Example 1: Dairy Products: Physio-chemicals and Microbiology
Agriculture is the single largest sector in the Pakistan, contributing 21. 8 to the gross domestic product and employing approximately 44. 7 of the workforce. Livestock is playing a vital role in the economy of Pakistan and account for 51. 8 % of the agriculture value added and 11. 3% of the national Gross Domestic Product. The milk production in country increased by 35. 6% from 1996 to 2007 (Anonymous, 2008).
Pakistan dairy sector is producing 41. 3 million tons milk and is the fifith largest milk producing country in the world. Its massive herd of 60. 8 million cows and buffaloes produced 40. 76 million tons of milk in the year 2007–2008. while 56. 70 million goats produced 0. 70 million ton (Anonymous, 2008). The role of livestock sector in the rural economy of Pakistan is important as 30–35 million rural population of the country derive their livelihood from livestock production as a primary or secondary activity (Anonymous, 2008),
Milk is defined as the whole, fresh, clean, lacteal secretion obtained by the complete milking of one or more healthy milk animals excluding that obtained within fifteen days before or five days after the calving or such period as may be necessary to render the milk practically colostrums free and containing the minimum prescribed percentage of milk fat and sold not fat (Goff and Griffth, 2006).
Milk is a dynamically balanced mixture and is also a perishable food. It is one of few foods consumed in the natural form throught the world. Milk contain 87% water 3. 9% fat, 3. 3% protein, 5% lactose and 0. 7% ash. Milk supply body building protein, bone forming minerals, health giving vitamin and energy giving lactose and milk fat. Besides providing certain essential fatty acids it contain all essential amino acid. All the properties of milk make it an important food for growing children, adults, adolescents, invalid, convalescents and patients (Khan et al., 2005).
There is a great potential for dairy industry but the sector operates mostly in the informal economy and needs a constituents effort to formalize and be able to contribute better to the national economy. There are nearly 5. 5 million small scale rural units owing less than 6 dairy herds. These small dairy holders produce 65% of all buffalos and cows milk. Out of total milk produced, 97% is in the informal sector (i. e. loose milk consumed in the village and or sold in the cities through ‘Gawalas’ in unhygienic condition and without any quality standard). The small scale milk collector collect 200–400 kg milk per day from different villages. Medium scale milk collectors collect 400–800 kg milk per day in a manner similar to the small milk collectors, but on a large scale, Large scale milk collectos collect 5-to 10 tons milk per day and supply milk the dairy factories (Garcia et al, 2003). There are hardly 15 milk processing plant (mainly UHT fluid milk, milk powder and yoghurt in Pakistan). Only about 3 % milk is being processed and 97% is consumed as a raw milk (Malik, 2008).
Milk and milk products are one of the most important food products with livestock origin which enjoy special significance in terms of its various nutritional properties such as protein, lactose, fat, minerals and vitamins. Many studies have been made on its constituents and physiochemical characteristics (Walstra et al, 1999).
Adulteration of milk and dairy products is one of the most serious issues in the dairy industry and causes economic losses and major health problem to consumers. Due to the limited number of large dairy farms, milk handling process in the traditional system are unhygienic and there is insufficient enforcement of standards, resulting in poor quality of milk products. In order to keep the milk safe, middleman add ice to the milk, in addition microbiological contamination occur due to addition of ice in the milk. The middleman increases the milk quality by adding water, vegitable oil, whey powder and other ingredients to increase the soilds of milk. Antibiotics and Hydrogen peroxide are often used as a preservatives (Garcia et al, 2003).
The adulterants in milk include water, starch, whey poxder, vegetable oil and hazourds substance such as antibiotics, caustic soda, urea, formaline, detergents and other chemicals preservatives. Adulteration in milk is a very serious issues in Pakistan. Keeping in view these facts, the present will be planned.
- To study the Physio-chemicals and microbiological quality of dairy products.
- To determine the adulterants and residues in the dairy products.
- To determine the relationship of physio-chemical parameters with adulterants.
- To make recommendation to the Govt of Punjab in the control of adulterants in milk and other food products.
Review of Literature:
A study conducted on physiochemical quality of UHT milk produced from whole milk powder and stored at 4°C and 25°C for 48 hours. They observed that non protein nitrogen content of UHT milk increased while pH decreased with storage and the rate of change being greater at higher storage temperature. Sediment increased with longer storage period, but independent of storage temperature. With longer storage at both 3+-1 C and 25-+ 1°C, greater sediment and lower pH were observed in UHT milk processed from older milk powder. The development of off flavors increased in UHT milk with a prolong storage period (Ernani et al, 1997).
Kuo et al. 2001 studied the effect of heat treatments on the meltability of cheese. They studied cheddar cheese of different composition and low-moisture. Cheese samples were heated to 60°C and held for 0, 10 and 20 min before allowing the melted cheese to flow. Mean meltabilities, over all ages of both Cheddar and Mozrella cheeses decreased significantly as holding time increased. Meltability of young cheese was scarcely affected by the holding time, in contrast to that of the old cheese where increasing the holding time greately reduced meltability.
Khan (2004) studied the physio-chemicals changes in UHT bottled milk and found that effect of treatments and storage on sedimentation, fat, pH, acidity and SNF was highly significant. Maaximum sedimentation was observed after 12 weeks of storage, pH gradually decreased and minimum value were found after 12 weeks. Maximum acidity was found after 12 weeks and minimum was noted in the first week.
Kumar and Mishara (2004), studied the effect of stabilizer addition on physiochemical, sensory, textural properties and stater culture counts of mango soy milk fortified yoghurt (MSFY). Three stabilizer namely gelatin, pectin and sodium alginate were used. The addition rate of stabilizer was 0. 2%, 0. 4% and 0. 6% w/w. Significant effect of type and addition rate on acidity, msture content and total solids ofMSFY were observed. Syneresis and acetaldehyde content of MSFY was reduced significantly. Lightness and yellowness of MSFY increased with gelatin and decreased with pectin and sodium alginate. Gelatin gave better effect on appearance and color, body and texture, flavor and overall acceptability in comparision with other stabilizer at 0. 4 % addition rate. Hardness, cohesiveness and adhesiveness of MSFY increased up to 0. 4 % stabilizer addition, while springiness and gumminess did not follow any trend. There was a significant effect of stabilizer addition on Streptococcus thermophillus and lactobacillus delbrueckii subsp. bulgaricus counts.
Griffiths et al 1988, manufactured low heat skim milk powder from raw farm bulk tank and creamery silo milk which had been stored at 2°C for 24 and 72 hours. During the storage period psychrotroph count increased by about 1log cycle after 24 hour aand 2 log cycle after 72 hours. There was no increase in thermoduric or spore counts of the milk under these storage condition. The powder manufactured from these milk was good bacteriological quality and conformed to ADMI recommendations regarding moisture content, titratable acidity and solubility. They concluded that storage of raw milk at 2 C had no deterintal effect on the heat stability of the powder manufactured from it when reconstituted to both 9 and 22% total solid concentrations.
Molska et al 2003 studied the microbiological quality of kefir (61 samples) and yoghurt (92 samples) purchased in retail network in Warsaw. The total number of bacteria in at least 90% of yoghurt and 73% of kefir was in the range of 10(7)-10(9) cfu/g. The domestic group of bacteria in kefir were mesophilic lactic acid streptococci and in yoghurt S. thermophillus. The number of L. delbrueckii in 40 % of sample was less than 10(7) cfu/g. More than 86 % of kefir and 97 % of yoghurt analysed were free from coliform bacteria., B. cereus, mould and yeast. About 48 % of kefir samples did not fulfilled the FAO/WHO requirements concerning the number of yeast.
Kessel et al 2004, determine the test for standard plate count (SPC) and fecal coliforms in the bulk tank milk in the inited state. As part of the 2002 survey, 861 bulk tank milk sample were collected from farms in 21 states, coliform were detected in 95 % samples. There were no apparent relationship between SPC and incidence of salmonella or L. monocytogenes. Although the prevalence of L. monocytogenes and salmonella was low, these pathogens represent a potential risk to consumers of raw milk and raw milk products.
Nero et al 2004 condcted a study to avaluate the microbiological quality and the presence of Listeria monocytogenes and Salmonella spp. In the raw milk produced in 210 small and medium farms located in four important milk producing Brazilian states. In 66% of the selected farms the milkng was manual. In 33 % of them, the milking was semi-automatic and only 1 % were equipped wit hfully automatic milking systems. All raw milk samples were negative for L. monocytogenes and salmonella spp. Mesophilic aerobes counts were higher than 10⁵ CFU /ml in 75. 7% of the samples. In 80. 4%, coliforms were over 10²CFU /ml. Escherichia coli were detected in 36. 8% of the samples.
Aygun et al 2005 purchased 50 randomly selected samples of Carra cheese, raw milk cheese, from different retail markets in the Antakya region and were investigated for microbiological quality and some chemical analyses. In their samples, the number of microorganisms were found as follows : Staphylococcus aureus 2. 51* 10³ cfu/g, coliform 1. 02*10⁴ cfu/g, E. coli 4. 27*10³ cfu/g, Salmonella were not detected in any of the samples. Mean moisture, salt and fat content of Carra cheese were found as 41. 26%, 7. 82% and 26. 77% respectively. The pH value of the samples varied b/w 4. 53 and 6. 32 with the mean of 5. 24. The microbiological finding showed the presence of high counts of microorganisms investigated and the poor hygienic quality of Carra cheese.
Little et al 2008 determined the microbiological quality of two retail fresh ripened and semi hard cheeses made from raw, thermized or pasteurized milk. Raw or thermized milk cheeses were of unsatisfactory quality due to level of Staphylococcus aureus st 10⁴ cfu/g, E. coli at 10⁵ cfu/g, whereas pasteurized milk cheeses were of unsatisfactory quality due to S. aureus at 10³ cfu/g and E. coli at 10³ cfu/g. Salmonella was not detected in any samples. They emphasize the need for applying and maintaining good hygeinc practices throught the food chain to prevent contamination and bacterial growth. Labelling of cheeses with clear information on whether the cheese was prepared from raw milk also requires improvement.
Sheppard et al 1985 demonstrated the application of various analytical methods to the detection, identification and quantitation of vegetable oil adulteration o ice cream. Total fat content, sterol, long and chain fatty acid, vit E, Reichert ââ‚¬”Meissle values and Polenske values were measured in ice cream. All method except total fat determination were capable of detecting vegetable oil adulteration. Sterol determination was the most effective and versatile measurement because it provided information not only on the detection and extent of adulteration but also on the possible identity of the adulterant.
Fox et al 1988, described a test for routine screening of Mozzarrella Cheese and butter for vegetable fat adulteration. Fat is extracted and sponified. The potassium salts of the fatty acids are measured through direct gas chromatographic analysis. A ratio, calculated from the concentration of butyric acid and oleic acid is used to avaluate the puroty of the samples. The test offers good precision and can detect less than 10% partially hydrogenated vegetable fat.
Kumar et al 2002, reported that adulteration in milk and milk products has reached an alarming stage. Milk fat is being mixed or replaced with cheaper vegetable oil. Therefore, often more than one test has to be employed to confirm the purity of milk fat. The various method for the detection of adulteration in milk at is based on the physical properties, chemical properties and presence or absence of specific constituents of either milk fat or adulterant fats.
Jha and Matsuoka 2004, conducted a study on the adulteration of natural milk by synthetic milk, prepared by mixing appropriate amount of vegetable oil, urea, detergent powder /shampoo, caustic soda, sugar /salt and skim milk powder to water. Detection of adulterants is difficult by a single method and sometimes more than two methods are required to confirm the presence. The potential of near-infrared spectroscopy were investigated (NIRS) in the wavelength range of 700–1124. 8 nm.
Material And Methods:
Collection of Samples:
The dairy products samples will be collected from the market and then analysis will be performed at Dairy Laboratory, National Institute of Food Science & Technology, University of Agriculture, Faisalabad.
Butter samples of three different brand namely Gourmet, haleeb and Nurpur Dairies will be collected. Three samples from each brand will be collected.
Yoghurt samples of three different brands namely Gourmet, haleeb and Nurpur Dairies will be collected. Three samples from each brand will be collected.
Cheese samples of three different brands namely Adams, Military dairy Factory and Nurpur Dairies will be collected. Three samples from each brand will be collected.
Milk Powder samples of three different brands namely Gourmet, haleeb and Nurpur Dairies will be collected. Three samples from each brand will be collected.
UHT milk samples of three different brands namely Gourmet, haleeb and Nurpur Dairies will be collected. Three samples from each brand will be collected.
Dairy product samples will be collected in clean sterilized container and put in ice chest, whereas milk powder will be collected in zip polyethylene bag. These samples will be transported for analysis to the Dairy Laboratory, National Institute of Food Science & Technology, University of Agriculture, Faisalabad.
All glassware like pipette, test tubes, petri dishes, beaker and flasks will be thoroughly cleaned and sterilized in an oven at 180 C for 2 hours. All media and solution will be prepared in distilled water and autoclaved at 121 C at 15 Ib pressure for 15 min using the procedure of AOAC (2000).
The samples will be subjected to different physic-chemical test which are detailed as under.
Melting Resistance and Melting Quality:
Melting Resistance and Melting Quality will be determined by the method as prescribed by Bhadari(2001).
Fat will be determined by using Gerber method as described by the Kirk and sawyer (1991).
The pH of all the treatments will be determined according to AOAC (2000) method no. 981. 12.
Moisture and ash
All the treatments will be analyzed for moisture and ash according to their respective methods mentioned in AOAC (2000).
Total solids called percent residues will be determined by drying the sample in hot air oven according to method described in AOAC (2000).
Protein content will be determined by using Kjeldhal method as described by AOAC (2000).
Dairy products samples will be tested for total plate counts, Coliform counts, Staphyloccus aureus and Yeast and Mould count by the method prescribed by AOAC (2000).
Chemicals Adulterants Detection Test:
Dairy products samples will be tested for the adulterants namely Formaldehyde, Boric acid, Hydrogen peroxide, Starch, Neutralizers (Sodium carbonate, bicarbonates, Sodium hydroxide by the method prescribed by AOAC (2000).
Results will be analyzed statistically to determine the level of significance (Steel et al., 1997).
Anonymous, (2008). Economic survey of Pakistan. Ministry of finance, economics adviser’s wing Islamabad.
AOAC, (2000). Official Method of Analysis International. 17th edition. Association of office analytical chemists Washington, DC.
AOCS, 1990. Official Methods and recommended practices of the American Oil Chemist Society.
Atlas, R. M. 2004. Handbook of Microbiology Media 3rd ed. New York. Pp 345–356.
Aygun, O. O. Aslantas and S. Oner, 2005. A survey on the microbiological quality of Carra, a traditional Turkish cheese. J. Food Eng 66(3): 401–404.
Bandyopadhyay, A. K. and P. K. Ghatak, 2007. Practical Dairy Chemistry. ISBN. 13 Kalyani Publishers, Iyall. book depot. New Delhi, India. PP 25–74.
Battu, S. R. B. Singh and B. K. Knag 2004. Contamination of liquid milk and butter with pesticides residues in the Ludhiana Distt. Of Punjab state, India. Ecotoxicology and Environmental Saftey, 59: 324–331.
Bhandari, V. 2001. Ice cream manufacture and technology. Tata McGraw Hill pub. co. Ltd. New Delhi.
Blake, A. J. , J. R. Powers, L. O. Luedecke and S. Clark 2005. Enhanced lactose cheese milk does not guarantee calcium lactate crystals in finished cheddar cheese. J. Dairy Sci. 88: 2302–2311.
Cheesebrough, M. 2002. District laboratory practice in tropical countries. UK. Cambridge Univesity Press. Pp: 382–389.
Ernani, L. , M. Lyer, Celestino and H. Roginski 1997. Reconstituted UHT treated milk, effects of raw milk, powder quality and storage condition of UHT milk on its physio-chemical attributes and flavor. Intl. Dairy J. , 7 (2) :129–140.
Fleet, G. H. , M. A. Mian 1987. The occurance and growth of yeast in dairy products. J. Food Micro. , 4(2): 145–155.
Flint, S. , J. L. Drocourt, K. Walker, B. Stevenson, M. Dwyer, I. Clarke and D. McGill 2006. A rapid, two hour method for the enumeration of total viable bacteria in samples from commercial milk powder and whey protein concentrate powder manufacturing plants. Intl. Dairy J. , 16(4):379–384.
Fox, R. J. , A. H. Duthie and S. Wulff 1988. Precision and sensitivity of a test for vegetable fat adulteration of milk fat. Journal of Dairy Science, 71 : 574–581.
Garcia, O. , K. Mahmood and T. Hemme 2003. Areview of milk production in Pakistan with Particular emphasis on small scale producer. International Farm Comparision Network FAO, Pp 11–21.
Griffiths, M. W. , J. D. Phillips, I. G. West, A. W. M. Sweetsur and D. D. Muir 1988. The quality if skim milk powder produced from raw milk stored at 2 C. Food Microbiology, 5(2) :89–96.
Guler, Z. 2007. Level of 24 minerals in local goat milk, its strained yoghurt and salted yoghurt (tuzlu yogurt). Small Ruminant Research, 71 (3): 130–137.
Kuo, M. I. , Y. C. Wang, S. Gunasekaran and N. F. Olson 2001. Effect of heat treatments on the meltability of cheeses. J. Dairy Sci. , 84(9): 1937–1943.
Leea, j. , H. J. Kima, Y. Yoona, J. Kima, J. S. Hamb, M. W. Byuna, M. Baekc, C. Jod, M. G. Shine 2009. Manufacture of Ice cream with improved microbiology safety by using gamma irradiation. 78 (7–8): 593–595.
Lin, T. Y. , C. W. Lind, C. H. Leeb 1999. Conjugated linoleic acid concentration as affected by lactic cultures and added linoleic acid. Food Chem. , 67 (1): 1–5.
Little, C. L. , J. R. Rhoades, S. K. Sagoo, J. Harris, M. Greenwood. , V. Mithani, K. Grant and J. McLauchlin 2008. Microbiology quality of retail cheeses made from raw, thermized or pasteurized milk in the UK. Food Micro. , 25 (2):304–312.
Malik, A. H. 2008. Dairy sector lacks policy focus. Net, Ed. Daily Dawn, Jan, 28.
Mayer. , H. K. 2001. Bitterness in processed cheese caused by an overdose of a apecific emulsifying agent. International Dairy Journal. 4(7): 533–542.
Molska, I. , R. Nowosielska and I. Frelik 2003. Changes in microbiological quality of kefir and yoghurt on the Warsaw market in the year 1995–2001. Rocz Panstw Zakl Hig. , 54 (2):145–152.
Murtaza, M. A. , M. Din, N. Huma, A. Shabbir, S. Mahmood 2004. Quality evaluation of ice cream prepared with different stabilizers /emulsifier blend. Inter J. Agri Bio. (1): 65–67.
Nero, L. A. , M. R. Mattos, V. Beloti, M. F. Barros, D. P. Netto, J. P. Minto, N. J. Andrade, W. P. Silva, Bernadette and D. G. M. Franco 2004. Hazards in non-pasteurized milk on retail sale in Brazil, prevalence of Slmonella spp, Listeria monocytogenes and chemicals residues. Braz. J. Microbiology. , 35 (3) :478–486.
Otero, J. L. , M. H ermida and A. Cepeda 1995. Determination of fat, Protein and total solids in cheese by near infrared reflectance spectroscopy. J. AOAC. Intl. 78 (3):802–806.
Examlpe 2: Immunological Responses to Malaria
Our immune system is comprised of many specialised components, which work collectively to defend the body from harmful foreign bodies. Knowledge of the immune response elicited during malarial infections mainly comes from research using small animal models such as rodents; Plasmodium berghei and Plasmodium yoelii are species of rodent malaria commonly used in studies. Although an immune response is elicited against malaria, in many individuals the parasite is not effectively eliminated, allowing the parasite to multiply and induce clinical symptoms. Due to the morphological transformations occurring, a different group of immune components will be stimulated at different stages of the life cycle.
Following immunisation of irradiated sporozoites, sterile protective immunity against malaria can be induced in all models studied, including humans (Nussenzweig et al., 1967; Edelman et al., 1993; Doolan & Hoffman, 2000). Rodent models have implicated antibodies as mediators of this protective immunity; Potocnjak et al. found that monoclonal antibodies against plasmodium berghei sporozoite proteins neutralised the parasite, blocking hepatocyte invasion and protecting mice from subsequent infection (Potocnjak et al., 1980). However, as discussed by Good & Doolan, parasite elimination in humans by antibodies is unlikely, as high levels of pre-circulating specific antibody would be required at sporozoite inoculation to prevent hepatocyte infection (Good & Doolan, 1999). In addition, studies have demonstrated that antibodies do not mediate protection and instead cell mediated responses are involved (Belnoue et al., 2004).
Schofield et al. highlighted the significance of a group of T lymphocytes called cytotoxic CD8+ T cells and the cytokine interferon-gamma (IFN-Î³). Mice immunised with attenuated sporozoites were not protected from malarial infection when depleted of CD8+ T cells, and when IFN-Î³ was neutralised mice were no longer immune (Schofield et al, 1987). Other studies have reported similar conclusions, suggesting CD8+ T cells and IFN-Î³ are important mediators of an immune response against pre-erythrocytic stages, as reviewed by Doolan & Martinez-Alier (Doolan & Martinez-Alier, 2006). However little is known of the activation or mechanism of CD8+ T cells in malarial infection. Rodent models have suggested naïve CD8+ T cells in the lymph nodes near the site of inoculation or in the liver become activated through coming into contact with antigen presenting cells called dendritic cells (DCs), which prime CD8+ T cells through cross presenting sporozoite antigens such as CSP. DCs internalise, process and present antigens in association with MHC class I molecules to CD8+ T cells. After specific interaction and co-stimulatory molecule signals, CD8+ T cells become activated and migrate to, or stay in the liver, where they can eliminate parasitised hepatocytes (Jung et al, 2002; Amino et al., 2006). Usually CD8+ T cells kill via cytotoxic mechanisms; however immunity to P. berghei sporozoites in mice was found to be independent of cytotoxicity molecules fas and perforin, which suggests the cytokine secretion of CD8+ T cells, eliminates parasites (Renglli et al., 1997). Evidence also indicates IL-12 and natural killer (NK) cells are important for CD8+ T cells to carry out effector functions (Doolan & Hoffman, 1999).
CD4+ T cells are essential for CD8+ T cell effector responses and optimal functioning; IL-4 secreting CD4+ T cells are crucial (Carvalho et al., 2002; Doolan & Martinez-Alier, 2006). Furthermore, CD4+ T cells have anti-parasitic functions; CD4+ T cells clones derived from mice immunised with irradiated sporozoites, provided protection against sporozoite infection in malaria-naïve mice (Tsuji et al., 1990). Belnoue et al. proved both CD4+ T cells and CD8+ T cells were important to eliminate pre-erythrocytic P. yoelii in mice; protection was mediated by IFN-Î³ production and dependent upon nitric oxide (NO) (Belnoue et al., 2004). The toxic effects of NO, suggest it is a critical mediator of effectively eliminating malaria.
The mechanisms remain undefined; studies have implicated many different immune components, which can singularly or collectively confer protection in rodent models, with parallel studies identifying different critical mediators.
Passive transfer studies provide evidence that antibodies are important in eliminating parasites; antibodies from malaria-immune individuals successfully treat individuals with malaria (Cohen S et al, 1961). Furthermore immunity in individuals living in malaria endemic areas may be mediated by high concentrations of antibody specific for a variety of erythrocyte stage parasitic antigens (Osier et al, 2008). As reviewed by Beeson et al., antibodies play a role and are likely to target merozoite proteins, such as MSP-1, to prevent erythrocyte invasion. Antibodies may also target parasitic ligands on the surface of PRBCs such as PfEMP-1. Antibody mechanisms may include inhibition of parasitic development or assist cell mediated destruction of PRBCs or merozoites through opsonisation or via the complement system (Beeson et al., 2008).
As discussed by Engwerda, the spleen is a primary site of cell mediated immune responses against erythrocytic parasites (Engwerda et al., 2005). Murine models have highlighted the significance of CD4+ T cells in eliminating malaria and suggest they are important for gamma-delta T cell (Î³Î´ T cell) expansion in the spleen during infection (van der Heyde et al.,1993). Research suggests that DCs internalise parasites, mature and migrate to the spleen, where they can present parasitic antigens in association with MHC class I molecules to naïve CD4+ T cells. The subsequent differentiation of CD4+ T cells, through IL-12 secretion from DCs, mediates protective immunity against erythrocytic malarial parasites. Th1 cells activate macrophages through the secretion of IFN-Î³ and Th2 cells assist B cell maturation for the production of antibodies through IL-4, IL-6 and IL-10 secretion (Taylor-Robinson, 1998; Good & Doolan 2010). The production of IL-12 is also believed to activate natural killer (NK) cells, which secrete IFN-Î³. Cytokine secretions from activated cells simulate a positive feedback loop, amplifying the immune response.
Using mice, Couper et al. demonstrated that monocytes/macrophages are crucial to eliminate malaria; the infection got worse in mice depleted of these cells. Evidence suggested there are other pathways of activating macrophages other than T cells and IFN-Î³ (Couper et al., 2007).
Activated macrophages secrete TNF-Î±, a mediator of inflammation, which is believed to participate in the pathogenesis of malaria. Macrophages destroy some PRBCs through phagocytosis and by the release of toxic free radicals such as NO (Good & Doolan, 2010).
Therefore antibodies, T cells, cytokines, macrophages and free radicals are likely to all play a role in the immune response against the symptomatic stage of the malaria life cycle.
Example 3: Artificial Insemination in Swine
The use of artificial insemination (AI) increased in these last years because it offers several advantages over natural mating. New genetics can be introduced into a herd with decreased health risks. The semen that is collected from the boar can be diluted in a semen extender and with one ejaculation multiple insemination doses can be created and can be used to breed several sows and gilts. This allows more extensive use of genetically superior boars, increasing the rate of genetic improvement within a herd. On farms employing artificial insemination few boars are needed, and as a consequence, feed, labour and housing costs are reduced. The major processes of AI are: semen collection, evaluation, and processing; detection of oestrus; and insemination.
Reproductive physiology of female swine
For successful artificial insemination, heat detection of the female swine is very important. Oestrus begins with the pituitary gland, which is a gland situated just below the brain. The pituitary gland secretes hormones into the bloodstream such as the luteinizing hormone (LH) and the follicle stimulating hormone (FSH), which are called gonadotropins. In immature gilts gonadotropin secretion is low, but at 6 to 8 months of age, when there is the 1st oestrus it increases dramatically. During the 2 to 3 day period just prior to oestrus, the increase of LH and FSH cause the follicles on each of the two ovaries to grow rapidly. The follicles secrete increased levels of estradiol, which is a hormone into the blood that causes changes in behaviour and physiology of the animal. These changes are associated with the oestrus. Each follicle contains an ovum. When the ovum is released and fertilised by a sperm cell, it develops into an embryo.
The increase of estradiol concentration in the blood reaches a threshold which triggers a large release of LH from the pituitary gland around the onset of oestrus. The release of ova from the follicles into the oviducts is stimulated by the LH. On average, ovulation occurs 40 hours after the onset of oestrus. Fertilization of the ova by the sperm cells occurs in the oviducts, the tubes between the ovaries and the horns of the uterus, and then the fertilised egg moves to the uterus. The sites on the ovaries from which ova are released, then form structures that are called corpora lutea. These corpora lutea secrete the hormone progesterone into the blood. During the luteal phase of the oestrus cycle, which is approximately between day 4 and day 16, progesterone inhibits the secretion of LH and FSH from the pituitary gland, inhibiting follicular growth.
When the ova are not fertilised during oestrus or embryos do not implant in the uterus, around day 16, the uterus starts t secrete the hormone prostaglandin-F2o into the blood. This hormone causes the falling off or death of the corpora lutea. This causes the progesterone level to decline and this allows the increase of LH and FSH levels, follicle growth, and the return of oestrus. In a female swine, oestrus occurs every 18 to 22 days, unless the cycle is interrupted by pregnancy, lactation, poor nutrition, disease, etc.
If fertilisation occurs and pregnancy is initiated, the prostaglandin-F2o is not released in the blood stream. The corpora lutea are maintained and secrete high levels of progesterone into the blood stream throughout gestation. Progesterone is essential in pregnancy, as it inhibits follicular growth and uterine contractions. Around day 114 of gestation, the uterus releases a large amount of prostaglandin-F2o into the blood, and this causes the corpora lutea to regress. The progesterone level is then decreased, uterine contractions commence and the foetuses are expelled.
During lactation, when the pigs are sucking from the sow, LH and FSH are not secreted. When the suckling are weaned, a stimulus allows the secretion of gonadotropin to increase and the follicles grow rapidly and there is the corresponding rise in the circulating levels of estradiol. The sows return in oestrus in seven days after weaning and estradiol elicits the surge of LH, causing ovulation.
The detection of oestrus is very important for successful artificial insemination. The oestrus duration is variable, but the average is 38 hours in gilts and 53 hours for sows. With the high concentrations of estradiol several sign can show that the sow or gilt is approaching or is in oestrus. These signs are: a red, swollen vulva and enlarged clitoris, mucous discharge from the vulva, nervous and restless behaviour, moving back and forth along pen partitions, frequent urination, increased vocalisation, decreased appetite, mounting other females and/or standing to be mounted by other females, elevation of ears, locking knees, and elevating the back.
The best indicator that female swine are in oestrus and ready to be mated is the immobilisation response. When in oestrus they exhibit the immobilisation response as a reaction to a combination of visual, auditory, olfactory and tactile stimuli originating from the boar. It is important to put a mature boar in contact with the female swine that are being checked for oestrus. The females should be checked at least twice a day, with 12 hours interval in between for more accurate determinations. When checking for oestrus, the female should be exposed to a boar for several minutes and observed closely for several signs. If the female re checked in the morning, this should be done before or at least one hour after feeding.
Considerable energy expenditure is required for maintaining the immobilization response. If a gilt or sow that is in oestrus becomes fatigued, it may become unresponsive to boar exposure and not resume an immobilisation response for several hours. During periods when not checking for oestrus, the boar should be kept away from the females, because this greatly increases the likelihood that sows and gilts in oestrus will display the immobilisation response when exposed to the boar during the oestrus check. The boar exposure during oestrus checking should be restricted to small group of females.
When the sows and gilts are housed in crates, a boar should be moved in the front of the females, while a second herdsman applies back pressure. If the female is in oestrus it move forward and assume immobilisation response and when pressure is applied to the back it will push back. This is an effective method of detecting oestrus.
Disposable AI equipment should be used and catheters should only be used ones, so different equipment is used on different female swine to protect plant health.
Before inseminating, the vulva should be cleaned with a paper towel and the tip of the catheter should be coated with a non-spermicidal lubricant. The lips of the vulva should be spread and the breeding catheter inserted. The catheter should be angled slightly upwards while moved through the reproductive tract. This helps prevent entry into the urethra, which is the tube leading to the bladder. After wards the catheter should be slid gently through the vagina until the operator feels resistance. The resistance indicates that the catheter has reached the cervix. With a spirette-type catheter the instrument should be turned counter-clockwise until it locks into the cervix. Then to remove the spirette, it should be turned clockwise while gently pulled outwards. With a foam-tipped catheter, firm forward pressure should be applied to the catheter until the bulbous tip is locked into the cervix, and to remove the bulbous catheter tip, it should be pulled outwards gently.
After the semen and extender is mixed gently, the semen bottle, tube, or bag should be connected to the open end of the catheter. The semen is dispensed by gently squeezing the container over a three to five minute period, taking care to avoid excessive back flow of the extended semen out of the vulva. The tip of the catheter may be blocked against cervical tissues occasionally, blocking the flow of the semen, and if this happens the catheter should be repositioned by turning it.
The insemination is easier if the female swine is exhibiting the immobilisation response, although it is not necessary. If a boar is placed in an adjacent pen, it can facilitate AI, but on the other hand, immobilisation response requires considerable energy expenditure and the female may become fatigued. When there is a large number of sows to be bred, some of them may become refractory to the boar stimuli prior to artificial insemination.
When the boar is present during artificial insemination, the sow’s pituitary gland releases oxytocin into the bloodstream, which a protein hormone. It stimulates muscles contractions of the uterus and oviducts, and these contractions cause the semen to be drawn into the reproductive tract during AI. This is also known as self insemination. If the AI technician applies firm back pressure and rubs the flank or udder of the sow during insemination, he will facilitate the self insemination.
Timing of insemination
Accurate oestrus detection is the success of artificial insemination. Timing of insemination is normally based on the time when oestrus is first detected. Insemination should be done prior to ovulation, i.e. maximum 24 hours before ovulation in sows and maximum of 12 hours before ovulation in gilts. Inseminating twice during oestrus increases the likelihood that one will occur during the optimum time. If female swine are in standing heat for 3 days, a third mating should be beneficial. The females that are not in oestrus should not be inseminated because reproductive performance will be adversely affected.
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