Calendula officinalis caused

significant reduction in the weight of the reproductive organs, serum testosterone

level which results in reduction the fertility of the male rats acting as an antifertility

agent (Kushwaha et al., 2007). The ethanolic extract of Tinospora cordifolia has a

reversible contraceptive effect in male albino rats (Singh et al., 2011). 50% ethanolic

leaf extract of Ficus bengalensis caused a suppression of spermatogenesis and has an

adverse effect on sperm functions resulting in reduced fertility (Gupta et al., 2012).

70% methanol seed extract of Strychnos potatorum caused a significant reduction in

the weight of the testis, sperm count, which results in suppression of fertility (Gupta et

al., 2006). 50% ethanolic seed extract of Nelumbo nucifera acts as anti estrogenic

agent in female rats. The hydro ethanolic extract of Stephania hernandifolia and

Achyranthes aspera caused a significant decrease in the epididymal sperm count,

serum testosterone level which can be used as an antifertility agent in male rats (Paul

et al., 2010). The chloroform seed extract of Carica papaya caused a significant

decrease in the sperm concentration, and an increase in the number of abnormal sperm

which resulted in azoospermia, cessation of the treatment resulted in gradual recovery

in the sperm parameters, thus acting as an antifertility agent in Langur monkeys

(Lohiya et al., 2002). The hydroalcoholic extract of Achillea santolina caused a

significant reduction in the weight of the testis and alteration in the spermatogenesis

acts as an antispermatogenic agent in mice (Golalipour et al., 2004). 50% ethanolic

extract of Tecoma stans leaves act as an antifertility agent causing reduction in the

weight of the reproductive organs, sperm count and finally the fertility rate in albino

rats (Mathur et al., 2010). The aqueous leaf extract of Aegle marmelos caused a

significant reduction in the weight of reproductive organs, testicular and epididymal

sperm count and an increase in the abnormal sperm count was noticed thus acting as

56

an antifertility agent in male albino rats (Sathiyaraj et al., 2010). The total alkaloids

isolated from the leaves of Aegle marmelos caused a significant decrease in the body

weight and sperm concentration in the treated animals suggesting the antifertility

activity in male rats (Kumar et al., 2011a). The aqueous extract of Ruta graveolens

caused a significant decrease in the weight of the reproductive organs, sperm density,

serum testosterone level, and the number of implantation site was reduced acting as an

antifertility agent (Khouri and El-Akawi., 2005). The methanolic leaf extract of

Morinda lucida acts as an antispermatogenic agent in male rats causing reduced

sperm count, increased sperm abnormality and reduced litter size is the final result

(Raji et al., 2005). The oral administration of fruit powder of Piper nigrum acts as an

antispermatogenic agent responsible for reduced litter size in male mice (Mishra and

Singh., 2009). The benzene and ethanolic extract of Terminalia bellirica caused a

significant decrease in the weight of the reproductive organs and sperm count, works

as an antifertility agent (Patil et al., 2010b). The ethanol and aqueous extract of stem

bark of Crataeva nurvala buch – hum acts as an antifertility agent in female rats

(Bhaskar et al., 2009). The methanolic extract of broom Spartium junceum acts as a

capable antifertility agent with complete reversible effect in male rabbits (Baccetti et

al., 1993). The petroleum and chloroform root extract of Cyclea burmanni acts as an

antifertility agent in female rats (Panda et al., 2003). The petroleum ether root extract

of Echinops echinatus caused a significant decrease in the relative weight of the

reproductive organs, serum testosterone level and sperm count level, thus acting as an

antifertility agent in male rats (Padashetty and Mishra., 2007). The methanol extract

of Ricinus communis caused a significant decrease in the weight of the reproductive

organs, sperm count, serum testosterone level and the litter size, thus affecting the

fertility of the male rats (Raji et al., 2006). The aqueous crude leaf extract of the

57

Bougainvillea spectabilis caused a significant decrease in the weight of the testis,

sperm count and testosterone level causing reduced fertility in male mice (Mishra et

al., 2009). The ethanolic seed extract of Abrus precatorious suppresses the

spermatogenesis after the treatment in male mice, thus affecting the fertility of the

mice (Jahan et al., 2009). The 50% ethanolic extract of the root bark of Cananga

odorata caused a significant reduction in the epididymal sperm motility and sperm

count, there was a significant increase in the abnormal sperm count, thus suggesting

its spermatotoxic effect in male rats (Anitha and Indira., 2006). The ethanolic root

extract of Plumbago indica and aerial parts of Aerva lanata reduced the percentage of

motile spermatozoa and implantation sites, resulting in reduced pregnancy rates in rats

(Savadi and Alagavadi., 2009). The methanolic extract of the stem bark of Nyctanthes

arbortristis caused a significant decrease in the weight of the reproductive organs,

sperm motility and density which resulted in total suppression of the fertility that

resulted in impairment in the testicular function and spermatogenesis in male rats

(Gupta et al., 2006). The aqueous leaf extract of Ocimum gratissimum acts as an

antifertility agent in male mice (Obianime et al., 2010). The ethanolic extract of Ruta

graveolens and Cannabis sativa acts as a spermatogenesis reducing property in male

Wistar rats (Sailani and Moeini., 2007). The benzene leaf extract of Ocimum sanctum

acts as an antifertility agent resulting in a reduced sperm count in male rats

(Reghunandanan et al., 1995). The aqueous leaf extract of Carica papaya acts as an

antiandrogenic agent causing reduced sperm count, sperm motility and serum

testosterone level in male rat (Akinloye and Morayo., 2010). The ethanolic extract of

Azadiracta indica shows a positive antifertility activity in adult male mice with a

considerable reversible activity of the plant extract in male mice (Deshpande et al.,

1980). The ethanolic seed extract of Nelumbo nucifera acts as an anti – estrogenic

58

agent without affecting the general physiology of the female rats (Mutreja et al.,

2008). Methanolic pod extract of Albizzia lebbeck (L) has shown inhibition of

spermatogenesis (Gupta et al., 2004). The crude aqueous extract of Allium sativum

bulb possesses sperm immobilization activity (Chakrabarti et al., 2003). Methanolic

extracts of Alstonia boonei stem bark have spermicidal action (Raji et al., 2005). The

leaf extract of Andrographis paniculata, when fed to male albino rats, caused the

arrest of spermatogenesis (Akbarsha et al., 1990). The seed oil of Azadirachata indica

when injected in single dose into the vas deferens of the rat showed an antifertility

response throughout 8 months of treatment and also reported to have

antispermatogenic properties (Purohit and Dixit 1991; Purohit, 1999). The leaf extract

of the same plant also showed antispermatogenic and antiandrogenic properties and

gradual withdrawal recovery (Aladakatti and Ahamad, 1991; Aladakatti et al., 2001;

Joshi et al., 1996). The leaf extract of Azadirachata indica has spermicidal activity in

male rats (Khillare et al., 2003). Isolated fractions of the Barleria prionitis root

methanolic extract showed a significant reduction of spermatogenesis (Verma et al.,

2002). The treatment of aqueous suspension of the roots of Cichorium intybus and

ethanolic leaf extract of Colebrokia oppositifolia showed notable depression of

spermatogenesis in mice and rats (Roy and Bhatt, 1996; Gupta et al., 2001). Crude

ethanolic extract of Citrulus colocynthis significantly reduced cauda epididymis

sperms (Chaturvedi et al., 2003). Crude extract of Curcuma longa in male rats shows

maximum reduction in spermatogenesis (Purohit et al., 2004). Powdered berries of

Embelia ribes administered to bonnet monkey affected the quality and quantity of

semen (Purandare et al., 1979). The rhizome of Zingiber officinale has shown

prominent antispermatogenic activity (Kamtchouing et al., 2002). The stem extract of

Bambusa arundinacea has shown loss of libido, reduced sperm motility, reduced

59

testis, epididymis, seminal vesicle and ventral prostate weights in rat (Kumari et al.,

1989). The root extract of Cyclea burmanni has shown estrogenic activity in female

albino rats (Panda et al, 2003). The root extract of Tripterygium hypoglaucum has

shown reduced sperm motility, count and reversible recursive effect in human (Qian

et al., 1988). The extract of Tylophora asthamatica has shown the antispermatogenic

and morphological changes of the seminiferous tubules in rats (Dikshith et al., 1990).

3.3.3.3. Materials and methodsMaterials and methodsMaterials and methodsMaterials and methods

3.1 Materials

Rearing of animals: Adult male and female Swiss albino mice weighing 30 – 40 g

were obtained from the Central animal facility, DOS in Zoology, University of

Mysore, Mysore. They were housed in polypropylene cages (three animals/ cage)

containing husk as the bedding material under 12 h light and 12 h dark schedule at

27±2°C and 70% humidity. The animals had access to food (mice chow pellets) and

water ad libitum during the experimentation period. The protocols of the experiment

approved by the Institutional Animal Ethics Committee under the guidelines of

CPCSEA, Government of India were followed for care and maintenance of animals.

3.2 Methods

Treatment: The experiment was conducted in two units and the treatment for the

animals was divided in the duration dependent manner. The first part of the

experiment was treatment of 250 and 500 mg/kg body weight (bw) selected were

treated for 30 days at an interval of 24 h with a recovery period of 30 days. The adult

mice were divided into three groups, while the control group received 0.2 ml distilled

water, the other two groups received 250 and 500 mg/ kg bw of the fruit extract in 0.2

60

ml of distilled water respectively per mouse. For recovery, few animals were kept

without treatment for 30 days, with food and water ad libitum for another 30 days.

Similar to the first part of the experiment all the treatment protocols are same, but the

treatment for the animals was given for 60 days with a recovery period also extended

to 60 days.

Toxicity test: Toxicity test for fruit extract was performed using Swiss male albino

mice. To this, animals were kept fasting for overnight providing only water. They

were divided into four groups containing three animals each. One group maintained as

control group and the other three treatment groups were then administered with the

extract.

Control group animals received 0.2 ml of distilled water whereas each animal

in 2nd, 3rd and 4th group received 200 mg (low dose), 400 mg (medium dose), and 600

mg (high dose) extract/ kg bw in 0.2 ml distilled water respectively. The extract with

the vehicle was administered to the animals through oral intubation by a smooth

plastic tube attached to a syringe. The treatment period was fixed to 30 days. Each

day after oral intubation the animals were observed for first 30 mins and were kept for

observation like sedation, convulsions, tremors, lethargy and death.

Weight of the body and reproductive organs: The body weight of each animal of

the entire group was noted before autopsy. Eight mice in each group were autopsied

and weights of the testes, epididymis, vas deferens, seminal vesicle and ventral

prostate were recorded. During autopsy epididymis of each mouse was carefully

separated from the testis and used for sperm count.

61

Histology and histometry: The testis and epididymis fixed in Bouin’s

solution were dehydrated with different grades of alcohol, cleared in chloroform,

infiltrated with molten paraffin wax and embedded in paraffin wax. Five micron thick

paraffin sections of the testis and epididymis were cut using the microtome. Testis and

epididymis sections were mounted on glass slides, stained with haematoxylin and

eosin. The stained sections were photographed using Olympus digital camera under

appropriate magnification. The diameter of seminiferous tubules and epididymal

tubules, diameter of the Leydig cell and the height of the secretory epithelium of

cauda epididymis were measured in 200 randomly selected cross sections of the testis

and epididymis per animal.

Sperm count: For the total sperm count, cauda region of the epididymis was

minced in 1 ml of buffered saline and filtered through muslin cloth. The filtrate was

taken in a leukocyte pipette up to 0.5 and filled up to the mark 11 with buffered

saline. The suspension was well mixed and charged to the Neubauer’s chamber. The

total number of spermatozoa present in 8 squares of 1 mm2 each was counted and

multiplied by 5×104 to express the number (millions) of spermatozoa/ epididymis

(Vega et al., 1988).

For the abnormal sperm count, the sperm suspension obtained for total sperm

count was mixed with aqueous eosin and kept for 30 min. A drop of the spermatozoa

suspension was taken on a clean slide as uniform smear and dried. One thousand

spermatozoa were screened per mouse for abnormal head shape like amorphous head,

hookless head, pin head, banana head, hammer head, folded head and double head

(Vega et al., 1988).

62

Fertility test: Eight adult female mice with proven fertility were used after each

treatment period for the fertility test. Four animals were taken from each group and

each male mouse from the experimental and control groups were kept with female

mice for 2 weeks. The female was examined for the presence of spermatozoa in the

smear every day and presence of spermatozoa in the vaginal plug confirmed the

mating (Al-Hamdani and Yajurvedi., 2010; Thejashwini et al., 2012). The fertility

parameters such as number of females conceived, average length of gestation, litter

size and litter weight of each group were recorded according to the procedure of

Kennedy et al., 1973, Adilaxmamma et al., 1994, Narayana et al., 2005 and Al-

Hamdani and Yajurvedi., 2012.

a. Fertility index of male = Number of fertile males × 100 Number of males used in the test

b. Fertility index of female = Number of pregnant mice × 100

Number of males used in the test

c. Parturition index = Number of females delivered × 100 Number of pregnant mice

d. Gestation index = Number of pups born alive × 100

Total number of pups born

e. Viability index = Number of pups born alive at day 4 × 100 Total number of pups born alive

f. Lactation index = Number of pups born alive at day 21 × 100

Number of pups born alive at day 4

Hormone assay: After the treatment period, 24 h after the last dose, animals were

killed by etherization and 1.5–2 ml blood were collected by cardiac puncture in dry

Eppendorf tubes. The blood samples were centrifuged and the serum was used for

63

testosterone assay. Levels of testosterone in the serum were determined using

radioimmuno assay (RIA) method (Wilke and Utley, 1978).

Biochemical parameters

Protein estimation: The protein content in testis and epididymis was estimated

according to the method of Lowry et al, (1951). The tissue was homogenized in 10 ml

of distilled water. The homogenate was centrifuged for 15 min and 1ml of supernatant

was collected in a test tube. To this 5 ml of alkaline copper reagent was added.

Alkaline copper reagent was prepared by adding 50 ml of solution A and 0.5 ml of

solutions B and C. Solution A was prepared by dissolving 2 g of sodium carbonate in

100ml 0.1 N NaOH, solution B was prepared by dissolving 1 g of copper sulphate in

100 ml distilled water. Solution C was prepared by dissolving 2 g of potassium

sodium tartarate in 100 ml distilled water (0.1 N NaOH can be prepared by dissolving

4g of NaOH in 1000 ml of distilled water). After 20 min 0.5 ml of Folin–Ciocalteu

reagent was added. After 30 min the optical density (OD) was read at 660 nm using

colorimeter. The calculations were made with reference to standard graph. For

preparation of standard graph, the standard protein solution was prepared by

dissolving 20 mg of Bovine Serum Albumin (BSA) in 100 ml of 0.2% NaCl (Saline).

This solution contains 200 mg protein per ml. From the standard protein solution

different quantities were taken and as mentioned above required reagents were added

and the optical density was measured at 660 nm by using a colorimeter. A standard

graph was plotted by using optical densities of standard protein solution versus

different concentration. Concentration of protein in the tissue was calculated by using

the following formula.

64

Amount of protein = OD of sample × Conc of standard × 1000 × 10 OD of standard Weight of tissue The concentration of protein was expressed as µg/ mg tissue.

Glycogen estimation: The tissue homogenate was prepared by using 10 ml 4% TCA

and centrifuged at moderate speed for 10 min. The supernatant was decanted and the

precipitate was discarded. To the 2 ml of supernatant 4ml of anthrone reagent (200

mg of anthrone dissolved in 100 ml of conc. H2SO4) was added. The tubes were

allowed to cool for 30 min. A blank is prepared by using distilled water. The optical

density was measured at 620 nm wavelength by using a colorimeter (Carrol, 1956).

Calculations were made using the formula

Concentration = OD of sample×Concentration of Standard×Volume of extract×1000×10

of glycogen OD of standard Weight of tissue

Results were expressed as µg / mg tissue

Cholesterol estimation: The total cholesterol content of various tissues was made

according to Libermann and Burchard. The method was described by Peters and

Vanslyke (1946). The organs were weighed and homogenized in 10ml of 3:1 alcohol-

ether mixture. The homogenate was centrifuged for 10 min and supernatant was

collected and dried at room temperature the residue was dissolved in 5 ml of

chloroform then 2 ml of the acetic anhydrate mixture prepared freshly by mixing 20

ml of acetic anhydride and 1 ml of conc. H2SO4 was added. The complete mixture

was kept in darkness for about 15 min for color development. The optical density was

measured using spectrophotometer at 660 nm wavelength. The calculation of tissue

cholesterol was made with reference to standard graph. For standard graph, five

samples of known concentration of cholesterol dissolved in 5 ml of chloroform were

used. To this 2 ml of the acetic anhydrite mixture was added and kept in a dark place

for color development. The OD of this sample was measured at 660 nm wavelength.

65

While processing the tissue for cholesterol estimation 1-2 standard samples were run

every time. The following formula was used for calculation.

Concentration of cholesterol = OD of sample × Conc. Of standard × 1000 × 10 OD of standard Weight of the tissue Results were expressed as µg/mg tissue.

Total ascorbic acid (TAA) estimation: Level of ascorbic acid was estimated by the

method of Roe and Kuether (1943). One homogenate was prepared in 10 ml of norit

reagent prepared by dissolving 2g of activated charcoal in 100 ml of 6% TCA. The

mixture was shaken well and allowed to stand for 15 min and then filtered through

Whatsman filter paper no.42. To 4 ml homogenate, 1 ml of 2, 4-dinitro

phenylhydrazine reagent (2 g of 2,4DNPH in 100 ml of 9N H2SO4) was added and

then a drop of 10% thiourea (10 g thiourea in 100 ml of 50% ethanol) was added in

order to activate the reaction. In blank tube, 4 ml of 6% TCA was taken instead of

homogenate. In the standard tube, 4 ml of ascorbic acid solution (50 mg of ascorbic

acid dissolved in 50 ml of 6% TCA, 1ml of this solution was diluted up to 100 ml

with 4% TCA) was taken. This contained 10 µg of ascorbic acid per ml. The contents

of the tubes were mixed well and were kept in boiling water bath for 15 min. And

thereafter were cooled in an ice-bath, after that the tubes were allowed to stand for 30

min and the O.D. was measured at 540 nm against blank of spectrophotometer. The

concentration of ascorbic acid was calculated by the following formula.

Concentration of = OD of sample × Conc. of standard x dilution × 1000 × 2 ascorbic acid OD of standard Weight of tissue

The results were expressed as µg/mg tissue weight.

66

Statistical analysis: The data were analyzed using the Statistical Package of Social

Science (SPSS) software under windows 11.5 version (SPSS Inc., Chicago, IL, USA).

All the data were computed following Duncan’s multiple range test of One-way

analysis of variance (ANOVA). The paired T-test was used to determine the toxicity

effect of treatment on the final body weight of the target animal.

4. 4. 4. 4. ResultsResultsResultsResults

4.1 Toxicity test

The toxicity study of the fruit O. elatior extract was examined using mice.

Interestingly, no variation in the external morphology and the body weight of mice

after the treatment with the extract was observed (Table 1). There was a gradual

increase in the final body weight of all control and treated group animals compared

with the initial body weight. The extract had neither significant increase nor

significant decrease in the final body weight of the treatment group.

Part A: Effect of 250 and 500 mg/ kg bw O.elatior fruit extract for 30 days

4.2 Body weight

The ethanolic fruit extract of O. elatior did not show any significant changes

in the body weight of the treated mice. No toxic effect of the fruit extract was

observed neither in low dose (250 mg/ kg bw) nor in high dose (500 mg/ kg bw)

treated animals with final body weight of the recovery group animals remains same

both at short and long durations (30 and 60 days) (Tables 2 & 13; Fig 1 & 13).

67

4.3 Reproductive organ weight

Significant (P≥0. 05) reduction in the mean relative weight of the testis

(561.62±8.07) and epididymis (180.25±7.217) was recorded at high dose (500 mg/ kg

bw) treated animals at short duration (30 days). No such reduction in the weights of

the vas deferens, seminal vesicle and ventral prostate was obvious, whereas a low

dose (250 mg/ kg bw) does not induce any significant reduction in the weight of testis

and other accessory reproductive organs of treated animals compared with controls.

The reduction in the mean relative weight of the testis and epididymis was 19.59%

and 25.43% respectively. However after cessation of the treatment the weight of the

testis and epididymis was recovered to 4.47% and 13.39% (Table 2; Fig 2a & b).

4.4 Histology and histometry of the testis

The light microscopic observation of the histoarchitecture of the testis is made

up of seminiferous tubules intactly arranged, each seminiferous tubule is lined by

germinal epithelium which consists of different germ cell types namely,

spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa.

In the lumen of each seminiferous tubule spermatozoa were present. The space

between the seminiferous tubules is filled with intercellular fluid, where the Leydig

cells are present. Administration of 250 mg/ kg bw extract to the animals did not alter

the histoarchitecture of the testis, the compactness of the seminiferous tubule did not

alter, the lumen was filled with spermatozoa, Leydig cells were intact in their

structure. However there was altered histological features observed in the testis after

the administration of 500 mg/ kg bw for 30 days. The compactness of the

seminiferous tubules was lost, the appearance of large or widened intercellular spaces

with regressed Leydig cells was observed. The germinal epithelium was found to be

68

ruptured in many tubules. The normal concentric arrangement of the germ cells was

absent, the germ cells were degenerated. The lumen was empty without spermatozoa

in many of the seminiferous tubules compared to control. However after cessation of

the treatment for 30 days, there was the reappearance of the normal histoarchitecture

of the testis (Fig 3a to d).

The mean diameter of seminiferous tubule per section of the cross section of

the testis and the diameter of Leydig cell nucleus was similar to control (171.70µm

and 6.13 µm) in 250 mg/ kg (173.45 µm and 6.30 µm) treated mice but a significant

reduction in the diameter of seminiferous tubule (138.00 µm) and Leydig cell nucleus

(3.99 µm) was observed at 500 mg/ kg bw. The percentage reduction in the diameter

of seminiferous tubule and Leydig cell diameter was 80.37 and 65.08 in 500 mg/ kg

bw. However there was a recovery in the diameter of the seminiferous tubules (161.05

µm) and the Leydig cell nucleus (5.19 µm) of the testis. The percentage of recovery

was 93.79 and 84.66 in the diameter of seminiferous tubule and Leydig cell nucleus

respectively (Table 3; Fig 5a & b).

4.5 Histology and histometry of the epididymis

The normal appearance of the epididymis was observed in the control group.

The cross section of the epididymis consists of many seminiferous tubules. Each

tubule is lined by a thick seminiferous epithelial layer made up of ciliated columnar

epithelial cells and the lumen is filled with numerous amount of spermatozoa. This

normal histology of the epididymis was also seen in the animals treated with 250 mg/

kg bw, but in the 500 mg/ kg bw treated animals the epithelial layer was ruptured and

69

there was a significant reduction in the epithelial height of the epididymis, the density

of the spermatozoa in the lumen was also reduced compared to control (Fig 4a to d).

The mean diameter of seminiferous tubule of the cauda epididymis per section

of the cross section of the epididymis and the epithelial height of the cauda

epididymis was similar to control (227.40 µm and 15.55 µm) in 250 mg/ kg (228.20

µm and 15.19 µm) treated mice but a significant reduction in the diameter of

seminiferous tubule (180.55 µm) and seminiferous epithelial height (11.76 µm) was

observed at 500 mg/ kg bw. The percentage reduction in the diameter of seminiferous

tubule and seminiferous epithelial height was 79.39 and 75.63 in 500 mg/ kg bw.

However there was a recovery in the diameter of the seminiferous tubules (206.05

µm) and the seminifeorus epithelial height (13.35 µm) of the testis. The percentage of

recovery was 90.61 and 85.85 in the diameter of seminiferous tubule and the

seminiferous epithelial height respectively (Table 3; Fig 5a & b).

4.6 Total sperm count

Animals treated with 250 mg/kg bw showed a slight decrease in the sperm

count, but the reduction in the total sperm count (5.4625±0.10078) was not considered

to be significant. However, a significant (P<0.01) reduction was observed in the total

sperm count of the animals treated with 500 mg/kg bw (4.75±0.14506). The reduction

in the total epididymal sperm count was 2.24%, 18.12% in the low-dose and high-

dose-treated animals. There was a recovery in the total sperm count after the cessation

of the treatment (5.2125±0.05543) for 30 days. The percentage of sperm count in the

recovery group was 6.712%. The recovery in the sperm count was nearly 11.40%

(Table 4; Fig 6a).

70

4.7 Abnormal sperm count

The percentage of abnormal sperms found in the animals treated with 250 mg/

kg bw was nearly similar (25.50±1.32288) to that of control. But there was a

significant (P<0.01) increase in the abnormal spermatozoa in 500 mg/ kg bw treated

animals. The increase in the abnormal spermatozoa count was 117.82%. The head

shape abnormalities observed was amorphous head, hookless head, pinhead, banana

head, hammer head and double head. The percentage of amorphous head shape

abnormality was found to be high compared to other abnormalities in 500 mg/ kg bw

treated animals. The percentage of total abnormal sperms were reduced to 14.85%

from 117.82% after the cessation of the treatment for 30 days (Tables 4 & 5; Fig 6b &

7).

4.8 Fertility parameters

The fertility index of male and female was 100% in both 250 and 500 mg/ kg

bw treated animals, similarly the gestation, viability, parturition and the lactation

indices were also 100% (Table 6). The litter size of the females cohabited with males

treated with 250 mg/ kg bw were 96.91% but the litter size of the females cohabited

with males treated with 500 mg/ kg bw were 63.92%. There was a 36.08 % decrease

in the litter size compared to control. However the litter size was increased to 87.63%

after the cessation of the treatment for 30 days. There was a 23.71 % increase in the

percentage of the litter size (Tables 7 & 8; Fig 8a).

4.9 Serum testosterone level

The serum testosterone levels were not significantly decreased in the 250 mg/

kg bw treated animals (723.4650±39.3274), but there was a significant (P<0.01)

71

decrease in the levels of the serum testosterone level (356.7925±48.0975) compared

with control. The reduction was 1.139%, 51.245% in 250 and 500 mg/ kg bw treated

animals respectively. There was an increase in the testosterone level

(548.5350±40.3770) to 25.043% in the recovery group after the cessation of the

treatment for 30 days (Table 8; Fig 8b).

4.10 Biochemical studies

4.10a Glycogen estimation

The glycogen content of the testis in the 250 mg/ kg bw treated animals did

not significantly reduced (1.80±0.084) compared with control (1.87±0.044). However,

in the 500 mg/ kg bw treated animals the glycogen content reduced significantly

(P<0.001) to 1.18±0.051. The reduced glycogen content was recovered (1.48±0.040)

after the cessation of the treatment in the recovery group for 30 days (Table 9; Fig 9).

In epididymis the glycogen content of the 250 mg/ kg bw treated animals did

not significantly reduced (4.50±0.105) compared to control (4.42±0.186). However,

in the 500 mg/ kg bw treated animals the glycogen content reduced significantly

(P<0.05) to 3.38±0.150. The reduced glycogen content was recovered (4.31±0.058)

after the cessation of the treatment in the recovery group for 30 days (Table 9; Fig 9).

4.10b Protein estimation

The protein content of the testis in the 250 mg/ kg bw treated animals did not

significantly reduced (9.65±0.388) compared to control (9.86±0.342). However in the

500 mg/ kg bw treated animals the protein content reduced significantly (P<0.01) to

6.77±0.221. The reduced protein content was recovered (8.66±0.172) after the

cessation of the treatment in the recovery group for 30 days (Table 10; Fig 10).

72

The protein content of the epididymis in the 250 mg/ kg bw treated animals

did not significantly reduced (14.33±0.369) compared to control (14.20±0.271).

However, in the 500 mg/ kg bw treated animals the protein content reduced

significantly (P<0.001) to 10.18±0.224. The reduced protein content was recovered

(12.07±0.359) after the cessation of the treatment in the recovery group for 30 days

(Table 10; Fig 10).

4.10c Cholesterol estimation

The cholesterol content of the testis in the 250 mg/ kg bw treated animals did

not significantly increased or decreased (5.26±0.466) compared with control

(5.28±0.251). However, in the 500 mg/ kg bw treated animals the cholesterol content

increased significantly (P<0.05) to 7.66±0.466. The increased cholesterol content was

recovered (6.30±0.373) after the cessation of the treatment in the recovery group for

30 days (Table 11; Fig 11).

The cholesterol content of the epididymis in the 250 mg/ kg bw treated

animals did not significantly increased or decreased (9.78±0.285) compared with

control (10.02±0.217). However, in the 500 mg/ kg bw treated animals the cholesterol

content increased significantly (P<0.01) to 12.00±0.304. The increased cholesterol

content was recovered (10.37±0.166) after the cessation of the treatment in the

recovery group for 30 days (Table 11; Fig 11).

4.10d Total ascorbic acid estimation

The estimation of total ascorbic acid was done only using the testis. There was

no any significant changes occurred in the 250 mg/ kg bw (5.10±0.110) and 500 mg/

73

kg bw (5.80±0.073) treated animals and the recovery group animals (5.26±0.250)

compared with control (5.13±0.616) (Table 12; Fig 12).

Part B: Effect of 250 and 500 mg/ kg bw O.elatior fruit extract for 60 days

4.11 Reproductive organ weight

Similar to the short duration there was a significant reduction in the mean

relative weight of the testis (728.62±13.256) and epididymis (279.00±6.842) at the high

dose treated (500 mg/ kg bw) animals at long duration (60 days). No such reduction in

the weights of the vas deference, seminal vesicle and ventral prostate was obvious,

whereas there were no significant weight differences was noticed in low dose treated

(250 mg/ kg bw) animals at long duration too when compared with control. The

reduction in the mean relative weight of the testis and epididymis was 19.33% and

31.004% respectively. However after cessation of the treatment after 60 days, the

weight of the testis and epididymis was recovered to 5.32% and 14.65% (Table 13;

Fig 14a & b).

4.12 Histology and histometry of the testis

Administration of 250 mg/ kg bw extract to the animals did not alter the

histoarchitecture of the testis, the compactness of the seminiferous tubule did not

alter, the lumen was filled with spermatozoa, Leydig cells were intact in their

structure. However, there was altered histological features observed in the testis after

the administration of 500 mg/ kg bw for 30 days. The compactness of the

seminiferous tubules was lost, the appearance of large or widened intercellular spaces

with regressed Leydig cells was observed. The germinal epithelium was found to be

ruptured in many tubules. The normal concentric arrangement of the germ cells was

74

absent, the germ cells were degenerated. The lumen was empty without spermatozoa

in many of the seminiferous tubules compared with control. The presence of vacuoles

in between the germ cells was significant. However after cessation of the treatment

for 60 days, there was the reappearance of the normal histoarchitecture of the testis

(Fig 15a to d).

The mean diameter of seminiferous tubule per section of the cross section of

the testis and the diameter of Leydig cell nucleus was similar to control (170.80µm

and 6.33 µm) in 250 mg/ kg (170.50 µm and 6.23 µm) treated mice but a significant

reduction in the diameter of seminiferous tubule (136.70 µm) and Leydig cell nucleus

(3.95 µm) was observed at 500 mg/ kg bw. The percentage reduction in the diameter

of seminiferous tubule and Leydig cell diameter was 80.03 and 62.40 in 500 mg/ kg

bw. However, there was a recovery in the diameter of the seminiferous tubules

(164.20 µm) and the Leydig cell nucleus (5.11 µm) of the testis. The percentage of

recovery was 96.13 and 80.72 in the diameter of seminiferous tubule and Leydig cell

nucleus respectively (Table 14; Fig 17a & b).

4.13 Histology and histometry of the epididymis

The normal histology of the epididymis was seen in the animals treated with

250 mg/ kg bw the tubules were intactly arranged, the lumen of the tubules were filled

with spermatozoa, the epithelium of the tubule were lined by ciliated columnar

epithelial cells, but in the 500 mg/ kg bw treated animals the compactness of the

seminiferous tubules was not found, the epithelial layer was ruptured and there was a

significant reduction in the epithelial height of the epididymis, the density of the

spermatozoa in the lumen was also reduced compared to control (Fig 16a to d).

75

The mean diameter of seminiferous tubule of the cauda epididymis per section

of the cross section of the epididymis and the epithelial height of the cauda

epididymis was similar to control (228.05 µm and 15.50 µm) in 250 mg/ kg (226.85

µm and 15.39 µm) treated mice but a significant reduction in the diameter of

seminiferous tubule (182.95 µm) and seminiferous epithelial height (11.83 µm) was

observed at 500 mg/ kg bw. The percentage reduction in the diameter of seminiferous

tubule and seminiferous epithelial height was 80.22 and 76.32 in 500 mg/ kg bw.

However, there was a recovery in the diameter of the seminiferous tubules (205.05

µm) and the seminifeorus epithelial height (13.49 µm) of the testis. The percentage of

recovery was 89.91 and 87.03 in the diameter of seminiferous tubule and the

seminiferous epithelial height respectively (Table 14; Fig 17a & b).

4.14 Total sperm count

Animals treated with 250 mg/ kg bw showed a slight decrease in the sperm

count, but the reduction in the total sperm count (5.4750±0.11087) was not considered

to be significant. However, a significant (P<0.05) reduction was observed in the total

sperm count of the animals treated with 500 mg/ kg bw (4.5375±0.18639). The

reduction in the total epididymal sperm count was 0.4%, 17.5% in the low dose and

high dose treated animals. There was a recovery in the total sperm count after the

cessation of the treatment (5.0625±0.04270) for 30 days. The percentage of sperm

count in the recovery group was 7.96%. The recovery in the sperm count was nearly

9.55% (Table 15; Fig 18a).

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4.15 Abnormal sperm count

The percentage of abnormal sperm found in the animals treated with 250 mg/

kg bw (32.00±3.71932) was 6.66% higher than the control (30.00±2.67706) but the

increase in the percentage of abnormal sperm was not considered to be significant.

However, there was a significant (P<0.001) increase in the abnormal spermatozoa in

500 mg/ kg bw (67.25±3.63719) treated animals. The increase in the abnormal

spermatozoa count was 125%. The head shape abnormalities observed was

amorphous head, hookless head, pinhead, banana head, hammer head and double

head. The percentage of amorphous head shape abnormality was found to be high

compared with other abnormalities in 500 mg/ kg bw treated animals. The percentage

of total abnormal sperms were reduced to 48.33% from 125% after the cessation of

the treatment for 60 days (Tables 15 & 16; Fig 18b & 19).

4.16 Fertility parameters

The fertility index of male and female was 100% in both 250 and 500 mg/ kg

bw treated animals, similarly the gestation, viability, parturition and the lactation

indices were also 100% (Table 17). The litter size of the females cohabited with males

treated with 250 mg/ kg bw were 100% but the females cohabited with males treated

with 500 mg/ kg bw did not show any signs of pregnancy, there was no occurrence of

spermatozoa in the vaginal plug even after 15 days after the treatment period and the

litter size was zero. However, the litter size was increased to 86.32% (P<0.001) after

the cessation of the treatment for 60 days. There was a 13.69 % increase in the

percentage of the litter size of the recovery group (Tables 18 & 19; Fig 20a).

77

4.17 Serum testosterone level

The serum testosterone levels were not significantly decreased in the 250 mg/

kg bw treated animals (735.96±24.757) compared with control (772.79±34.590), but

there was a significant (P<0.001) decrease in the levels of the serum testosterone level

(175.22±32.114) compared with control. The reduction was 4.77%, 77.33% in 250 and

500 mg/ kg bw treated animals respectively. There was an increase in the testosterone

level (529.78±22.196) to 31.45% in the recovery group (P<0.01) after the cessation of

the treatment for 60 days (Table 19; Fig 20b).

4.18 Biochemical studies

4.18a Glycogen estimation

The glycogen content of the testis in the 250 mg/ kg bw treated animals did

not significantly reduced (1.72±0.092) compared with control (1.87±0.045). However,

in the 500 mg/ kg bw treated animals the glycogen content reduced significantly

(P<0.001) to 0.70±0.042. The reduced glycogen content was recovered (1.21±0.084)

after the cessation of the treatment (P<0.01) in the recovery group for 60 days (Table

20; Fig 21).

In epididymis the glycogen content of the 250 mg/ kg bw treated animals did

not significantly reduced (4.63±0.219) compared to control (4.78±0.278). However,

in the 500 mg/ kg bw treated animals the glycogen content reduced significantly

(P<0.05) to 3.16±0.279. The reduced glycogen content was recovered (4.32±0.197)

after the cessation of the treatment in the recovery group for 60 days (Table 20; Fig

21).

78

4.18b Protein estimation

The protein content of the testis in the 250 mg/ kg bw treated animals did not

significantly reduced (9.51±0.468) compared with control (9.83±0.302). However, in

the 500 mg/ kg bw treated animals the protein content reduced significantly (P<0.001)

to 6.67±0.162. The reduced (P<0.05) protein content was recovered (8.20±0.172)

after the cessation of the treatment in the recovery group for 60 days (Table 21; Fig

22).

The protein content of the epididymis in the 250 mg/ kg bw treated animals

did not significantly reduced (14.34±0.285) compared with control (14.10±0.261).

However, in the 500 mg/ kg bw treated animals the protein content reduced

significantly (P<0.001) to 9.31±0.212. The reduced (P<0.01) protein content was

recovered (11.97±0.281) after the cessation of the treatment in the recovery group for

60 days (Table 21; Fig 22).

4.18c Cholesterol estimation

The cholesterol content of the testis in the 250 mg/ kg bw treated animals did

not significantly increased or decreased (5.52±0.427) compared with control

(5.39±0.350). However, in the 500 mg/ kg bw treated animals the cholesterol content

increased significantly (P<0.05) to 8.45±0.537. The increased cholesterol content was

recovered (6.96±0.095) after the cessation of the treatment in the recovery group for

60 days (Table 22; Fig 23).

The cholesterol content of the epididymis in the 250 mg/ kg bw treated

animals did not significantly increased or decreased (10.09±0.085) compared with

control (10.04±0.202). However, in the 500 mg/ kg bw treated animals the cholesterol

79

content increased significantly (P<0.01) to 11.94±0.221. The increased cholesterol

content was recovered (9.97±0.179) after the cessation of the treatment in the

recovery group for 60 days (Table 22; Fig 23).

4.18d Total ascorbic acid estimation

The estimation of total ascorbic acid was done only using the testis. There was

no any significant changes occurred in the 250 mg/ kg bw (5.39±0.137) and 500 mg/

kg bw (5.54±0.307) treated animals and the recovery group animals (5.45±0.155)

compared with control (5.42±0.258) (Table 23; Fig 24).

5.5.5.5. DDDDiscussioniscussioniscussioniscussion

Medicinal plants have been of age long remedies for human diseases because

they contain components of therapeutic values (Nostro et al., 2000; Britto and

Gracelin., 2011). Therapeutic properties of the green parts of the Opuntia plant the

cladodes, have very long been known in the traditional medicine (Cornett., 2000;

Knishinsky., 1971). Recently the potential activities of the fruit and the nutritional

benefits has been explored recently, that made the cactus pear fruits a health

promoting food and food supplements. However a systematic research is in need to

confirm the benefits of these fruits to document health effects and claims (Livrea and

Tesoriere., 2006). Preliminary phytochemical screening revealed the presence of

alkaloids and flavonoids, phenolics, saponins, and tannins (Chauhan., 2010;

Ramyashree et al., 2012). As these compounds are associated with nutritional and

health promoting aspects, the fruits of Opuntia are also considered to be of therapeutic

value (Stintzing et al., 2001). The presence of flavonoids in the hydroalcoholic extract

of Mikhania glomerata was responsible for the antifertility activity (Silveira e Sá et

80

al., 2003). The presence of different phytoconstituents of pharmacological importance

of fruits of O. elatior, offered further investigations.

The toxicity tests of O. elatior fruit extract revealed no toxic side effect on the

external morphology and the body weights of the mice till a dose level of 600 mg/ kg

bw (Ramyashree et al., 2012), thus dose levels less than LD50 were selected in the

present study. The body weight of the extract treated animals remained unchanged

which indicate no toxic effect of O. elatior on growth and metabolic processes of the

treated animals which in conformity with the observation of D’Cruz and Mathur

(2005), where piperine treated mice did not show any significant changes in the body

weight.

In the present study treatment with the O.elatior fruit extract did not cause any

changes in the body weight of any treated groups. All the animals were in good

health. No evidence of mortality of the animals was observed. Body weight changes

may lead to impaired reproductive functions, Gupta et al., (2011) concluded that no

alteration in the body weight after the treatment of ethanolic extract of Opuntia

dillenii refers to normal reproductive functions. Body weight is also one of the criteria

to assess the toxicity of the sample. The treatment with ethanolic bark extract of C.

albidum shows an increase in the body weight, thus Onyeka et al (2012) concluded

that increase in the body weight indicates that the extract may have a toxic effect on

the treated animals. The oral administration of ethanolic extract of Lepidagathis

alopecuroides did not cause a significant reduction in the body weight of the treated

rats indicating non-systemic toxicity of the extract in rats (Orlu et al., 2012). Body

weight is also associated with metabolic process of the treated animals. D’Cruz and

81

Mathur (2005) reported that the piperine did not cause any changes in the body

weight, which shows that the dose selected were not toxic and the metabolic process

of the treated animals were normal. Monitoring of body and organ weights gives

information on the general well being of the animal. The aqueous leaf extract of

Oldenlandia affinis did not alter the body weight of the male rats, which is important

in the interpretation in the reproductive data (Sewani-Rusike and Gundidza., 2011).

Thus no alteration in the body weight refers to normal reproductive functions and

metabolic process and non toxic effect of the plant extract. Similar results were

observed in the animals treated with Strychnos potatorum (Gupta et al., 2006), Ficus

bengalensis (Gupta., 2012), Cyamposis psoralioides (Thejashwini et al., 2012)

A significant difference in the weight of the reproductive organs in high dose

treated group animals compared with that of the control was observed both in short

and long durations, wherein the weight of the testes and epididymis was declined to

561.62 mg/100 g bw and 180.25 mg/100g bw in the high dose (500 mg/ kg bw)

treated animals compared to that of control in short duration. The reduction in the

mean relative weight of the testis and epididymis was 19.59% and 25.43%. But no

such significant changes observed in weight of vas deferens, seminal vesicle and

ventral prostate. No reductions in the weight of the testes and other accessory

reproductive organs were observed in the low dose (250 mg/ kg bw) treated animals

compared with control. Similar to short duration there was a significant reduction in

the weight of the testis and epididymis in the animals treated with 500 mg/ kg bw.

The weight of the testes and epididymis was declined to 587.75 mg/ 100g bw and

192.50 mg/ 100g bw. The reduction in the mean relative weight of the testis and

epididymis was 19.33% and 31%. However, no such significant changes observed in

82

weight of vas deferens, seminal vesicle and ventral prostate. No reductions in the

weight of the testes and other accessory reproductive organs were observed in the low

dose (250 mg/kg bw) treated animals compared with control.

The mammalian testis is a complex organ which serves two important

functions : the synthesis and secretion of steroid hormones, and the production of

spermatozoa. It is well known that the normal testicular development and the

maintenance of spermatogenesis are controlled by gonadotrophins and testosterone

whose effects are modulated by a complex network of factors produced locally and

among them (Carreau et al., 2007). Seminiferous tubules make up about 90% of the

wet weight of the normal testis thus testicular weight loss may be attributed to the

spermatogenic disruption and degeneration of germinal elements (Mishra and Singh.,

2008). Reduction in the weight of the reproductive organs is correlated to reduced

circulating androgen level according to Gupta (2006), where the methanolic extract of

Strychnos potatorum seeds was responsible for reduced weight of testis and accessory

reproductive organs, which might be due to low levels of androgen. Similar

impairment in the reproductive activity of testis and accessory reproductive organs

was also mainly because of circulating androgen (Raji et al., 2006; Rahim et al.,

2010). The methanolic seed extract of Ricinus communis caused a significant decrease

in the weight of the reproductive organs, which is mainly due to decreased levels of

testosterone (Raji et al., 2006). The reproductive organ weight reduction is the clear

indication of structural and functional alteration in the testes and epididymis due to

drugs. The ethanolic extract of Tinospora cordifolia stem and the methanol extract of

Momordica dioica induced reduced reproductive organ weight (Singh et al., 2011;

Kachchawa et al., 2010). For the normal functioning, growth and development of

83

reproductive organs testosterone are a key element, whereas 50% ethanolic extract of

Calendula officinalis flower showed a weight loss of reproductive organs (Kushwaha

et al., 2007). As these organs are androgen dependent, decrease in the serum

testosterone level leads to decreased weight of reproductive organs (D’Cruz and

Mathur., 2005), the oral administration of piperine caused a significant reduction in

the weight of the testis and accessory reproductive organs. Cell degradation in

reproductive organs and accessory sex organs and significant decrease in their weight

and sperm production showed the effects of the extract on cellular and physiological

functions of the testes and epididymis (Gupta et al., 2011), the administration of

ethanolic extract of Opuntia dillenii caused a significant reduction in the weight of the

testes, epididymis, vas deferens, seminal vesicle and ventral prostate. Depression in

spermatogenesis is usually accompanied by reducing testicular weight as the bulk of

testicular weight is made up of spermatids and spermatozoa (Sewani-Rusike and

Gundidza., 2011). Decreased testicular weight may be due to germ cell loss due to

testosterone withdrawal and a part of this weight loss might represent a decrease in

seminiferous tubular fluid production, as the weight of testes is largely dependent on

the mass of differentiated spermatogenic or may be due to reduced tubule size,

spermatogenic arrest and inhibition of steroid biosynthesis of Leydig cells, a site of

steroid biosynthesis. (Shahinturk et al., 2007; Joshi et al., 2012; Joshi and Bansal

2012; Raji et al., 2011). Oral administration of 2,4-Dchlorophenoxyacetic acid was

responsible for the reduced weight of reproductive organs, as 2,4-D reduce the

spermatogenic potential by reducing their number of Sertoli cells (Joshi et al., 2012).

Decrease weight of accessory sex glands in the ethanolic extract of Calendula

officinalis indicate the atrophy of glandular tissue and diminished secretary's ability

reflects the decrease level of testosterone as these organs are androgen dependent

84

(Agarwal et al., 2011). Decrease in seminiferous tubule diameter reflects tubular

shrinkage, which may occur due to cell death or sloughing of epithelial cells (Gupta et

al., 2006). The significant decrease in the organ weights of the treated animals is

indicative of the toxic effect of the extract. The highest significant decrease observed

in the weights of testis and epididymis may be due to loss of spermatogenic elements

in the testis and the absence of sperm in the epididymis (Asuquo et al., 2012), the oral

administration of Spondias mombin leaf extract caused a significant decrease in the

weight of the reproductive organs. The observed reduction of the testicular weight

may be due to the altered production of seminiferous tubular fluid. The petroleum,

chloroform and ethanolic extract of Momordica charantia caused a significant

reduction in the weight of the reproductive organ, which was mainly because of

altered seminiferous tubular fluid. The protein content of any organ is directly

proportional to the growth rate (Gupta et al., 2006; Patil and Patil., 2007; Kachchawa

et al., 2010). The reduction in protein content of the testis in extract treated mice may

also be another reason for a reduction in the weight.

Similar results were observed in the treatment with alcoholic extract of Citrus

limonum (seed) (Kulkarni et al., 2012), Feronia limonia (fruit pulp) (Dhanpal et al.,

2012), U and D bitter herbal supplement (Ejeatuluchukwu and Orisakwe., 2011),

Bulbine natalensis (stem) (Yakubu and Afolayan., 2009), Oldenlandia affinis

(Leaves) (Sewani-Rusike and Gundidza., 2011), Cissampelos Pereira (Leaves)

(Harisha., 2012), Cuminum cyminumin (seeds) (Muthu and Krishnamoorthy., 2011),

Lepidagathis alopecuroides (Leaves) (Orlu et al., 2012), Madhuca latifolia (seeds)

(Gopalakrishnan and Shimpi., 2011), Bacopa monnieri (Singh and Singh., 2009),

Trigonella foenum graecum (Kassem et al., 2006),

85

Oral administration of artesunate caused a significant reduction in the weight

of the reproductive organs (Raji et al., 2011), tetrazamacrocyclic compounds caused a

structural and functional alterations of reproductive organs (Chaudhary and Singh.,

2006), oral administration of dicofol caused a significant decrease in the weight of

testis and epididymis (El - Kashoury et al., 2009).

The disruption of testicular cytoarchitecture has adversely affected the Leydig

cell number and function probably led to a decrease in the serum levels of testosterone

(Raji et al., 2006). The affected tubules in the testes show intraepithelial vacuolation,

exfoliation of germ cells, presence of spermatids of different stages of the

spermatogenic cycle in the same tubule and phagocytosis of elongated spermatids.

Formation of multinucleated giant cells, containing round or elongated spermatids in

seminiferous tubules is connected with each other by intercellular bridges and any

alterations in the intercellular bridge results in the formation of multinucleated giant

cells by fusion of germ cells (Gupta., 2012). Examination of testes section mainly

revealed disruption in the arrangement of seminiferous tubules and very low Leydig

cell count. The low Leydig cell number was also noticed. In high dose groups the

seminiferous tubules were lined by only few necrotic germ cells with scattered Sertoli

cells which suggest that the treatment caused alteration in the kinetics of

spermatogenesis (Jahan et al., 2009). DG caused distortion/ destruction of the

architecture and structure of the testicular histology, characterized by edema, reduced

spermatogenesis and maturation arrest of spermatozoa at different stages of germ cell

developments. The histopathological result was corroborated by a significant

reduction in the mean testicular weight (Obianime et al., 2010). Leydig cell

86

destruction leads to the generation of little or no testosterone (Shahinturk et al., 2007).

Reduction in the number of Sertoli cells results in decreased number of

spermatogonia, as Sertoli cells play a critical role in spermatogenesis by providing the

physical support, nutrients and hormonal signals, necessary for successful

spermatogenesis. Sertoli cells can seriously reduce their supportive capacity and result

in increased elimination of germ cells (Gupta et al., 2006).

The epididymis is a site that can be exploited for male contraception without

undue side effects. It has been identified as the site where the essential post testicular

sperm maturation and storage occurs. Nevertheless, little is known about the process

of sperm maturation and factors affecting it. Understanding the physiology of the

epididymis could provide new possibilities for infertility treatment on one hand, and

could suggest new strategies for the development of a new non hormonal male

contraceptive on the other. (Dehghan et al., 2005). Androgen deficiency causes a

significant reduction in the seminiferous tubular diameter, regression in the

epididymal epithelium and decline in the number of spermatozoa (Ahmed et al.,

2008).

Any chemical agent that can affect reproductive activity will as well affect the

quality and quantity of the sperm (Ezeonwu., 2011). Depletion in the sperm count

may be one of the reasons for altered spermatogenesis and reduced fertility. Normal

male fertility relies on normal spermatogenesis (O’Donnell et al., 2001).

Spermatogenesis is the process of male gamete production, wherein the

spermatogonia transform into highly specialized matured spermatozoa within testis

(Wistuba et al., 2007) which is regulated by gonadotrophins and testosterone in

mammals (Jones., 1991). This process takes place within the seminiferous tubules of

87

the testis, in close association with the somatic cells of the seminiferous epithelium

(O’Donnell et al., 2001). The sperm count is one of the most sensitive tests for

spermatogenesis and it is highly correlated with fertility (Joshi and Bansal 2012).

Maturation of sperm is also one of the important events that take place in epididymis

where the sperm is nurtured by epididymal secretion (Jones., 1991; Cooper., 1999). A

reduction in sperm count suggests alterations in sperm maturation and sperm

production (Dhanpal et al., 2012). Decrease in the sperm concentration may be due to

the inhibition in spermatogenesis which has been reflected here by the low count

(Onyeka et al., 2012). The reduced concentration of caudal spermatozoa may be due

to the suppressive effect of the extract on spermatogenesis (Jahan et al., 2009; Gupta.,

2012). Biologically active gonadotropins are essential for normal sperm production,

growth, development and maturation of testes and cauda epididymis. The suppression

of gonadotropin may decrease sperm density in testes and cauda epididymis. (Joshi et

al., 2012). The decrease in sperm function of the treated rat supported the dose

dependent reduction in serum testosterone levels and its consequent effects on the

testis and accessory reproductive organs (Raji et al., 2006; Singh and Bansode.,

2010). The reduction in the number of spermatozoa is due to inhibition of androgen

synthesis and as a result there is a reduction in weights of testis and accessory

reproductive organs. (Saratchandiran et al., 2007). Reduced weight of the testis and

androgen level is one of the reasons for depletion of sperm count (D’Cruz and

Mathur., 2005; Gupta et al., 2006). Epididymis normally provides a favorable milieu

for acquisition of fertilizing ability and viability of spermatozoa (Raji et al., 2006;

Gupta., 2012). The spermatozoa produced in the testis attain further development,

motility and physiological maturation in the microenvironment of the epididymis. The

decrease in the cauda epididymal sperm count may not only due to decrease in the

88

testicular spermatogenic process, but also due to the altered microenvironment of the

epididymis (Patil and Patil., 2007; Kushwaha et al., 2007). Low sperm counts have

been associated with reduced fertility. A significant decrease in the sperm count is

supported by the various degrees of degeneration in the histologic sections of the

testes (Raji et al., 2005; Gopalakrishnan and Shimpi., 2011; Oyekunle et al., 2012).

Reduction in the total number of epididymal sperm count could also be a result of

disruption of seminiferous tubule as observed in the histological section of the testis

(Raji et al., 2006). The decrease in the sperm count is likely due to direct damages to

the Leydig and Sertoli cells which are directly involved in the production of

spermatozoa (Obianime et al., 2010). Significant decrease in the total sperm count is

now evident that extract induced oxidative stress exerts toxic effects on organs such

as testis. Thus extract induced oxidative results in a reduced sperm count (Gupta et

al., 2011; Onyeka et al., 2012). The administration of the ethanolic extract of

Rauwolfia vomitoria caused a significant reduction in the sperm count, which

suggests that the prolonged exposure of the extract crossed the blood testes barrier

and interfered with the functioning of testicular epithelium, which is the site of sperm

production causing reduction in the sperm count (Oyekunle et al., 2012). The

spermicidal effect may be due to the effects of triterpenes and saponins components in

the plant which are deleterious to sperm cells. The toxicity of the saponins may be

related to their astringent actions on the cell surfaces of sperm cells causing a

disruption of the cell membrane, which could result in the reduction in sperm motility,

as well as the inhibition of specific enzymes necessary for the sperm synthesis

(Obianime et al., 2010). Most plants derived spermicides that caused sperm

immobilization in animals and humans were confirmed to contain saponins. The

aqueous ethanolic extract of Lawsonia inermis leaves has a spermicidal effect

89

(Hyacinth et al., 2012). Decrease in the mean sperm count was observed in animals

treated with Iranian neem seed oil (Dehghan et al., 2005). Ethanolic extract of

Trachyspermum ammi (fruits), caused a significant reduction in the sperm count

(Kumar et al., 2011b). The alcoholic extract of Citrus limonum (seeds) also showed

the similar results (Kulkarni et al., 2012).

The oral administration of ethanolic extract of Feronia limonia (fruit)

(Dhanpal et al., 2012), Calendula officinalis (Agarwal et al., 2011), Rauwolfia

vomitaria (bark) (Oyekunle et al., 2012), Cissampelos pereira (leaves) (Harisha.,

2012), Madhuca latifolia (seed) (Gopalakrishnan and Shimpi., 2011), Chrysophyllum

albidum (bark) (Onyeka et al., 2012), Calendula officinalis (flower) (Kushwaha et al.,

2007), Ficus bengalensis (leaves) (Gupta., 2012), aqueous extract of Bulbine

natalensis (stem) (Yakubu and Afolayan., 2009), Ricinus communis (root)

(Sandhyakumary et al., 2003), Austroplenckia populnea (leaves) (Mazaro et al., 2002)

resulted in reduced sperm count. Similar results were observed after the oral

administration of monocrotophos (Joshi and Bansal 2012), Benzene (Singh and

Bansode., 2010).

The occurrence of the high number of abnormal sperm indicates interference

with testicular spermatogenesis (Parveen et al., 2003; Kumar et al., 2011b). The

functional deficiencies of the testis and accessory reproductive organs can be

attributed to the morphological abnormalities of the spermatozoa. The irregular

mitochondrial sheaths, aberrant attachment of tails and deviant head shapes impair

their motility, whereas the lack or malformation of the acrosomes as well as the

anomalous head shapes precludes fertilization (Gupta et al., 2011). The abnormality

90

in the sperm morphology (secondary abnormality) was associated with epididymal

functions of transportation, maturation and storage of sperm cells during which period

the spermatozoa develop motility (Raji et al., 2006). The treatment with ethanolic

extract of Iranian neem seed oil attributed to a significant increase in the head shape

abnormalities (Dehghan et al., 2005). The alcoholic extract of Trachyspermum ammi

was responsible for increased levels of abnormal sperms, which may attribute it

toward the antifertility property (Kumar et al., 2011b).

Interference at the level of testicular androgens, arrests the process of

spermatogenesis in the testis, without disturbing general metabolism (Kushwaha et

al., 2007). The weight of the testis and reproductive organs are dependent on

testosterone levels. Thus reduced weight decreased number of spermatozoa is directly

dependent on androgen levels (Singh et al., 2011; Gopalakrishnan and Shimpi., 2011).

The protein content of the reproductive organs is also dependent on the androgen

levels. Thus the mode of action was therefore through the pituitary gonadal axis,

confirmed by the decreased serum testosterone level (Kushwaha et al., 2007; Onyeka

et al., 2012). Testosterone in association with FSH normally acts on the seminiferous

tubules to initiate and maintain spermatogenesis. As all organs of male reproduction

are androgen dependent, they serve as indicators of the Leydig cell function or

androgen action (Raji et al., 2005; Muthu and Krishnamoorthy., 2011). The

impairment in the reproductive activities is mainly due to decrease in the testosterone

secretion. Testosterone was necessary for the development, growth and normal

functioning of the testes and male accessory reproductive glands. Low serum

testosterone levels have been reported to adversely affect the structure, weight and

functions of the testis and accessory reproductive organs. The spermatogenesis in

91

mammals depends on testosterone production by Leydig cells in response to

stimulation of FSH and LH. FSH increases Sertoli cell synthesis of an androgen

binding protein needed to maintain high concentrations of testosterone. LH stimulates

testosterone production by interstitial cells of the testes (Joshi and Bansal 2012; Raji

et al., 2011). The arrest of spermatogenesis possibly occurred as a consequence of

reduction in serum testosterone, which has been shown to be essential for the

completion of meiotic division during spermatogenesis. The low level of testosterone

arrests spermatogenesis. Reduction in the FSH and LH affects the development and

function of the testes and inhibit the development of spermatogenesis and

seminiferous tubule. (Joshi et al., 2012; Kachchawa et al., 2010). Disruption in the

cytoarchitecture affected the Leydig cell number and function which probably led to a

decrease in the serum levels of testosterone (Oyekunle et al., 2012). Significant

inhibition of androgenic function is evidenced by reducing the size and morphology

of Leydig cells and significant decline in accessory sex organ (Singh and Bansode.,

2010). Furthermore, regulation of testicular secretion occurred via a negative

feedback system involving the hypothalamus-pituitary-testicular axis. The

hypothalamus released pulses of gonadotrophin releasing hormone, which stimulated

the anterior pituitary to secrete and release luteinizing hormone and follicle

stimulating hormone in a pulsatile manner. LH acted on the Leydig cell activate the

synthesis of pregnenolone from cholesterol. Synthesized testosterone had a paracrine

effect on the sertoli cells, where it played an essential role in the facilitation of

spermatogenesis. Disruption of this pathway could deprive the Leydig cells of LH,

and its stimulatory action leading to a reduction in the secretion and release of

testosterone (Raji et al., 2006; Sewani-Rusike and Gundidza., 2011). Testosterone

withdrawal has been shown to cause DNA fragmentation by stimulating caspase

92

activation in Sertoli cells in vitro, which indicated that decreased testosterone levels

can stimulate apoptotic pathways (D’Cruz and Mathur., 2005). The testosterone level

in serum and plasma correlate with sperm concentration and sperm motility (Onyeka

et al., 2012). Cholesterol is the principal precursor for the formation of androgens in

the biogenic pathway in the testis and involved in steroidogenesis in the testes

(Dhanpal et al., 2012; Chaudhary and Singh., 2006). The increased level of

cholesterol in the testis inhibited synthesis of testosterone a potent androgen, the main

hormone involved in the control of fertility of animals including rats (Patil and Patil.,

2007; Joshi and Bansal 2012; Agarwal et al., 2011). The reduction in the testosterone

concentration may explain the reduced mating success observed with the highest dose

as testosterone is considered to contribute to improvement in sexual function, libido,

and penile erection . This is an indication of a potential negative effect of the extract

on the male hormone at this dose (Yakubu and Afolayan., 2009).

Similar results were observed after the treatment with ethanolic extract of

Spondias mombin (leaves) (Asuquo et al., 2012), Momordica charantia (seeds) (Patil

and Patil., 2007), Citrus limonum (Kulkarni et al., 2012), Rauwolfia vomitaria (bark),

(Oyekunle et al., 2012) methonolic extract of Nyctanthes arbortristis (Gupta et al.,

2006), Momordica dioica (seeds) (Kachchawa et al., 2010), Feronia limonia

(Dhanpal et al., 2012), Calendula officinalis (Agarwal et al., 2011), Cuminum

cyminamin (seed), (Muthu and Krishnamoorthy., 2011), Madhuca latifolia (seed)

(Gopalakrishnan and Shimpi., 2011), aqueous extract of Bulbine natalensis (Yakubu

and Afolayan., 2009), Sida cardifolia (Sewani-Rusike and Gundidza., 2011), 2-4,

dichlorophenoxyacetic acid (Joshi et al., 2012), Monocrotophos (Joshi and Bansal.,

2012), Artesunate (Raji et al., 2011), Benzene (Singh and Bansode., 2010).

93

Cholesterol is the principal precursor for the formation of androgens in the

biogenic pathway in the testis. (Saratchandiran et al., 2007; Dhanpal et al., 2012).

Cholesterol is involved in steroidogenesis in the testes. Mammalian cells require

cholesterol which plays an important role in acting as a precursor molecule in the

synthesis of steroid hormone. Androgens are synthesized from cholesterol (Chaudhary

and Singh., 2006). An increased level of cholesterol is attributed to decreased

androgen concentration, resulting in impaired spermatogenesis (Sathiyaraj et al.,

2011; Kushwaha et al., 2007). The conversion of cholesterol to pregnenolone in

Leydig cells depends upon the availability of LH. The increased level of cholesterol in

the testis of treated rats in the present study indicates inhibited synthesis of

testosterone a potent androgen, in the testis (Patil and Patil., 2007). The increased

concentration of cholesterol in the testes may be the result of its non-utilization

leading to the reduction of the production of testosterone, the main hormone involved

in the control of fertility of animals including rats (Joshi et al., 2012; Muthu and

Krishnamoorthy., 2011; Joshi and Bansal., 2012). A high accumulation of cholesterol

in the testes of the extract treated rats may be due to decreased steroidogenesis (Gupta

et al., 2006). The accumulation of cholesterol may partly result from degeneration of

the germinal epithelium (Watcho et al., 2001). Increased levels of cholesterol may be

due to decreased androgen production, which resulted in accumulation of cholesterol

in testes and thus impaired spermatogenesis. (Agarwal et al., 2011). Administration

of aqueous extract of Terminalia chebula caused a significant increase in the

cholesterol levels in the testis (Krishnamoorthy et al., 2007). The ethanolic seed

extract of Crotolaria juncea brought about a significant increase in the cholesterol

level in the testis (Vijaykumar et al., 2003). Administration of Momordica charantia

94

seed extract caused a significant increase in the cholesterol level in the testis (Nassem

et al., 1998).

Proteins are the most important and abundant macromolecules playing a vital

role in the architecture and physiology of the cell and cellular metabolism. The

changes in protein suggested that there is a reduction in the synthetic activity in testes

in the functional ability of spermatozoa (Joshi and Bansal., 2012). Reduced testicular

and epididymal protein content could be correlated with the absence of spermatozoa

in the lumen, since the luminal fluid of epididymis contains a number of proteins,

some of which remain bound to spermatozoa (Sathiyaraj et al., 2011). It is evident

that testicular function would be altered by reduced protein content (Gupta et al.,

2006). The reduced protein content may be another reason as the growth rate of any

organ is proportional to its protein content. (Vijaykumar et al., 2003; Kachchawa et

al., 2010). The protein content of the reproductive organs was significantly decreased,

again perhaps due to a low level of androgens (Kushwaha et al., 2007). The decrease

in the protein content of the cauda epididymis may affect the glycoproteins secreted

by the epididymis and coated on the sperm to stimulate its motility, thus causing

reduced fertility in rats (Sinha., 1990). Protein is involved in the alteration of almost

every physiological system and the total protein runs parallel to the growth and is

sensitive to estrogen and androgen, respectively. The protein level directly correlates

with the secretory activity of the epididymis, which in turn depends on the androgen

levels (Chaudhary and Singh., 2006). The protein content in testes and other sex

organs significantly decreased following Strychnos potatorum seed extract

administration probably due to the absence of the spermatogenic stages in the testes.

95

(Gupta et al., 2006). Administration of aqueous extract of Terminalia chebula caused

a significant decrease in the protein levels in the testis (Krishnamoorthy et al., 2007).

Glycogen, a reserve carbohydrate found in Sertoli cells and spermatogonia in

testis, serves as a source of glucose, which is an energy supplier to the tubular cells.

Glycogen might represent a source of nourishment for the spermatozoa during its

development and maturation (Chaudhary and Singh., 2006). The glycogen content in

the cell indicates energy storage. Sertoli cells and spermatogonia often contain

glycogen and secrete substrate from the blood and provide sources of reserve

carbohydrates for seminiferous tubular cells and the glycogen level is found to be

directly proportional to the steroid hormones (Vijaykumar et al., 2003; Gupta et al.,

2006). The glycogen content in the testicular cell indicates energy storage. Sertoli

cells and spermatogonia often contain glycogen and secrete substrate from the blood

and provide sources of reserve carbohydrates for seminiferous tubular cells and the

glycogen level is found to be directly proportional to the steroid hormones (Jahan et

al., 2009). A significant decrease in glycogen content after the treatment with aqueous

extract of Aegle marmelos leaves possibly could be explained by an inhibition of

glycolysis during spermatogenesis (Sathiyaraj et al., 2011). The reduced glycogen

content of the testis observed after the treatment of M. charantia seed extract in the

present study may be due to impaired glycolysis (Patil and Patil., 2007). Reduction in

glycogen level after the administration of 2, 4-inhibited the glycogen synthesis which

eventually decreases spermatogenesis. (Joshi et al., 2012). Reduced level of glycogen

may be due to interference in the enzymes of the glycolytic pathway, TCA cycle,

glucogenesis and oxidative phosphorylation which affects the maturation process of

spermatozoa and their motility. Inhibition of glycogen synthesis eventually decreased

96

spermatogenic process. (Joshi and Bansal 2012). The decrease glycogen content of

the testes after the administration of Nyctanthes arbortristis stem bark extract may

reduce the energy source for spermatogenic activity which might have resulted in

spermatogenic arrest (Gupta et al., 2006). Reduced glycogen reflects a decreased in

the number of post-meiotic germ cells which are thought to be the sites of glucose

metabolism. A decrease in glycogen content in testes indicative of decreased number

of post meiotic germ cells (spermatids), a site for glucose metabolism (Agarwal et al.,

2011). The glycogen content was significantly decreased in testes after the

administration of methanolic Cuminum cyminumin seeds extract treated rats may

reduce the energy source for spermatogenic activity which might have resulted in

spermatogenic arrest (Muthu and Krishnamoorthy., 2011). The ethanolic seed extract

of Crotolaria juncea brought about a significant decrease in the glycogen level in the

testis (Vijaykumar et al., 2003).

After the administration of both the doses the libido of the experimental males

remained unimpaired even after 30 days, but the fruit extract at high dose for 60 days

showed loss of libido, none of the females cohabited with the males treated with 500

mg/ kg bw for 60 days were pregnant after a stipulated period also. Reduced fertility

and reduction in the rate of pregnancy in the female mice shows the incapacities of

the male mice to successful mating (Al-Majed et al., 2006). It is well known that the

production of spermatozoa able to fertilize and develop a normal progeny result from

normal sperm maturation in the epididymis (Rahim et al., 2010). The litter size and

growth of the pups born indicated lack of teratogenecity. Normal newborn animals

suggest absence of any teratogenic effect of the extract (Kulkarni et al., 2012). 30%

reduction in the litter size in 500 mg/ kg bw treated animals, due to low androgen

97

concentration (Dohle et al., 2003), which might be sufficient for the normal mating

behavior, but insufficient for the maintainance of fertilizing ability of the epididymal

spermatozoa (Sharma and Jacob., 2002; Thejashwini et al., 2012). The ethanolic

extract of Cyamposis psoralioides caused upto 50% reduction in the litter size, which

was mainly due to reduced testosterone level (Thejashwini et al., 2012). The

treatment with aqueous extract of Mentha arvensis to male mice did not impair the

sexual behavior and libido of the experimental mice, but none of the females were

pregnant after successful mating (Sharma and Jacob., 1996). 50% reduction in the

fertility rate has been noted after the administration of Kalanchoe gastonis bonnieri to

adult male rats (Miranda – Beltrán et al., 2003).

All these factors thus brought about functional sterility in 500 mg/ kg bw

treated mice. However, the induced infertility was completely reversed after

withdrawal of treatment of another period of 30 days. The present study shows that

treatment with O. elatior fruit extract had no impact on the libido of extract-treated

males in the short duration, though, the number of live implants decreased

significantly in females impregnated by males treated with 500 mg/kg bw. However,

the high dose treatment in long duration the extract affected the libido of the treated

mice as a result the litter size was zero in high dose treated mice for long duration.

Low sperm count and high percentage abnormal spermatozoa level each have been

associated with reduced fertility (Raji et al., 2006) as obvious in the present study.

98

Table 1: Toxicity study on the body weight of the Opuntia elatior treated mice

Initial body weight (g) Final body weight (g)

Control 33.36±0.560 39.06±0.887

Low dose (200 mg/ kg bw) 33.93±0.898 39.40±0.700

Medium dose (400 mg/ kg bw) 34.46±1.068 39.26±0.895

High dose (600 mg/ kg bw) 34.73±0.811 39.63±0.796

Note: Values represented as Mean±SE (n=3).P value >0.05, thus considered non

significant

99

Table 2: Effect of Opuntia elatior fruit extract treatment for 30 days on the body and reproductive organs weight of mice

Treatment

groups

Mean weight (mg)/ 100g body weight ± SE

Initial body

weight

Final body

weight Testis Epididymis

Vas

deferens

Seminal vesicle Ventral

prostate

Control 27.93±0.33 38.18±0.47

698.5±22.61b

241.75±3.39c

74.12±2.97ab

531.25±27.77

16.87±2.22

Low dose

(250mg /kg bw) 28.18±0.25 39.06±0.33 696.25±17.81

b 254.12±5.62c 75.87±6.25

ab 500.00±30.91 13.25±1.91

High dose

(500mg /kg bw) 27.96±0.30

38.93±0.42

561.62±8.08a

180.25±7.22a

65.62±3.43a

429.87±51.54

15.72±1.19

Recovery of

500mg /kg bw 27.75±0.27 38.56±0.59

667.25±36.48b

209.37±11.96b

79.25±2.17b

466.37±52.46

14.12±1.86

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple range test. Values with the same superscript

are not significantly different whereas those with different superscript are significantly different from each other.

100

Table 3: Effect of Opuntia elatior fruit extract treatment for 30 days on the histometry of

testis and epididymis of mice

Treatment groups

Diameter of ST of

testis

(µm±SE)

Leydig cell nuclear

diameter

(µm±SE)

Diameter of ST of

cauda epididymis

(µm±SE)

Seminiferous

epithelial height

of cauda

(µm±SE)

Control

171.70±1.247

c 6.13±0.071

c 227.40±3.242

c 15.55±0.217

c

Low dose

(250 mg/ kg bw) 173.45±1.515

c 6.30±0.065

c 228.20±3.373

c 15.19±0.261

c

High dose

(500 mg/ kg bw) 138.00±3.373

a 3.99±0.151

a 180.55±5.139

a 11.76±0.352

a

Recovery for high

dose 161.05±1.934

b 5.19±0.122

b 206.05±1.642

b 13.35±0.400

b

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with the same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

Table 4: Effect of Opuntia elatior fruit extract treatment for 30 days on the total sperm

count and abnormal spermatozoa count

Treatment groups Total sperm count

(Millions/ epididymis)

Total percentage of abnormal

spermatozoa (%)

Control 5.5875±0.07465 c 25.00±1.35401

a

Low dose (250 mg/ kg bw) 5.4625±0.10078 bc

25.5000±1.32288 a

High dose (500 mg/ kg bw) 4.575±0.14506 a 54.5000±3.59398

b

Recovery of high dose 5.2125±0.05543 b 29.0000±1.95789

a

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with the same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

101

Table 5: Effect of Opuntia elatior fruit extract treatment for 30 days on the count of

abnormal spermatozoa

Types of

abnormalities

Mean number per 1000 spermatozoa ± SE

Control Low dose

(250mg/kg bw)

High dose

(500mg/kg bw) Recovery group

Amorphous head 16.75±1.10868 a 16.75±0.85391

a 28.25±3.06526

b 17.25±0.47871

a

Hook less head 2.5±0.64550 a 4.0±0.70711

a 13.25±2.17466

b 3.25±0.62915

a

Pin head 1.5±0.64550 a 1.0±0.40825

a 3.5±0.64550

b 2.75±0.95743

ab

Banana head 1.0±0.40825 a 0.75±0.25

a 2.5±0.5

b 1.75±0.47871

ab

Hammer head 3.25±0.85391 a 3.0±1.08012

a 6.75±1.10868

b 4.0±0.40825

ab

Double head Nil Nil 0.25±0.25 Nil

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

Table 6: Effect of Opuntia elatior fruit extract treatment for 30 days on the fertility

parameter of mice

Parameters Control Low dose High dose Recovery

Fertility index

(m) 100 (4) 100 (4) 100 (4) 100 (4)

Fertility index (f) 100 (8) 100 (8) 100 (8) 100 (8)

Parturition index 100 (4) 100 (4) 100 (4) 100 (4)

Gestation index 100 (94) 100 (94) 100 (62) 100 (85)

Viability index 100 (94) 100 (94) 100 (62) 100 (85)

Lactation index 100 (94) 100 (94) 100 (62) 100 (85)

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

102

Table 7: Effect of Opuntia elatior fruit extract treatment for 30 days on the litter size of

mice

Treatment

groups

No of males

mated/ female

No of pregnant

females Litter size Fertility rate

Control 4/8 8 97 100%

Low dose

(250 mg/ kg bw) 4/8 8 94 96.91%

High dose

(500 mg/ kg bw) 4/8 8 62 63.92%

Recovery for

high dose 4/8 8 85 87.63%

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

Table 8: Effect of Opuntia elatior fruit extract treatment for 30 days on the litter size and

serum testosterone level of mice

Treatment groups Litter size (No of pups/ female) Testosterone level (ng/ dl ± SE)

Control 12.1250±0.125 c 731.7975±36.8076

c

Low dose (250mg/ kg bw) 11.7500±0.16366 c 723.4650±39.3274

c

High dose (500mg/ kg bw) 7.500±0.26726 a 356.7925±48.0975

a

Recovery group 10.2500±0.16366 b 548.5350±40.3770

b

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

103

Table 9: Effect of Opuntia elatior fruit extract treatment for 30 days on the glycogen level

in the testis and epididymis of the mice

Treatment groups Testis

(µg/g)

Epididymis

(µg/g)

Control 1.87±0.044 c 4.42±0.186

b

Low dose

(250 mg/ kg bw) 1.80±0.084

c 4.50±0.105

b

High dose

(500 mg/ kg bw) 1.18±0.051

a 3.38±0.150

a

Recovery for high

dose 1.48±0.040

b 4.31±0.058

b

Note: Mean values were compared by One- way ANOVA followed by Duncun’s

multiple range test. Values with same superscript are not significantly different

whereas those with different superscript are significantly different from each other.

Table 10: Effect of Opuntia elatior fruit extract treatment for 30 days on the protein level

in the testis and epididymis of the mice

Treatment groups Testis

(µg/g)

Epididymis

(µg/g)

Control 9.86±0.342 c 14.20±0.271

c

Low dose

(250 mg/ kg bw) 9.65±0.388

c 14.33±0.369

c

High dose

(500 mg/ kg bw) 6.77±0.221

a 10.18±0.224

a

Recovery for high

dose 8.66±0.172

b 12.07±0.359

b

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas those with

different superscript are significantly different from each other.

104

Table 11: Effect of Opuntia elatior fruit extract treatment for 30 days on the cholesterol

level in the testis and epididymis of the mice

Treatment groups Testis

(µg/g)

Epididymis

(µg/g)

Control 5.28±0.251 a 10.02±0.217

a

Low dose

(250 mg/ kg bw) 5.26±0.466

a 9.78±0.285

a

High dose

(500 mg/ kg bw) 7.66±0.466

b 12.00±0.304

b

Recovery for high

dose 6.30±0.373

a 10.37±0.166

a

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas those with

different superscript are significantly different from each other.

Table 12: Effect of Opuntia elatior fruit extract treatment for 30 days on the total ascorbic

acid level in the testis of the mice

Treatment groups Testis

(µg/g)

Control 5.13±0.616

Low dose

(250 mg/ kg bw) 5.10±0.110

High dose

(500 mg/ kg bw) 5.80±0.073

Recovery for high

dose 5.26±0.250

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

105

Table 13. Effect of Opuntia elatior fruit extract treatment for 60 days on the body and reproductive organs weight of mice

Treatment

groups

Mean weight (mg)/ 100g body weight ± SE

Initial body

weight

Final body

weight Testis Epididymis

Vas

deferens

Seminal vesicle Ventral

prostate

Control 27.72±0.38 38.49±0.75

728.62±13.26

b 279.00±6.84

c 127.12±3.06

b 799.87±16.62

16.37±1.35

Low dose

(250mg /kg bw) 28.30±0.32

39.12±0.23 725.50±38.82

b 288.00±7.94

c 127.50±3.19

b 788.37±28.63

15.07±1.61

High dose

(500mg /kg bw) 28.08±0.28 38.81±0.65 587.75±28.52

a 192.50±13.27

a 115.00±4.20

a 799.25±26.66

15.82±1.24

Recovery of

500mg /kg bw 27.89±0.38

39.06±0.41

689.87±18.88

b 238.12±10.18

b 119.00±4.35

ab 793.25±26.48

15.31±1.78

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple range test. Values with same superscript are

not significantly different whereas those with different superscript are significantly different from each other.

106

Table 14: Effect of Opuntia elatior fruit extract treatment for 60 days on the histometry of

testis and epididymis of mice

Treatment groups

Diameter of ST of

testis

(µm±SE)

Leydig cell nuclear

diameter

(µm±SE)

Diameter of ST of

cauda epididymis

(µm±SE)

Seminiferous

epithelial height

of cauda

(µm±SE)

Control

170.80±2.009

b 6.33±0.111 c 228.05±3.262 c 15.50±0.256 c

Low dose

(250 mg/ kg bw) 170.50±2.233

b 6.23±0.100

c 226.85±3.558

c 15.39±0.283

c

High dose

(500 mg/ kg bw) 136.70±3.592

a 3.95±0.167

a 182.95±5.317

a 11.83±0.356

a

Recovery for high

dose 164.20±2.441

b 5.11±0.151

b 205.05±3.407

b 13.49±0.392

b

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

Table 15: Effect of Opuntia elatior fruit extract treatment for 60 days on the total sperm

count and abnormal spermatozoa count

Treatment groups Total sperm count

(Millions/ epididymis)

Total percentage of

abnormal spermatozoa (%)

Control 5.5000±0.19039 b 30.00±2.67706

a

Low dose (250 mg/ kg bw) 5.4750±0.11087 b 32.00±3.71932

a

High dose (500 mg/ kg bw) 4.5375±0.18639 a 67.25±3.63719

c

Recovery of high dose 5.0625±0.04270 b 44.50±3.52373

b

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

107

Table 16: Effect of Opuntia elatior fruit extract treatment for 60 days on the count of

abnormal spermatozoa

Types of

abnormalities

Mean number per 1000 spermatozoa ± SE

Control Low dose

(250mg/kg bw)

High dose

(500mg/kg bw) Recovery group

Amorphous head 17.50±1.554 a 17.75±1.701

a 30.75±3.449

b 22.75±2.322

a

Hook less head 3.25±0.75 a 5.75±1.493

ab 17.5±2.327

c 9.5±1.707

b

Pin head 2.5±1.040 a 2.25±1.436

a 7.0±0.9128

b 3.75±0.4787

a

Banana head 1.25±0.25 1.5±0.645 1.75±0.250 2.0±0.4082

Hammer head 5.5±0.6455 a 4.75±0.8539

a 10.25±1.436

b 6.5±0.6455

a

Double head Nil Nil Nil Nil

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

Table 17: Effect of Opuntia elatior fruit extract treatment for 60 days on the fertility

parameter of mice

Parameters Control Low dose High dose Recovery

Fertility index

(m)

100 (4) 100 (4) 100 (4) 100 (4)

Fertility index (f) 100 (8) 100 (8) 100 (0) 100 (8)

Parturition index 100 (4) 100 (4) 100 (0) 100 (4)

Gestation index 100 (96) 100 (97) 100 (00) 100 (83)

Viability index 100 (96) 100 (97) 100 (00) 100 (83)

Lactation index 100 (96) 100 (97) 100 (00) 100 (83)

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

108

Table 18: Effect of Opuntia elatior fruit extract treatment for 60 days on the litter size of

mice

Treatment

groups

No of males

mated/ female

No of pregnant

females Litter size

Percentage

fertility

Control

4/8 8 95 100%

Low dose

(250 mg/ kg bw) 4/8 8 97 100%

High dose

(500 mg/ kg bw) 4/8 0 00 0%

Recovery for

high dose 4/8 8 82 86.32%

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

Table 19: Effect of Opuntia elatior fruit extract treatment for 60 days on the litter size and

serum testosterone level of mice

Treatment groups Litter size (No of pups/

female)

Testosterone level (ng/

dl ± SE)

Control 11.875±0.125 c 772.79±34.590

c

Low dose (250mg/ kg bw) 12.125±0.2265 c 735.96±24.757

c

High dose (500mg/ kg bw) -Nil- a 175.22±32.114

a

Recovery group 10.25±0.1636 b 529.78±22.196

b

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

109

Table 20: Effect of Opuntia elatior fruit extract treatment for 60 days on the glycogen level

in the testis and epididymis of the mice

Treatment groups Testis

(µg/g)

Epididymis

(µg/g)

Control 1.87±0.045

c 4.78±0.278

b

Low dose

(250 mg/ kg bw) 1.72±0.092

c 4.63±0.219

b

High dose

(500 mg/ kg bw) 0.70±0.042

a 3.16±0.279

a

Recovery for high

dose 1.21±0.084

b 4.32±0.197

b

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

Table 21: Effect of Opuntia elatior fruit extract treatment for 60 days on the protein level

in the testis and epididymis of the mice

Treatment groups Testis

(µg/g)

Epididymis

(µg/g)

Control

9.83±0.302

c 14.10±0.261

c

Low dose

(250 mg/ kg bw) 9.51±0.468

c 14.34±0.285

c

High dose

(500 mg/ kg bw) 6.67±0.162

a 9.31±0.212

a

Recovery for high

dose 8.20±0.172

b 11.97±0.281

b

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

110

Table 22: Effect of Opuntia elatior fruit extract treatment for 60 days on the cholesterol

level in the testis and epididymis of the mice

Treatment groups Testis

(µg/g)

Epididymis

(µg/g)

Control

5.39±0.350

a 10.04±0.202

a

Low dose

(250 mg/ kg bw) 5.52±0.427

a 10.09±0.085

a

High dose

(500 mg/ kg bw) 8.45±0.537

c 11.94±0.221

b

Recovery for high

dose 6.96±0.095

b 9.97±0.179

a

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

Table 23: Effect of Opuntia elatior fruit extract treatment for 60 days on the total ascorbic

acid level in the testis of the mice

Treatment groups Testis

(µg/g)

Control 5.42±0.258

Low dose

(250 mg/ kg bw) 5.39±0.137

High dose

(500 mg/ kg bw) 5.54±0.307

Recovery for high

dose 5.45±0.155

Note: Mean values were compared by One- way ANOVA followed by Duncun’s multiple

range test. Values with same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

111

Fig 1: Vertical bars showing the changes in the body weight of the mice compared to

the initial body weight following the treatment for 30 days with 250 and 500

mg/ kg bw fruit extract with control and recovery group. Mean values were

compared by One-way ANOVA followed by Duncun’s multiple range

test.Values with the same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

112

Fig 2(a) and (b): Vertical bars showing the mean weight of the testis, epididymis (2a)

and vas deferens, seminal vesicle and ventral prostate (2b)

following the treatment for 30 days with 250 and 500 mg/ kg bw

fruit extract with control and recovery group. Mean values are

compared by One-way ANOVA followed by Duncun’s multiple

range test. Values with the same superscript are not significantly

different whereas those with different superscript are significantly

different from each other.

Fig 3(a) – (d): Photomicrographs of cross sections of testis:

a) Control mouse showing the seminiferous tubules with normal

spermatogenesis, with the normal arrangement of spermatogenic cells and

with intact basement membrane.

b) Mouse treated with 250 mg / kg bw fruit extract for 30 days showing the

seminiferous tubules with normal spermatogenesis similar to control group.

c) Mouse treated with 500 mg / kg bw fruit extract for 30 days showing the

disorganization of the spermatogenic cells, degeneration of the Leydig cells,

shrinkage of the seminiferous tubules and spermatogenic cells, and reduced

number of spermatozoa in the lumen.

d) Mouse after cessation of the treatment with 500 mg / kg bw for 30 days

showing the regeneration of the Leydig cells and recovery of the number of

spermatozoa in the lumen.

All figures: 200X, Eosin-Haematoxylin staining, PS – Primary Spermatocytes, SS–

Secondary Spermatocytes, ST – Spermatozoa, LC – Leydig Cells.

113

Fig 4(a) – (d): Photomicrographs of cross section of cauda epididymis

a) Control mouse showing the lumen of the cauda epididymal tubule filled with

sperms and the columnar epithelial cells.

b) Mouse treated with 250 mg / kg bw fruit extract for 30 days showing the

intactness of the tubules as in control group filled with sperms and the

columnar epithelial cells.

c) Mouse treated with 500 mg / kg bw fruit extract for 30 days showing the

shrinkage of the epithelial height of the columnar epithelial cells of the cauda

epididymis.

d) Mouse after cessation of the treatment with 500 mg / kg bw for 30 days

showing the recovered columnar epithelial cells.

All figures: 200X, Eosin-Haematoxylin staining, CE – Columnar Epithelial

cells, SP - Sperms

114

115

Fig 5(a) and (b): Vertical bars showing the mean diameter of the seminiferous

tubules of testis, cauda epididymal tubules (5a), and the mean

diameter of the Leydig cell nucleus, the epithelial height of the

cauda epididymis (5b) following the treatment for 30 days with 250

and 500 mg/ kg bw fruit extract with control and recovery group.

Mean values are compared by One-way ANOVA followed by

Duncun’s multiple range test. Values with the same superscript are

not significantly different whereas those with different superscript

are significantly different from each other.

116

Fig 6(a) and (b): Vertical bars showing the total spermatozoa count (6a) and

abnormal spermatozoa count (6b) following the treatment for 30

days with 250 and 500 mg / kg bw fruit extract with control and

recovery group. Mean values are compared by One-way ANOVA

followed by Duncun’s multiple range test. Values with the same

superscript are not significantly different whereas those with

different superscript are significantly different from each other.

117

Fig 7: Vertical bars showing the mean number of abnormal spermatozoa following the treatment for 30 days

with 250 and 500 mg/ kg bw fruit extract with control and recovery group. Mean values are compared

by One-way ANOVA followed by Duncun’s multiple range test. Values with the same superscript

are not significantly different whereas those with different superscript are significantly different from

each other.

118

Fig 8(a) and (b): Vertical bars showing the mean litter size (8a) and serum

testosterone levels (8b) following the treatment for 30 days with

250 and 500 mg/ kg bw fruit extract with control and recovery

group. Mean values are compared by One-way ANOVA followed

by Duncun’s multiple range test. Values with the same superscript

are not significantly different whereas those with different

superscript are significantly different from each other.

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Fig 9: Vertical bars showing the mean glycogen concentration in the testis and

epididymis following the treatment for 30 days with 250 and 500 mg/ kg bw

fruit extract with control and recovery group. Mean values are compared by

One-way ANOVA followed by Duncun’s multiple range test. Values with the

same superscript are not significantly different whereas those with different

superscript are significantly different from each other.

Fig 10: Vertical bars showing the mean protein concentration in the testis and

epididymis following the treatment for 30 days with 250 and 500 mg/ kg bw

fruit extract with control and recovery group. Mean values are compared by

One-way ANOVA followed by Duncun’s multiple range test. Values with the

same superscript are not significantly different whereas those with different

superscript are significantly different from each other.

120

Fig 11: Vertical bars showing the mean cholesterol concentration in the testis and

epididymis following the treatment for 30 days with 250 and 500 mg/ kg bw

fruit extract with control and recovery group. Mean values are compared by

One-way ANOVA followed by Duncun’s multiple range test. Values with the

same superscript are not significantly different whereas those with different

superscript are significantly different from each other.

Fig 12: Vertical bars showing the mean total ascorbic acid concentration in the testis

following the treatment for 30 days with 250 and 500 mg/ kg bw fruit extract

with control and recovery group. Mean values are compared by One-way

ANOVA followed by Duncun’s multiple range test. Values with the same

superscript are not significantly different whereas those with different

superscript are significantly different from each other.

121

Fig 13: Vertical bars showing the changes in the body weight of the mice compared

to the initial body weight following the treatment for 60 days with 250 and

500 mg/ kg bw fruit extract with control and recovery group. Mean values

were compared by One-way ANOVA followed by Duncun’s multiple range

test. Values with the same superscript are not significantly different whereas

those with different superscript are significantly different from each other.

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Fig 14(a) and (b): Vertical bars showing the mean weight of the testis, epididymis

(14a) and vas deferens, seminal vesicle and ventral prostate (14b)

following the treatment for 60 days with 250 and 500 mg/ kg bw

fruit extract with control and recovery group. Mean values are

compared by One-way ANOVA followed by Duncun’s multiple

range test. Values with the same superscript are not significantly

different whereas those with different superscript are significantly

different from each other.

Fig 15(a) – (d): Photomicrographs of cross sections of testis:

a) Control mouse showing the seminiferous tubules with normal

spermatogenesis, with the normal arrangement of spermatogenic cells and

with intact basement membrane.

b) Mouse treated with 250 mg / kg bw fruit extract for 60 days showing the

seminiferous tubules with normal spermatogenesis similar to control group.

c) Mouse treated with 500 mg / kg bw fruit extract for 60 days showing the

disorganization of the spermatogenic cells, degeneration of the Leydig cells,

shrinkage of the seminiferous tubules and spermatogenic cells, and absence of

spermatozoa in the lumen.

d) Mouse after cessation of the treatment with 500 mg / kg bw for 60 days

showing the regeneration of the Leydig cells and recovery of the number of

spermatozoa in the lumen.

All figures: 200X, Eosin-Haematoxylin staining, PS – Primary Spermatocytes, SS–

Secondary Spermatocytes, ST – Spermatozoa, LC – Leydig Cells.

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Fig 16(a) – (d): Photomicrographs of cross section of cauda epididymis

a) Control mouse showing the lumen of the cauda epididymal tubule filled with

sperms and the columnar epithelial cells.

b) Mouse treated with 250 mg / kg bw fruit extract for 30 days showing the

intactness of the tubules as in control group filled with sperms and the

columnar epithelial cells.

c) Mouse treated with 500 mg / kg bw fruit extract for 30 days showing the

shrinkage of the epithelial height of the columnar epithelial cells of the cauda

epididymis.

d) Mouse after cessation of the treatment with 500 mg / kg bw for 30 days

showing the recovered columnar epithelial cells.

All figures: 200X, Eosin-Haematoxylin staining, CE – Columnar Epithelial

cells, SP - Sperms

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125

Fig 17(a) and (b): Vertical bars showing the mean diameter of the seminiferous

tubules of testis, cauda epididymal tubules (17a), and the mean

diameter of the Leydig cell nucleus, the epithelial height of the

cauda epididymis (17b) following the treatment for 60 days with

250 and 500 mg/ kg bw fruit extract with control and recovery

group. Mean values are compared by One-way ANOVA followed

by Duncun’s multiple range test. Values with the same superscript

are not significantly different whereas those with different

superscript are significantly different from each other.

126

Fig 18(a) and (b): Vertical bars showing the total spermatozoa count (18a) and

abnormal spermatozoa count (18b) following the treatment for 60

days with 250 and 500 mg / kg bw fruit extract with control and

recovery group. Mean values are compared by One-way ANOVA

followed by Duncun’s multiple range test. Values with the same

superscript are not significantly different whereas those with

different superscript are significantly different from each other.

127

Fig 19: Vertical bars showing the mean number of abnormal spermatozoa following the treatment for 60 days with 250 and 500 mg/ kg bw

fruit extract with control and recovery group. Mean values are compared by One-way ANOVA followed by Duncun’s multiple range

test. Values with the same superscript are not significantly different whereas those with different superscript are significantly different

from each other.

128

Fig 20(a) and (b): Vertical bars showing the mean litter size (20a) and serum

testosterone levels (20b) following the treatment for 60 days with

250 and 500 mg / kg bw fruit extract with control and recovery

group. Mean values are compared by One-way ANOVA followed

by Duncun’s multiple range test. Values with the same superscript

are not significantly different whereas those with different

superscript are significantly different from each other.

129

Fig 21: Vertical bars showing the mean glycogen concentration in the testis and

epididymis following the treatment for 60 days with 250 and 500 mg/ kg bw

fruit extract with control and recovery group. Mean values are compared by

One-way ANOVA followed by Duncun’s multiple range test. Values with the

same superscript are not significantly different whereas those with different

superscript are significantly different from each other.

Fig 22: Vertical bars showing the mean protein concentration in the testis and

epididymis following the treatment for 60 days with 250 and 500 mg/ kg bw

fruit extract with control and recovery group. Mean values are compared by

One-way ANOVA followed by Duncun’s multiple range test. Values with the

same superscript are not significantly different whereas those with different

superscript are significantly different from each other.

130

Fig 23: Vertical bars showing the mean cholesterol concentration in the testis and

epididymis following the treatment for 60 days with 250 and 500 mg/ kg bw

fruit extract with control and recovery group. Mean values are compared by

One-way ANOVA followed by Duncun’s multiple range test. Values with the

same superscript are not significantly different whereas those with different

superscript are significantly different from each other.

Fig 24: Vertical bars showing the mean total ascorbic acid concentration in the testis

following the treatment for 60 days with 250 and 500 mg/ kg bw fruit extract

with control and recovery group. Mean values are compared by One-way

ANOVA followed by Duncun’s multiple range test. Values with the same

superscript are not significantly different whereas those with different

superscript are significantly different from each other.

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SummarySummarySummarySummary

The treatment was given in two doses and in two durations. Low-dose

treatment neither affected the body weight nor the reproductive function of the male

mice, neither in the short nor in the long duration. The high-dose treatment did not

affect the body weight but in turn affected the testis and epididymis weight, a

significant decrease in the sperm count, testosterone level, fertility rate, concentration

of protein, glycogen and cholesterol was evident. The litter size was reduced to

63.92% in high-dose-treated mice for short duration, but there was loss of libido in the

high-dose-treated mice for longer duration. A significant increase in the number of

abnormal spermatozoa was also noteworthy. However, no changes were observed in

the total ascorbic acid content. Thus there was a dose-dependent response in the

extract treated mice. Duration did not play a significant role, except in terms of

testosterone level and fertility rate. After the cessation of the treatment both in short

and long durations, i.e., for 30 and 60 days, the affected parameters were returning to

normalcy. The plant extract thus has a tendancy to return to the normal conditions

after the cessation of the treatment.

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ConclusionConclusionConclusionConclusion

Prickly pear is considered to be a wild fruit in most of the regions of India and

confirms the presence of many phytoconstituents that are biologically important. The

phytochemical analysis is evident for the presence of alkaloids, carbohydrates, fats

and oil, flavonoids, steroids and tannins. Further, the GC–MS analysis revealed the

presence of 14 compounds, out of which one is considered to be a 5-

hydroxymethylfurfural with an unidentified functional group. The literature survey

regarding Opuntia elatior fruits shows scarce number of results, but the scientific data

regarding the GC–MS analysis of the fruit are nowhere published as per my

knowledge. Thus, this is the milestone in the further analysis of the fruit extract for

human benefits. Further investigation of the unidentified functional group and

structural analysis of the compound is being done.

The reproductive toxicity is also the initiating step taken by us, as no work has

been published and no literature is available regarding the fertility-regulating effect of

the O. elatior fruits. The ethanolic fruit extract is found to regulate the fertility of

male Swiss albino mice at a dose of 500 mg/kg body weight (bw) for 30 as well as 60

days. Treatment for the shorter duration affects only the litter size by reducing the

fertility rate to 63.92%. However, the treatment for long duration not only affects the

litter size but also affected the libido of the treated mice. No mating occurred between

the fertile virgin female and the high-dose-treated mice for longer duration.

The fertility-regulating effect of the fruit extract may be due to the presence

and activity of the 5-hydroxymethylfurfural with an unidenfied functional group.

Further, the presence of furans and its derivatives, which cause reproductive toxicity,

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may be responsible for the antifertility activity of the fruit extract. The presence of

furans in high percentage in the fruit extract, which is actually considered to be

genotoxic, mutagenic, is responsible for lethality of the animals, but none of the

animals died in the experimental groups (both low and high dose) throughout the

experimental period.

To conclude, first the dosage at which the fruit extract is showing its activity

for fertility regulation, i.e., 500 mg/kg bw is very high for human consumption (for a

minimum body weight of 65 kg man, 32.5 g of the extract has to be given). Second, as

the fruit extract is responsible for the significant decrease in the testosterone level in

500 mg/kg bw treatment both in short and long durations and the libido of the treated

mice in long duration has been affected. Thus O. elatior fruit cannot be considered as

an ideal contraceptive herb. The synergistic effect of the whole fruit ethanol extract

may affect the hormone level, but a single compound isolated from the fruit extract

may be the novel compound that has the ability to reduce the fertility rate without

disturbing the libido of the treated male. Thus, the present study has paved a way for

ample of opportunities for further investigations in the field of pharmacy and

pharmacology.