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Ultrasonography has an importance place in human practice and veterinary f ield especially in canine practice. Since many years two dimensional ultrasound has been used for scanning purposes both in human and animal practice. But the scenario has changed in human practice with development of three dimensional technology and later four dimensional ultrasonography has developed which shows movements of the objects being scanned especially in the case of fetuses. Nowadays it has been common to use these advanced technology in the human practices. Yet these technologies are in the childhood stage in the veterinary field. High cost of 3D/4D ultrasound equipment, operational difficulties in non sedated animals & lack of specific knowledge about the operation of volume scanning hinder the use of this technology in the veterinary field. Better understanding of the different application of these technologies can be made to use in research areas for the better understanding of the abnormalities, post therapeutics as well in the diagnosis of the diseases

Principle of Three Dimensional and Four Dimensional Ultrasonography

Sound waves of frequency higher than human audible frequency usually ranges from 2-13 MHz are used in ultrasonography. Depending upon on depth of tissue from the scanning surfaces frequency is selected. Three dimensional ultrasonography gives three dimensional images of the volume being scanned .it works on the same principle of 2D ultrasonography but ultrasonographic beams are sent different angles to get three dimensional image of object being scanned. Volume information is obtained either directly using 2D array probe or by reconstructing three dimensional volume from a series of continuous or non- continuous 2D images by using software (Fenster and Downey, 2000). An advancement of 3D technology is 4D ultrasound. Four dimensional ultrasound adds the fourth dimension as time and gives a dynamic real-time 3Dultrasonography of the concerned organ with a continuous volume image acquisitioned rendering process. (Khurana and Dahiya, 2004).

In Doppler imaging, the reflections are from circulatory vessels, while in 2D ultrasonic image the reflection is from a tissue(s) or organ(s) In 2D ultrasound strength of reflection in B-mode depends upon density of the organ.On other hand the reflection in Doppler imaging depends upon direction of the circulation. If circulaltion is coming towards transducer, the signal will be strong, while it looses its strength while flowing away from the transducer. As shown in the diagram, the position of transducer on curved surface decides the strength or amplitude of signal. If it is positioned on curve end from where circulation is coming, the signal will be of higher amplitude, but when position is changed to other end of curved surface, where circulation is going down the amplitude of signal will decrease accordingly and is shown in reverse direction.

Principle of Doppler

Movement of blood in vessels is detected, which is depicted by color in some ultrasound machines. The flow coming towards transducer is shown above baseline, while flow going down i.e. away from transducer is shown below baseline. If position of transducer is changed from where the arterial flow is going away from transducer then, the arterial flow will be shown below baseline and venous flow that is coming towards transducer will be shown above baseline. Some machine shows red and blue color of the flow. The red color shows flow coming towards and blue color shows flow going away from transcer. A beginner ultrasonographer may think that the red color is from artery and blue is from vein. This is not true, this shows direction of flow. Since ultrasonic image of flow in Doppler is formed from series of pulses returning from vessels, the magnitude of pulse changes with variation in flow. A stationary flow will not show any height of magnitude.

Diagnostic techniques are routinely used for diagnosing different surgical conditions. The most frequently used imaging techniques in veterinary practice are radiography and ultrasonography. Radiography and ultrasonography are not suitable for each organ in large animals. In small animals most organs are suited to ultrasound images except bones and gascontaining structures such as lungs etc. Bone and gas reflect ultrasound and do not allow the sound waves to form image therefore radiography gives better diagnostic images for these organs. The liver, spleen, kidneys, prostate, gravid uterus and fluid containing organs like urinary bladder, gall bladder and major blood vessels can be better imaged with ultrasound. In buffaloes in surgical section important clinical conditions relate to urinary and digestive systems, mammary gland and perineal area.

1. Urinary System: In bovine, the cases of obstructive urolithiasis are commonly observed. These types of cases are more common in bullocks and in buffalo calves. In these cases radiography is not so useful. Sonography can be tried to take the image of ruptured urethra in bullock and can be compared with radiography. Due to ruptured urethra there will be accumulation of urine in the s/c tissue of prepuce. Due to fluid density the image will be radioopaque and will appear white and no detail of the organ can be seen. The ultrasonogram (USG) will show images of glans penis and its coverings. Different diameters of urethra and a point of rupture just behind to glans penis can be make out. Due to obstruction, The B.calves either will pass drop wise urine or there will be complete stoppage of the urine. These cases can also be easily diagnosed by ultrasonography.

The ultrasonography is based on echo-principal i.e. reflection of sound waves. The ultrasound waves, commonly termed as ultrasound beam is generated from the pizzo-electric crystals, under electric stimulation. The sound beam travels and strike to the organs/tissues on its way and get either reflected or reabsorbed by these structures. The reflected beam is received back by the transducer, which are processed and displayed on the monitor of ultrasound scanner. The display of signal is in form of shades of grey scale i.e. from bright to black picture. If these sound beams strike to organ such as bone, then major part of the beam is reflected and it appears bright (white) on screen. If the sound waves strike to fluid, these get absorbed and are not reflected back, therefore, the image of fluid appears as black on the screen. Other organs appear in between black and bright white. In fact, the reflected sound beam is not proportionate to frequency and velocity of emitted ultrasound beam. The strength of ultrasound beam (grey scale) or brightness of the image depends upon the acoustic impedance of organ or tissue. In simple term acoustic impedance is the capacity of tissue or organ to reflect or absorb these sound beams that again depends upon density or composition of these structures. The reflection of sound beams also depends upon, the angle at which the ultrasound beam strikes the structure on its way. The distance of the organ from the transducer also affects image brightness.

Ultrasonography in goat is becoming popular in the field of reproduction and replacing the other old-age techniques, viz. abdominal ballotment, recto- abdominal probes, X-rays and radioimmuno-assays. The reasons are numerous such as unsatisfactory results, hazardous effects to animal, operator or environment, high cost, long delay in diagnostic results and requirement of sophisticated labs. The ultrasound system has low operating cost and does not need special labs as it can be done at farm itself. Ultrasonography is considered safe to the animal and the operator and provides quick and reliable results. The popular opinion about ultrasonography is that it is noninvasive imaging system and is rapid, safe and practical means of diagnosis of pregnancy and reproductive disorders in sheep and goat. The B-mode real time ultrasonographic system particularly portable system is popular among researcher and practitioners. In goat, B-mode real time ultrasonography was used in 1971, using 5 MHz frequency transducer. A transducer of switchable frequency from 7.5 to 5.0 is more popular in small ruminant research and practice.

Ultrasonography in sheep is becoming popular in the field of reproduction and replacing the other old-age techniques, viz. abdominal ballotment, recto- abdominal probes, X-rays and radioimmuno-assays. The reasons are numerous such as unsatisfactory results, hazardous effects to animal, operator or environment, high cost, long delay in diagnostic results and requirement of sophisticated labs. The ultrasound system has low operating cost and does not need special labs as it can be done at farm itself. Ultrasonography is considered safe to sheep and the operator and provides quick and reliable results. The popular opinion about ultrasonography is that it is noninvasive imaging system and is rapid, safe and practical means of diagnosis of pregnancy in sheep. The B-mode real time ultrasonographic system particularly portable system is popular among sheep breeders. In sheep, B-mode real time ultrasonography was used in 1971, using 5 MHz frequency transducer. A transducer of switchable frequency between 7.5 to 5.0 is more popular in sheep practice.

Transrectal Procedure in Sheep

The procedure is similar to goat, but sheep remain quite during transrectal ultrasonography than goat. Following are some of the steps to obtain good quality image:

Introduction Ultrasonography is well-established technique in human sciences, particularly for diagnosis of pre-natal defects of fetus. In veterinary sciences, this technique is emerging an important tool. This tool is being employed in the small animal practices, particularly bitches for diagnosis of various reproductive disorders and estimation of gestational status. In the past, veterinarians had limited assistance for assessing reproductive status of this animal. Pregnancy diagnosis in a bitch was possible either with the use of X-rays or transabdominal palpation, but that too at an advanced stage of gestation. These methods could provide information either about presence of pups in vitro, or their number, but assessment of fetal viability was not accurately predicted with these traditional methods. With the use of X-rays, there was always risk of harmful effects on the health of mother and fetus. As far as reproductive disorders are concerned, most of them remained either undiagnosed due to the lack of proper access to the internal organs or were diagnosed with a guess. Diagnostic ultrasound is a valuable alternative imaging system that can provide more accurate information about the canine pregnancy and reproductive disorders in comparison to traditional methods.

The Liver

The liver is a large and an irregular organ of the body, which extends from the sixth to the eleventh intercostal space. It extends much more caudally on the right side as compared to the left side. Its most caudal part is the caudate lobe whose small portion is visualized encircling the right kidney dorsally at the eleventh intercostal space. The liver of a dog is divided into various lobes namely the left lateral, left medial, quadrate, right medial, right lateral and the caudate lobes which are not identifiable ultrasonographically, except when f luid is present in the abdomen. Otherwise, the position of the different liver lobes can only be approximated. The gallbladder is located to the right of the midline between the quadrate and the right medial lobes of the liver.

Proper preparation of the animal is important to obtain images of abdominal orgnas including that of liver. It is better to keep animal off fed for at east six hours prior to ultrasound scanning. This will avoid interference of gases and solid contents of the gastrointestinal tract. If stomach is taken as landmark of scanning then enough fluid should be given prior to scanning to get good images of stomach. The liver can be easily visualized on one side of stomach. One should be liberal in applying good amount of gel over transducer and body for better image of liver.

Ultrasonographic images of stomach are required to diagnose ingestion of any abanormal materials by dog. When content of stomach are dry or there is gas in stomach then, it is difficult to get proper image of stomach. Offering water to animal or oral administration of water with the help of syring is also helpful in getting the images of content of stomach. Some time, while doing ultrasonography, movement of contents of stomach can be visualized. Stomach can be images between liver and spleen. The size depends upon contents or intake of food by dog. For proper image of gastrointestinal tract including stomach, there should not be gas in GIT. One should remember that ultrasound beam does not travel in gas. The best medium of ultrasound beam is liquid.

Scanning Technique

If possible, fasting for 12 hours is good to diminish the amount of gastrointestinal gas. However, non-fasted dogs, when offered water may show adequate image quality. The water may be administered in quantity of 15 ml per kg of bodyweight. Removing the stomach gas using an orogastric tube has been recommended tto enhance the visualization of lesions of the stomach, however, this is not in practice in India.

Ultrasonography in bull is helpful in diagnosis of problems of testis and accessory glands. This can also be used in assessment of development of these organs. Such developmental assessment, when combined with endocrine data, is useful to know age of puberty, sexual development and dimension of these organs. The ultrasonographic images and data are available for testis, bulbourethral gland, prostate and seminal vesicles in the bull. Ultrasonography of testis of bull can be useful to assess developmental changes, testicular dimensions and pathological conditions of the testis viz. tumors, lesions, abscess etc. Epididymis can also be examined for change in lumen, inflammation, granuloma etc. The accessory sex glands can be better viewed in ultrasonographic scanning to assess their dimensions, normal developmental changes and alterations in their morphology. Before, ultrasonography is undertaken, one should thoroughly revise the knowledge of sectional anatomy and location of these organs.

Testis

Location: The testis, in the bull, is located in the scrotum. In abnormal conditions, the testis might be retained in the inguinal canal or abdomen. Before, ultrasonography, location of the testis should be assured. For this, the testis should be palpated to assess their pliability and rule out adhesions with the scrotal wall. In normal condition, the testis should be freely movable in the scrotum, soft of palpation and both the testis should touch the bottom end of scrotum.

Real time ultrasonography provides means to directly image the reproductive tract. An extensive amount of information can be gained from even a single examination, including determination of ovarian (luteal or follicle detection) and uterine status, detection and monitoring of ovarian and uterine pathology, monitoring the post-partum mare, detection of twins, prediction of ovulation, fetal growth and development and determination of sex of fetus. When first introduced the use of ultrasonography was primarily limited to early pregnancy diagnosis only, with advancement of knowledge and interpretive skills, the diagnostic ultrasonography has a much more application in reproduction. Some of the applications are discussed in this lecture.

Procedure

The procedure and precautions for intrarectal ultrasonographic examination are similar to those for rectal palpation, and no additional restraint is required. The transducer should be protected by the examiner’s hand to prevent trauma to the rectal wall, and the transducer should be well-lubricated. Care should be taken to avoid fecal material attaching to the transducer. After evacuating fecal material from the rectum, the probe is introduced and moved across the reproductive tract in the following pattern: uterine body, right uterine horn, right ovary, right uterine horn, uterine body, left uterine horn, left ovary, left uterine horn, uterine body then cervix. Good contact must exist between the transducer and rectal wall. Air in the rectum or a gas or fluid-filled loop of bowel will result in a distorted image. To minimize scanning errors, principally those of omission, it is recommended to conduct the same scanning procedure during each examination.

Corpus luteum, one of the biological clock of the estrous cycle and pregnancy, is known foremost for its production of progesterone that blocks the pituitary release of gonadotrophins and prepare the uterus for pregnancy. The ovarian follicle wall consists of granulosa cell and theca cells vascularised and luteinize after ovulation to from a corpus luteum. Corpus luteum is temporary steroid- producing gland undergoes marked structural and functional changes far a short time span during its development functional life and regression. During estrous cycle, the corpus luteum undergoes an initial period of growth (metestrus), followed by a period of maximal function (diestus) and finally by a period of regression (proestrus) preceding the next ovulation (Robert, 1986). The most prominent cell in the CL is the large luteal steroidogenic cell.

Ultrasound imaging has been used to detect and measure the corpus luteum throughout the estrous cycle and corpus luteum has been characterized during pregnancy and non pregnancy. Changes in corpus luteum diameter as detected by ultrasonography are highly correlated with progesterone production. A high correlation has been found in the ultrasonographic detection of bovine corpora lutea (Lean et al., 1992) and between luteal tissue area measured ultrasonographically and plasma progesterone concentration (Bergfelt et al., 1989).

This paper primarily considers the general principles of ultrasound and instrumentation and its application in camel reproduction.

Ultrasound waves are mechanical pressure waves similar to audible sound waves and must have a medium in which to audible sound waves and must have a medium in which to propagate. The “frequency” (f) is the number of high- or low- pressure regions crossing each area of tissue each second. The frequencies of the sound waves used in medical ultrasound are much higher than the human audible range (20 to 16000 Hz); hence the waves are called ultrasound. Ultrasound instrumentation

The gray scale imaging equipment uses only time delay and echo amplitude information in creating the image. Most equipment contains the same basic building- block circuits; transmitter, receiver, display and scan converter.

Transmitter

The first circuit building block, the transmitter circuit produces either a high- amplitude, short- duration voltage shock pulse or a driving voltage waveform. Then an output control attenuates the amplitude of the shock pulse or the driving voltage wave form before it is applied to the transducer.

Mammary glands are important part of body which synthesize and store the milk in between milkings or suckling by the calf. Therefore it attains prime importance in any dairy animal. However, it has received little attention in buffaloes till now. Buffalo presents several unique characteristics in terms of milk production, length and shape of teats and arrangement of duct system in the mammary glands. Development of udder may be related to milk production and the knowledge of changes in its structure in relation to different physiological stages may also have practical implication.

Transducer Selection and Preparation of Scanning

Preparation of the site to be imaged will enhance the quality of the image. Therefore, any loose hair and dirt must be clean off. Ultrasound gel is applied to the area of interest. The gel acts as a coupling agent to prevent air from trapping between the transducer and skin. A 7.5 MHz transducer is ideal for imaging the teats. A 5.0 MHz transducer provides an adequate image. The mammary gland should be scanned with a 5.0-7.5 MHz transducer to give adequate penetration. All structures should be scanned in cross section and longitudinal planes.

Ultrasonography of the GI tract presents problems and challenges for the sonographer. Sonography of the GI viscera was once throught to be impossible because of artifacts created by gas within the lumen but with the technical advancement in the ultrasonography now the problem has been solved. If the animal is kept of feed and adlib water is given to the dog with I/v fluid than ultrasonography becomes helpful in the diagnosis of gastrointestinal tract disorders. Other conditions such as esophageal carcinomas can also been diagnosed by ultrasonographic endoscopy. in human beings.

The normal ultrasonography of the liver, kidney, stomach & spleen in dogs has been standardized and its use in evaluation of pathological lesions has been reported but ultrasonographic evaluation of oesophagus & intestine, both in normal animals and diseased conditions is yet to be established. Therefore normal ultrasonography of the oesophagus & small intestine in dogs and the importance of diagnostic capability of ultrasonography in diagnosis of pathology of these organs have been compiled.

Veterinary ultrasonographer has to write report of ultrasonography in different animals. The proper ultrasound report writing is the moral duty of the vet ultrasonographer. The accuracy of diagnosis must be ensured. The poor resolution/quality of machine may lead to erroneous diagnosis. Therefore, veterinarian must ensure good quality of machine and proper diagnosis. The report might be different in different animals e.g. report writing about uterus will be different in bitch, cows, buffaloes, camel, horse, goat and sheep. However following might be guidelines in writing ultrasound report of uterus. fetus, endometrium

Uterus: Position of uterus, contents in the uterus, shape and size in initial pregnancy and infections, appearance of myometrium, thickening of uterine layers, presence or absence of fluied/pus/masses, left and right horns and their contents. Observations on the images of uterine fluid and its echoicity, membrane appearance in pregnant animals. Special attentions should be paid to endometrium.

Endometrium: Appearance and thickness; any growth on the surface of endometrium. Images of retained portion of placenta can be ascribed in the cases of retained placenta.

Ovary: Ovaries are scanned to assess cyclic structures, size, shape, its Position/ location and internal echo pattern of follicles. This echo pattern will indicate whether follicle is normal, cystic or with any other abnormalities of follclular f luid. Ultrasound reports also include shape and size of corpus luteum. In cows and buffaloes, presence of cavity, looseness/compactness of luteal cells are also mentioned.

Cell culture refers to removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been establwashed.

Primary culture refers to the stage of culture after the cells are isolated from tissue and proliferated under appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of drug discovery process can save time and money both by eliminating least beneficial NCEs.

Environmental agents (xenobiotics) that are injurious to cells trigger a spectrum of responses that can range from cell death to adaptation, repair, and proliferation. Cell death, in turn, can take the form of apoptosis (a programmed form of cell death). Xenobiotics have been implicated in toxic cell injury, among them free radicals (nitrogen, oxygen, and carbon derivatives) are known to interact with cellular constituents including membrane lipids and chromagen materials.

Cytotoxicity

Cytotoxic: Toxic to cells, cell-toxic, cell-killing. Any agent or process that kills cells. The prefix cyto- denotes a cell. It comes from the Greek “kytos” meaning hollow, as a cell or container. Toxic is from the Greek “toxikon” = arrow poison.

In nutshell, cell cytotoxicity refers to the ability of certain chemicals or mediator cells to destroy living cells. By using a cytotoxic compound, healthy living cells can either be induced to undergo necrosis (accidental cell death) or apoptosis (programmed cell death).

Measuring cytotoxicity can prove to be very valuable tool in identifying compounds that might pose certain health risks in humans and animals. Cytotoxicity assays are widely used by pharmaceutical industry to screen for cytotoxicity in compound libraries from initial high-throughput drug screens for unwanted cytotoxicity. This can be of vital importance and prove to be quite indispensable during research phase of developing therapeutic new pharmaceutical products to ensure the safety of end-users. Additionally, understanding the mechanisms involved in cytotoxicity can likewise give researchers a more in-depth knowledge on the biological processes (both normal and abnormal) governing cell growth, cell proliferation and death. Compounds that have cytotoxic effects often compromise cell membrane integrity. Therefore, assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. There are three commonly used methods which can be employed to assess in vitro cytotoxicity (disruption of cell integrity) in cell cultures:

Safety is a prime concern for humans and animals. Without evaluating the safety of a chemical, no regulatory authority in the world allows the chemical to be used by humans. There are stipulated guidelines for toxicity evaluations. Among the toxicity assays genotoxicity assays are important and have occupied centre stage due to increase in understanding of the genome.

Genotoxicity describes the property of chemical agents to damage the genetic information within a cell to cause mutations, which may even lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic; however, not all genotoxic substances are mutagenic. Generally and depending on the agent, a cytotoxic agent may be genotoxic (causing DNA damage), mutagenic (causing gene mutation) or it may be harmful for cell organelles (e.g. cell membrane) or it may even be teratogenic. Therefore, every cytotoxic agent is not genotoxic.

Why we Need to Evaluate Genotoxicity?

Toxicity can be at any level according to the nature of chemicals and type of exposure. Duration of toxicity can also be short lived or long lived within a generation or continue to next generation. Genotoxicity effects are able to continue in next generation(s), in which the exposed population may not suffer seriously but the next generation may suffer without being exposed to the same chemicals. Therefore, genotoxicity evaluations need serious attention.

Apoptosis is triggered by extrinsic and intrinsic stimuli as radiation, oxidative stress and genotoxic chemicals. It is defined by characteristic changes in the nucleus morphology including chromatin condensation and fragmentation, overall cell shrinkage/rounding and formation of apoptotic cell bodies. Among the many markers of apoptosis, DNA fragmentation is considered a hallmark. The DNA ladder assay described here is a simple, sensitive, cost-effective and rapid method for estimating apoptosis in cells.

Isolation of Genomic DNA

Genomic DNA from primary cells culture of PMNC and splenocytes of rats is isolated by using standard phenol-chloroform DNA isolation protocol of Sambrook and Russel (2001) as described below:

1. Preparation of primary cells culture of different cells, namely-PBMNCs, thymocytes and splenocytes form Wistar rats and viability checked by trypan blue dye exclusion test.

2. 106 cells/ml of primary cell culture is used and seeded in 24 well culture plates containing along with treatment with xenobiotics at different µM concentration.

3. Cell culture plates are incubated in CO2 incubator at 37°C with 5% CO2 for 12 hrs.

4. The cells are collected in 1.5 ml of micro centrifuge tube and centrifuged at 3200 rpm for 10 min at 4°C; supernatant is discarded and pellet is washed with PBS (pH 7.2).

5. DNA extraction buffer (500 µl/tube) is added in tubes. Gently tap to disperse the cell pellet in extraction buffer and keep in water bath for 1.0 hr at 37 °C (it is to ensure that pellet is completely suspended so that cells are accessible to SDS and Proteinase K).

6. 10 % SDS is added (20 µl/ml) to cell suspension and the tubes are gently mixed by inverting the tube. The contents of tube become viscous which indicates lysis of cells. After that sample is handled gently from onward to avoid shearing of DNA.

7. Proteinase K (15 µl) of 20 mg Proteinase K/ml of buffer is added in tubes in two pulse i.e. half the requirement is added to tube in 1st pulse, mixed gently end to end and kept in water bath at 50°C. After 3-4 hrs, the second pulse of the remaining amount of Proteinase K is added and tubes are incubated at 50°C overnight.

8. Equal volume of equilibrated phenol (Tris saturated phenol pH> 7.8 ) is added in the tubes next day morning. Mix the contents of tube gently by inverting the tube for 15 min till a light coffee coloured uniform solution without any balls of phenol is formed and then centrifuge at 3400 rpm for 15 min.

The concept of single cell micro-gel electrophoresis (SCGE)/comet assay is f irst introduced by Ostling and Johanson (1984) as a method to measure DNA single-strand breaks under neutral conditions. Singh et al. (1988) modified this technique by using alkaline conditions (>13 pH) of electrophoresis, which enabled detection of not only frank strands breaks but also alkali labile sites, DNA cross- linking and incomplete excision repair sites. Now-a-days, this technique is extensively used to study the genotoxic effects of various chemicals. The advantages of SCGE technique include: (1) collection of data at the level of individual cell, allowing for more robust types of statistical analysis; (2) need for small number of cells per sample (<10,000); (3) its sensitivity for detecting DNA damage; and (4) that virtually any eukaryotic cell population is amenable to analysis. The idea is to combine DNA gel electrophoresis with fluorescence microscopy to visualize migration of DNA strands from individual agarose-embedded cells. If the negatively charged DNA contained breaks, DNA supercoils are relaxed and broken ends are able to migrate toward the anode during a brief electrophoresis. If the DNA is undamaged, the lack of free ends and large size of the fragments prevented migration. Determination of the relative amount of DNA that migrated provided a simple way to measure the number of DNA breaks in an individual cell.

Genotoxic effects of any xenobiotics in PMNC and splenocytes of Wistar rats can be studied according to the working protocol of Dhawan et al.

Genotoxicity potential of any environmental pollutants can be investigated using the micronucleus (MN) test. It is among the most sensitive DNA damage indicators and it has been applied to several organisms and tissues for evaluation of environmental contaminants (Lemos et al., 2011). Micronuclei (MN) assay is more rapid and simpler than chromosomal analysis (Gandhi et al., 2003). Compared to the chromosome aberrations, the final result is relatively easily scored and thus requires less time to make an assessment of the clastogenicity of a chemical (Garriott et al. 2002). An in vitro MN assay can detect the clastogens and aneugens as well as mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction (Corvi et al., 2008). Although, micronuclei constitute well-characterized biomarkers of chromosomal damage (Salazar et al. 2009) but in vitro MN test does not provide information on the origin of micronuclei (Corvi et al., 2008).

Experimental Procedure

Micronuclei assay is carried out in PMNC and splenocytes as per method of Hayashi et al. (1983).

Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Here we perform the apoptotic assay test by TUNEL assay kit to identify the apoptotic property of any agent or chemical after treatment of primary cells culture at different concentration as per protocol provided with TUNEL Assay Kit and apoptotic cells are identified by using fluorescent microscope. Detailed procedure of TUNEL assay is as follows:

1. Preparation of primary cells culture as described in earlier methods.

2. Induction of apoptosis in primary cells culture is done by treatment of primary cells culture with different concentration of xenobiotics and incubation in CO2 incubator for 12 h as mentioned in micronuclei assay method.

3. The positive and negative control cells provided with the kit are already f ixed. However, negative control cells are prepared from control sample with absence of inducing agent.

4. Cell Fixation: Cell fixation is a required step in the TUNEL assay and it is done according to the procedure mentioned in the supplied kit by using paraformaldehyde solution. Suspend 1–2 × 106 cells (treated) in 0.5 ml of phosphate-buffered saline (PBS) and the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.

5. Centrifuge the cells for 5 minutes at 300 × g and discard the supernatant.

Spontaneous contractions of the uterus are controlled and coordinated for various reproductive functions. The non-pregnant uterus facilitates ova and sperm transport, provides adequate embryo placement for implantation, and contributes to the expulsion of menstrual debris through increased contractility, or periods of relative quiescence (Hutchings et al., 2009). Uterine dysmotility has been implicated in the pathogenesis of certain infertilities, implantation failure, endometriosis, dysmenorrhea, and abnormal menstrual events (Kunz and Leyendecker, 2002; Aguilar and Mitchell, 2010). Although these conditions may not contribute to mortality but can significantly affect patient’s reproductive health, and considerably alter their quality of life (Jones et al., 2004; Weissman et al., 2004; Chachamovich et al., 2010). Dysmenorrhoea refers to the occurrence of painful cramps in the lower abdominal region during menstruation and is one of the unresolved gynecological disorders in adolescent girls and women of reproductive age (Wang et al., 2004; Harel, 2008). This condition is mainly characterized by cramping pain in the lower abdomen immediately before or during menstruation, which does affect the quality of their life and work (Davis and Westhoff, 2001).

Introduction

Acute lung injury (ALI) is the manifestation of an inflammatory response of lung resulting from severe sepsis, bacterial or viral pneumonia, trauma, and burns in animals (Ali et al., 2020).It is distinguwashed by disruption to the alveolar capillary membranes, atelectasis of the pulmonary airspaces, and neutrophil and protein-rich fluid infiltration into the alveolar space (Li et al., 2019). Although advances in treatment, the disability and fatality rates of ALI remain high, that is around 30-40% (Ali et al., 2020). At the moment, new therapeutic alternatives are urgently required to treat ALI. As a result, this is a very active area of research to understand the molecular principles underlying pulmonary inflammation and ALI (Reiss et al., 2012). The use of animal models of inflammatory lung has allowed for the probing of pathways of inflammation responsible for injury. Specially, rodent models may serve a link between in vitro research to large animals and clinical studies by allowing for high quantities of samples under physiological circumstances at low expenses (Domscheit et al., 2020).LPS, found in the outer membrane of gram-negative bacteria, consists of a polar lipid head group (lipid A) and a chain of repeating disaccharides (Gao et al., 2023). LPS induced lung injury model, closely mimic sepsis and inflammatory lung diseases, is a well-establwashed model in this field of research (Reiss et al., 2012). To mimic acute lung injury, LPS can be administered either directly through intratracheal or intranasal route (direct pulmonary insult) or indirectly through intraperitoneally and intravenous routes (indirect extra pulmonary insult) (Perl et al., 2011). This chapter focuses mostly on rodent model of acute lung injury caused by lipopolysaccharide (LPS), both direct and indirect. Additionally, it also includes the characteristics and underlying molecular mechanism of LPS induced lung injury along with applications in drug development.

Mesenchymal Stem Cells

Recent advances in the field of regenerative medicine have opened up newer therapeutic modalities using cell-based and cell-free therapies, particularly stem cells, as potential candidates. As per the definition, stem cells are the undifferentiated progenitor cells that can regenerate and can also differentiate into various specialized cells (Hoang et al., 2022). They can be classified as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and postnatal adult stem cells (Locke et al., 2009). Among postnatal adult stem cells, mesenchymal stem cells (MSCs) have emerged as a popular choice of candidate for therapeutics due to the relatively easy availability, simple isolation techniques, low risk of teratogenicity, minimal ethical and legal concerns unlike embryonic stem cells, hence, are easy to apply in clinical research and practice (Voga et al., 2020). Among MSCs of different origin, bone marrow and particularly adipose tissue-derived stem cells (ADMSCs) received more attention and are one of the most common and promising stem cell types. These cells can be simply obtained from subcutaneous and omental fat or liposuction aspirates for the treatment of diverse clinical applications (Kim &Heo, 2014). These cells can also be expanded vigorously in vitro and differentiated into specific cell lineages such as adipocytes, osteoblasts, chondrocytes, and neurocytes and are even genetically stable in long-term cultures (Miana& González, 2018). Mesenchymal stem cells (MSCs) are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the (ISCT) International Society for Cell and Gene Therapy (Dominici et al., 2006).

Injudicious and indiscriminate use of several agrochemicals viz. pesticides, herbicides, insecticides, dyes and medicaments in the field of agriculture and veterinary practices threatens the human and animal population for the toxicity of these environmental deterrents. These agrochemicals have been associated with several episodes of accidental poisoning and mortality among human and animal population. The episode of Mass ethion poisoning in Gujarat is one of the commonest example of inclusion of pesticides or agrochemicals in the environmental pollutants. There are modern tools to evaluate and analyze these chemicals in the environmental pollutants of human and animal use. The sensitivity of these modern highly sophisticated tools has now immensely changed the analytical environmental pollutants industry. Gas chromatography and Mass Spectrophotometry i.e. (GC-MS) technique is employed to detect the volatile residues from environmental pollutants. Whereas, High performance liquid chromatography i.e. HPLC is employed to analyse and detect non volatile drug residues. Gas Chromatography/Mass Spectrometry (GC/MS) instrument separates chemical mixtures (the GC component) and identifies the components at a molecular level (the MS component). It is one of the most accurate tools for analyzing environmental samples.

Flow Cytometry is a tool used to detect and measure chemicophysical characteristics of a population of cellular particles. Modern day biomedical research needs the molecular assembly of tools to proceed in the advent of biomedical research with real time diagnostic, prognostic and experimental detection of markers. For the same, a naïve tool is flow cytometry. In this technique, a sample having cells or particles is suspended into a fluid and injected into instrument. The ability to perform these measurements in a very rapid time span is one of the key advantages of the flow cytometry process. A single-cell suspension must first be prepared in order to analyse solid Tissues. A flow cytometer is look alike to microscope, except that, instead of producing an image of the cell, it generally offers a cell-by-cell basis high-throughput, automated quantification of specified optical parameters.

They can quantify up to three to six properties or components are quantitated in a single sample, cell by cell, for about 10,000 cells, in less than one minute. Sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. These cells are tagged with fluorescent markers in order to absorb the light and then emitted them in the form of band of wavelengths. Average ten thousand cells can be quickly examined and the data gathered are processed by a computer.

High-performance liquid chromatography or high-pressure liquid chromato graphy (HPLC) is a chromatographic method that is used to separate a mixture of compounds in analytical chemistry and biochemistry so as to identify, quantify or purify the individual components of the mixture. It involves the injection of a small volume of liquid sample into a tube packed with tiny particles (1.8- to 5-micron (μm) particle size called the stationary phase) where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.

Aflatoxins are toxic and carcinogenic compounds produced as metabolites by many Aspergillus species. This fungus invades habitats such as soil, grain, nuts, or decaying vegetation when- ever suitable conditions such as high moisture and temperature are met. B1 is the most toxic of at least 14 different types of naturally occurring aflatoxins. Therefore, many countries and regulatory agencies impose strict limits on B1 as well as other common aflatoxins such as B2, G1, and G2 despite exhibiting lower toxicity. The U.S. FDA set the action level to20 μg/kg (ppb) for human food and to 300 μg/kg (ppb) for animal feed. Aflatoxin B1 is extremely widespread and is present in food and feed products such as cottonseed, corn, and peanuts. It is highly toxic and has been classified as a group 1 carcinogen by the WHO. The aflatoxins B2 , G1 , and G2 are usually found accompanying B1 , in lower concentrations in the contaminated samples. The order of toxicity is AFB1 > AFG1 > AFB2> AFG2.

Introduction

Minerals are present in all body tissues and fluids to carry out the physicochemical processes of life; both in animals and human. It is broadly classified into two categories: (1) macro and (ii) micro or trace elements. The macro elements include calcium, phosphorus, potassium, magnesium, sulfur, sodium and chloride, while elements such as iron, copper, cobalt, iodine, zinc, manganese, molybdenum, selenium and chromium come under micro elements. Animals need large amounts of calcium for construction and maintenance of bone and normal function of nerves and muscles. Phosphorus is a vital constituent of adenosine triphosphate (ATP) and nucleic acid and is also essential for acid-base balance, bone and tooth formation. Iron is an important component of the cytochromes for carrying out cellular respiration in addition to its vital role in red blood cells. Magnesium, copper, selenium, zinc, iron, manganese and molybdenum are important co-factors found in the structure of certain enzymes and are indispensable in numerous biochemical pathways. Animals need iodine to make thyroid hormones. Sodium, potassium and chlorine are important in the maintenance of osmotic balance between cells and the interstitial fluid. Excessive intake of some minerals can upset homeostatic balance and cause toxic side effects.

Isolated tissue preparations are widely used in physiological or pharmaco logical research to delineate the mechanism of action of drugs or any active biomolecules. Traditionally it is used as a tool for bioassay of drugs. The tissue used for such pharmacodynamics studies may be isolated from different animal models viz. frog, rodents, rabbits, sheep, goat, buffalo, chicken etc. Evaluation of drug-receptor interaction and underlying mechanism of action is then performed using isolated organ bath or myograph where the isolated tissues are mounted under optimum resting tension and changes in the tension following exposure of drug is recorded with the help of recording device. With the advancement of science and technology, the recording devices are also gradually upgraded from smoked drum to student’s physiograph to the most advanced Data Acquisition System (DAS) based polyphysiograph.

The isolated tissue preparations (uterine strips, arterial rings, tracheal chains etc.) need essential and appropriate nutrients and environment during experimental which is generally provided by using suitable salt solutions (Physiological Salt Solutions) which provide ionic balance, energy supply, maintain pH and tissue tonicity. The composition of physiological salt solutions is such that it provides an artificial media simulating with the inorganic composition of blood plasma together with a buffer mechanism to maintain an optimum pH of 7.0 to 7.4 and glucose to facilitate tissue metabolism. Commonly used PSS are Frog ringers, tyrode, Kreb’s, de Jalon, Ringer-Locke, modified Kreb’s Henseleit solution etc.

The cytological insult due to exposure of different environmental pollutant viz. pesticides, herbicides etc. impose great loss to the vitality and health of the individuals. The basic pathophysiological distrubances following the expo sure of these deterrents to the different visceral organs leads to dysfunction of these organs. Imbalance between body’s antioxidant defense and peroxidation of the lipid layer of cells’ causing free radical generation is defined as Oxidative stress. Free radicals are the molecules or entity that contains one or more than one unpaired electron. Pathophysiology of several diseases are associated with the generation of oxidative insult and many xenobiotics associated toxicity are also a result of oxidative damage and generation of reactive oxygen species and reactive nitrogen species. Alzheimer’s Disease is one of the fine example of oxidative damage associated neuronal degeneration.

Oxidative stress can be estimated using following parameters:

1. Lipid peroxidation (Paula et al., 2005)

2. Reduced glutathione (Sedlak and Lindsay, 1968)

3. Superoxide dismutase (Madesh and Balasubramanian, 1998)

4. Catalase (Aebi, 1983)

Tissue Collection

Rats are most commonly employed as test subjects to conduct the experiments related to drug metabolizing enzyme assays involving various enzymes i.e. microsomal and non microsomal enzymes. Rats are humanely sacrificed at the end of the exposure period of 28 days of any therapeutic agents or drug and the liver of all the animals has to excise and immediately wash with ice-cold normal saline, weigh and perfuse with ice-cold 1.15% KCl solution containing 0.05 mM EDTA. Livers are processed further for preparation of microsomes for determination of microsomal drug metabolizing enzymes.

Preparation of Microsomes

Livers tissues are homogenized in 3 volumes (1:3) of ice-cold (1-4o C) homogenization buffer containing 1.15% potassium chloride in 50 mM potassium phosphate buffer solution (pH 7.4). The homogenates are centrifuged at 15000 g for 30 min (Hitachi cs120GX II). The mitochondrial supernatant (8 ml) is transferred to ultracentrifuge tubes and spun at 105000 g for 1 hour in ultracentrifuge to sediment the microsomal pellet. Cytosolic fractions are collected from the middle separating the lipid layer on top in separate micro centrifuge tubes. The microsomal pellet is then washed by adding 8 ml of resuspension buffer (homogenization buffer containing 0.05 mM EDTA) and spun again 105000 g for 15 min. The microsomal pellet so obtained is resuspended in 4 ml of resuspension buffer and 1 ml aliquots are stored immediately at -80o C until assayed for drug metabolizing enzymes.

Acetylcholinesterase activity in brain is measured by the method described by Ellman’s et al. (1961).

Reagents

1. Phosphate buffer (0.1M) (pH 8.0) Solution A: 5.22 g of K2 HPO4 and 4.68 g of NaH2 PO4 are dissolved in 150 ml of distilled water. Solution B: 6.2 g of NaOH is dissolved in 150 ml of distilled water. Solution B is added to solution A to get desire pH 8.0 and then finally the volume is made up to 300 ml with distilled water.

2. DTNB reagent 39.6 mg of DTNB with 15 mg of NaHCO3 is dissolved in 10 ml of 0.1 M phosphate buffer.

3. Acetylthiocholine (ATC) 21.67 mg of Acetylthiocholine is dissolved in 1 ml of distilled water.

Procedure

The brain tissue is weighed and 10% homogenate is made in 0.1M Phosphate buffer (pH 8.0). Take 0.4 ml aliquot of homogenate in a cuvette containing 2.6 ml phosphate buffer (0.1M) and 100 µl DTNB. The contents of the cuvette are mixed thoroughly by bubbling air and absorbance is measured at 412 nm in a spectrophotometer. When absorbance reached a stable value, it is recorded as the basal reading .To this 20 µl of acetylthiocholine is added and change in absorbance is recorded for a period of 10 min at intervals of 2 min. Change in the absorbance per minute determined.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using a large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of the drug discovery process can save time and money both by eliminating least beneficial NCEs.

Preparation of Reagents

Culture media

For the culture of hepatocytes medium M199 (GibcoBRL, USA) is used. One pouch of the dry media is dissolved in 1 liter of filtered distil water. The pH of the media is adjusted by adding 0.4% sodium bicarbonate to be around 7.4. The media is than sterilized by filtration through seitz filter (0.22 mm) and stored in 50 ml sterilized conical flasks.

Isolation of Peripheral Blood Lymphocytes

Chemicals

1. Heparin

2. Sterile PBS (pH 7.2)

3. Histopaque-1077

4. RPMI-1640 Media

Protocol

Collect 5 ml of blood from the animal in 1 ml (20 IU/ml of blood) of diluted heparin and add 2 ml of PBS. Layer over the diluted blood on 5 ml of histopaque-1077 in 15 ml centrifuge tube and centrifuge at 1500 rpm for 40 min. Collect the buffy coat at the interface of plasma and histopaque in separate centrifuge tubes. Wash the cells thrice with PBS and suspended in 2 ml of RPMI-1640 media supplemented with 10% fetal calf serum. Determine the percentage cell viability by 0.1% trypan blue exclusion test, using hemocytometer. Finally dilute the cell suspension to 106 cells/ml in RPMI 1640 medium.

LD 50- It is defined as the concentration/dose that kills 50 % of the experimental population.

Acute toxic studies have been conducted in order to calculate LD50. There are several methods to calculate LD50, some are as follows:

Methods to Determine LD 50

• Karber’s method.

• Miller & Tainter method

• Lorke method

• Fixed dose method

• Acute Toxic Class method

• Up & down procedure

Karber’s Method

This method involves the administration of different doses of test substance to various groups, which has five animals each. The first group of animals is administered with the vehicle in which the test substance was dissolved or diluted

Introduction

The immense use of chemicals and pharmacological entities has put the public health at brisk of potential health hazards. These substances in modern days are presented in the form of common constituents of food, medicines, beverages and junk foods etc. However, long term consumption of these substances might be responsible for chronic toxicity, but the accidental ingestion of large quantities of these substances may result into adverse acute toxic reactions. Hence an experimenter should have the acumen to develop and perform the acute toxic tests in order to find out the lethal dose 50 i.e. LD50 of the substances.

Acute Toxicity Studies

Acute toxicity studies helps to establish the dose dependent adverse effect including mortalities. The LD50 has been used as a major parameter in screening of pharmacological agents for acute toxic potential. Apart from mortality, other biological effects and the time of onset, duration and degree of recovery on survived animals, are also important in evaluation of acute toxicity. Hence, acute toxicity study solely gives information about LD50, therapeutic index and the degree of safety of a pharmacological agent.

Acute toxicity is defined as adverse effects occur following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours. Oral administration is the most common form of acute systemic toxicity testing.

Epididymal Total Sperm Count

The epididymal sperm is obtained as described above, incubated at 35°C, which is the optimum temperature of rat epididymal sperm. The epididymal f luid is then diluted to a volume of 5 mL of pre-warmed (32°C) the spermatozoa are counted by hemocytometer using the Improved Neubaur (Deep 1/10mm. LABART, Germany) chamber as described by Pant and Srivastava (2003).

Total viable cell/ml = (Average number of sperm per chamber) x 103 x (Dilution Factor)

Liveability and abnormal sperm

Per cent live spermatozoa is estimated by differential staining technique using Eosin-Nigrosin stain (NE) (Campbell et al., 1953). These slides are also used for estimating the per cent abnormal sperm morphologically on the basis of observable abnormalities of head, neck, mid-piece and trail region of the spermatozoa.

The percent live spermatozoa are determined adopting the differential staining technique using Eosin-Nigrosin stain.

• A drop of epididymal semen is taken on a clean, grease free pre warmed glass slide.

• 4-5 drops of Eosin-Nigrosine stain (1% Eosin and 5% Nigrosine in 3% sodium citrate dehydrate solution) is placed near the semen drop.

• Epididymal semen and stain are mixed gently using a blunt fine glass rod.

• After 30 seconds to 1 minute a thin smear is made on a clean, grease free glass slide.

• The smear is examined under oil immersion objective.

Human beings came into existence 1 million years ago. Insects which constitute 70-90% of all animals present in this world came into existence 250- 500 million years ago.Insects are highly specialized group of invertebrates belonging to phyla arthropoda characterized by having a bilateral symmetrical body covered by Exoskeleton chitin; with jointed appendages provided with independently movable muscle and inserted in to special pockets of exoskeleton; with a haemocoelic body cavity containing the circulating blood in which are bathed the internal organs; with a dorsal heart and decentralized ventral nerve cord; and with tracheal system for respiration; metamorphosis direct or indirect.

The insects are bilaterally symmetrical tracheate Arthopods is which the body is divided into head, thorax and abdomen. A single pair of antennae is present and head also bears a pair of mandibles, a pair of maxillae,. The thorax possess three pairs of legs and usually two pairs of wings. The abdomen is devoid of any ambulatory appendages and the genital opening is situated near the posterior end of the body. Post embryonic development is rarely direct and metamorphosis usually occurs.

Insect definition

Insects (from Latin insectum, a calque of Greek ?ντομον [éntomon], “cut into sections”) are a class of invertebrates within the arthropod phylum that have a chitinous exoskeleton, a three-part body (head, thorax and abdomen), three pairs of jointed legs, compound eyes and one pair of antennae.

American Ecologist Whittaker (1969) proposed the five kingdom system of classification. In his ‘Five Kingdom System’, he added three more kingdoms in to Linnean model of two kingdom system. These were: Monera, Protista and Fungi. Whittaker succeeded to overcome the difficulties as well as demerits of two, three and four kingdom systems and to represent the living organisms according to the evolutionary relationships among themselves. He also defined the kingdoms by a number of special characteristics such as whether the organisms possessed a true nucleus or not. Whittaker’s five kingdom system of classification is based on (a) mode of nutrition (b) cell structure and complexity (c) phylogenetic relationship (d) body organization and (e) reproduction.

The large branches within the Animal Kingdom evolutionary tree are ‘phyla’ (the singular being ‘phylum’). ‘Animal phyla’ outlines thirty three phyla, although there are still disputes and disagreements concerning how many phyla are needed to classify the less well-known species. The most easily recognised are Porifera, Cnidaria, Arthropoda, Nematoda, Annelida, Mollusca, Platyhelminthes, Echinodermata, and Chordata. Animal phyla each comprise a branch of the evolutionary tree and these branches are always connected to other branches.

Body Segmentation

In general, insect body is divided in to a series of segments, which in primitive arthropods are known as “somites” or “metameres”. During the process of evolution, these somites gets fused with each other in different ways forming the body parts of the existing arthropods. The type of arrangement of these body segments in embryonic stage is known as primary segmentation while in adult insects is known as the secondary segmentation which differ from primary in having a sclerotized membranous intersegmental region.

In adult and nymphal insects, and hexapods in general, one of the most striking external features is the amalgamation of segments into functional units. This process of tagmosis has given rise to the familiar tagmata (regions) of head, thorax, and abdomen. In this process the 20 original segments have been divided into an embryologically detectable six-segmented head, three-segmented thorax, and 11-segmented abdomen (plus primitively the telson), although varying degrees of fusion mean that the full complement is never visible.

Types of body segmentation.

(a) Primary segmentation, as seen in soft-bodied larvae of some insects. (b) Simple secondary segmentation. (c) More derived secondary segmentation. (d) Longitudinal section of dorsum of the thorax of winged insects, in which the acrotergites of the second and third segments have enlarged to become the postnota. (After Snodgrass 1935)

Endoskeleton of the Head (Tentorium)

• The tentorium forms during development when 2-4 pairs of apophysis/ apodemes (finger-like invaginations of exoskeleton) fuse internally to create a “bridge”.

• The major components of the tentorium are the anterior tentorial arms, the posterior tentorial arms, the dorsal tentorial arms, and the tentorial bridge.

• The anterior tentorial arms originate as invaginations at the lateral limits of the frontoclypeal suture. The coalescence of the anterior tentorial arms forms a more or less solid structure, the tentorial bridge.

• The posterior tentorial arms arise from pits at the ventral ends of the post occipital suture and they unite to form a bridge running across the head from one side to the other.

• In many insects, e.g. Orhtoptera, the central part of the composite tentorium enlarged to form a broad plate referred to as the Corporotentorium.

• The tentorium serves as an internal “truss” that reinforces the head capsule, cradles the brain, provides attachment for muscles and provides a rigid origin for muscles of the mandibles and other mouthparts. The antennal muscles also arise from the tentorium.

Antennae are paired, highly mobile, segmented appendages present between or below or behind the compound eyes. The antennae arise from the 2nd or antennal segment of the head possessing nerves coming from deutocerebrum of the brain. Antennae are absent in order protura and class Arachnida whereas 2 pairs of antenna (antennules) are present in class Crustacea.

Antennae are set in a socket of the cranium called antennal socket (anetnnifer) and connected by an articulatory membrane which allows free movement of antenna. Antennal socket is provided with an antennal suture. Although antennae vary widely in shape and function, all of them can be divided into three basic parts:

• Scape

• Basal segment, larger than other segments and articulates with the head capsule. It is provided with intrinsic muscles.

• Pedicel

• Second segment of antennae. In this, mass of cells called Johnston organ (auditory) is present (eg. houseflies). The above two segments have intrinsic muscles ie., their own muscles. In honey bee, wasps pedicel forms the pivot between scape and flagellum.

• Flagellum/Clavola

• The remaining segments or annuli or flagellomeres are called flagellum which lack individual muscles. Flagellum is supplied with many sensory receptors that are connected by nerves to brain. Flagellum is further divided into three parts

Light spectrum holds rich information about the world. The spectrum of natural illumination changes with daily cycle and objects differ in spectral reflection or emission properties. Like many other animals, insects use chromatic information to f ind favorable habitat, to efficiently locate food sources and to identify conspecific mates using the spectral information. Pollinating insects such as many species of bees and butterflies use their color vision to maximize success in foraging. They detect flowers, memorize the colors and patterns of rewarding flowers, and preferentially collect nectars from the flowers in their later visits. Visual guidance of behaviour is challenging when photons, the elementary particles of light, are scarce. To produce a reliable representation of the surroundings, the visual system must (i) ensure absorption of a sufficient number of photons into a photoreceptor, (ii) house photoreceptors that efficiently convert each photon absorption into a neural signal, and (iii) process these signals appropriately. Insects are a numerous and diverse class of arthropods that have evolved to occupy ecological niches from the brightest to the darkest.