Maintaining protein nutrition in chickens

Maintaining protein nutrition in chickens - Vinayak IngredientsNecrotic enteritis in broiler chickens can be caused due to the increased level of protein, 2-6 weeks post hatching. This is because of the over growth of 
C. perfringens in the small digestive system, increasing from a typical level of 104 CFU to 107 or 109 CFU per gram of digesta and causing harmful diseases.

With the increased dietary protein level, there will likewise be an increased activity of the trypsin in the small digestive system. This will, thusly, prompt to speedier arrival of coccidia from their oocytes which will lead so dynamic as to be less responsive to immunization.

In situations where such bacterial and protozoan are probably going to prevail, it might then be advantageous, among different measures, to reduce the supply of protein and maintain it underneath the prescribed range. It is likewise important to consider the amino acid balance of the protein source to be utilized. Methionine and glycine, for instance, have been known to empower growth and establishment of C. perfringens and different pathogens in the gut. Accordingly, the utilization of protein sources having over the top measures of these amino acids ought to be limited.

Additionally, there are some protein sources, for example, cottonseed meal, raw soybean, and flax cakes, which contain varying measures of anti-nutritional factors, for example, trypsin inhibitors, gossypol, and glucosides. At the point when ingested by the bird, these components would then apply some damaging consequences for the small digestive tract, in this manner impeding the safe mechanical assembly having nearby as well as systemic defensive capacities. Excessive utilization of such protein sources in the diet ought to, therefore, be avoided as well.

Is It Necessary To Premix?

Premix in livestock feed - Vinayak IngredientsA premix is a blend of minerals, vitamins, feed supplements and diluents. The premix business is charged of the obligation of assembling a fantastic premix reliably, effectively and monetarily. The principle objective in feed mixing is to guarantee that an animal gets the majority of its figured supplement remittances consistently. Most sustain producers utilise the coefficient of variety (CV) to gauge blender execution and blend consistency. The CV is characterised as 100 * standard deviation/mean. A 5% CV is the business standard for generally fixings. An ingredient mix CV of 5% grants that an animal get no less than 90% of its planned dietary stipends 95% of the time. Be that as it may, the size of an adequate CV will change contingent upon the logical accuracy for measuring the fixing and the fixing proportion in the eating routine. The CV for a fixing test (repeatability of the explanatory system) ought to be not exactly the wanted CV for blender proficiency. With respect to ingredient ratio, the lower the ingredient concentration in the mix, the higher the CV.  Hence, the CV is generally higher for follow mineral, vitamins and medications in light of the fact that their fixing proportions are low (under 1:10,000).

A quality premix can be made just through a stringent quality affirmation program and current great assembling rehearses (cGMP). Quality affirmation is a proactive, persistent framework for observing reproducibility and dependability of an item. It incorporates all the activities undertaken to guarantee foreordained benchmarks of a quality premix. Good manufacturing practices covers covers all the areas of the production process like staff, offices, crude materials, quality affirmation checks, stock control, preparing, blending, packaging and conveyance.

Guidelines for feed formulation

1) Premix: Premix micro ingredients, for example, antibiotic growth promoters, vitamins, and minerals with a reasonable diluent before their consideration in a supplement. Diluents serve to dilute the micro ingredient and consequently encourage the rate of blending. Diluents ought to be dry with a specific end goal to allow a more uniform scattering of individual miniaturised scale fixing particles. Dampness must be maintained a strategic distance from as it might bring about entrainment and bunching (hygroscopic mixes, for example, urea are not appropriate diluents). Nonetheless, it can be preformed all the more effectively and proficiently by method for a small portable cylinder mixer (cement mixer). Defensive apparel, gloves and clean veil ought to be worn when when handling micro ingredients.

2) Supplement: This supplement will contain the premix, an appropriate Carriers, and the remaining minor dry ingredients in the diet routine including minerals, urea, and supplemental protein sources. Carriers are feed ingredients which join with the small scale fixings in the premix to change their physical attributes. By adsorbing to the carrier, the fine particles of the micro ingredients are permitted to move all the more quickly and consistently through the blend. This quick development of micro ingredients through the blend is vital to guarantee satisfactory dispersion before addition of molasses. The carriers ought to have physical properties practically identical to ground grain or oil seed dinners. For sure, both of these might be utilised as carriers. Be that as it may, the adsorptive properties of ground grain and oil seed suppers are low. This constraint might be overcome by first consolidating 2% fat to the ground grain or oil seed feast before mixing with the premix. In preparing the supplement, first include the carrier, and then include other major ingredients until they achieve the focal shaft line, then include the premix and other minor ingredients, lastly include the remaining major ingredients.

3) Finished feed: Finished feed might be prepared as follows: 1) include the grain portion of the diet to the blender; 2) add the dry supplement  to the centre of the blender (if possible, include supplement the inverse end of the blender to where the feed is released); 3) permit feed to blend for at least 1 minute; 4) add forage part of the diet; 5) add fat segment of the diet; 6) add molasses of the diet; 7) permit to blend for the time determined for the blender.

4) Adding Molasses: Molasses is a typical ingredient in diet formulation. However, it is very viscous and this introduces a few issues in feed mixing. Surely, if added to the diet despicably it can bring about marked increments in the CV of the micro ingredients. Dark strap molasses is especially viscous. The proficiency of mixing dark strap molasses with other dietary fixings will be improved in the event that it is initially weakened with water. The consistency of molasses is notably diminished by warming. Molasses ought not be warmed to temperatures in abundance of 43 degree Celsius, except for very short periods of time, as this may cause carmelization.

Here are some of the benefits of premixing

Accuracy of the weighting of the micro-ingredients

Micro-ingredients would require a much higher precision of weighting than macro-ingredients. That would request adjusted hardware that ought to be committed and set up particularly for micro-ingredients

Savings of blending time

As specified over, the preparation of premixes can decrease the preparing time. In fact, the weighting of the micro-ingredients can be extremely tedious and it would be more profitable to sort out this procedure in parallel to the preparation of the macro-ingredients.

Reduction of crossed contamination

The addition of diluted micro-ingredients into the system will leave lower traces than if the same micro-ingredients were incorporated pure. Also, it is simpler to clean up the premixing equipment because of its smaller size and easier access.

The Science of Feed Formulation

The Science of Feed FormulationFeed formulation requires top to bottom information of animal nutrition, especially the supplement necessities and the nutritional composition of the animal feed. It requires nutritionist with good knowledge of using certain proportions of some ingredients that affect on problems such as free flow through the mill, pellet quality of the diet, digestion of the animal feed or total gut well being of the animal.

Feed industry worldwide use 4 basic steps to produce animal feed.

  1. Receiving raw materials from supplier.
  2. Generating a feed formula based on previous authentic research.
  3. Blending all the ingredients together to prepare feed.
  4. Packaging and labelling feed to be shipped for commercial or retail purposes.

Quality control is an arrangement of methodology followed to guarantee quality of a product and all factors involved. Quality assurance starts with examination of incoming ingredients. Damage by heat, molds, climate, insects, adulterant and contaminants can be detected by trained technical supports. Blending quality can likewise be assessed quickly. The presence or absence of micro ingredients, minerals and vitamins are established by observation or by micro spot-testing.

Laboratory testing is a critical part of any quality control program since it quantifies particular segments of a feed/ingredient sample to guarantee that it meets quality determinations. Tests include physical, chemical, biological and other electronic measurements to determine the quality of product in contrast with a prescribed standard.

Liquid feed is used in dairy industry to feed cattle and provide a well-balanced mixture of nutritional supplements in a molasses base, which can be distributed in a feed taken or added to another form of feed, is that the proteins promote the probiotic microorganism in the rumen, which contribute to the breakdown of fiber and forage.

Feed manufacturers must follow regulations represented by the U.S. Food and Drug Administration. FDA is in charge of the regulation of feed and implementation of policies through the state government and the Association of American Feed Control Officials. Various feed certification programs—such as the American Feed Industry Association’s Safe Feed/Safe Food Certification Program— are likewise accessible for additional compliance and regulation.

Micro Emulsions in Poultry

Micro Emulsions in PoultryMicro emulsions in Poultry are dispersion’s of oil and water with an emulsifier. They are clear, thermodynamically stable, isotropic liquid mixtures. They are super solvents which improves stability and thermodynamic activity of formulation. Micro emulsions are beneficial to be used because it increases efficacy of the formulation allowing dose reduction. The average particle size of micro emulsion is 0.1 micrometer which helps in increasing the inter facial area thereby allowing active ingredient to get released easily. In poultry, micro emulsions are designed to include natural essential oils cell wall which in turn binds to mycotoxins to protect animals against mycotoxosis.

Vinayak Ingredients have introduced micro emulsion which is an alternative to antibioticsnamed as Herbofloxin. It is of natural origin prepared from essential oil of syzygium, citronella, thymus, eucalyptus. Herbofloxin has a particle size less than 0.1 micrometer which makes it easily soluble in water. It maintains poultry gut’s pH-6.5 to 6.7 which is slightly acidic. As it is a micro emulsion it has better dispersion in water, stable at 45 degree Celsius temperature and has a longer shelf life. All these factors makes it safe to be consumed by poultry without having any side effects which are otherwise usually caused by using antibiotics. Herbofloxin is natural replacer for antibiotic growth promoters.

Mechanism of action: Herbofloxin being a micro emulsion when mixed with water forms nano emulsion due to which particle size decreases further making it easier to penetrate the bacterial cell wall and disrupt it. Disruption of cell wall leads to killing of bad bacteria such as Escherichia coli, Campylobacter, Clostridium and Salmonella. Thus it acts like a bacteriostatic. It also acts as an anti-inflammatory by improving mucin coverage which is a first line of defence in poultry.

Herbofloxin as a micro emulsion replaces antibiotics such as tetracycline’s, fluoroquinolones, amino glycosides and selectively modulates poultry gut to promote the beneficial microflora.

Neonatal Poultry Nutrition

Neonatal Poultry NutritionOver the last five decades improvements in nutrition and genetic selection have reduced the time required to produce a 2 Kg broiler within 1.7 FCR. The neonatal period is defined as the first seven days of the production cycle after hatch. It is a crucial time when the chick requires special management and nutrition. Efforts to control metabolic disorders such as ascites and leg problems have led to recommending early feed restriction during the first two weeks post-hatch. Thus, it is essential to know the effect of poultry management practices on subsequent chick development. A paper presented in the ohio university explains the importance of the relationship of neonatal nutrition to muscle development. Muscle growth and development can be divided into two distinct periods: hyperplasia and hypertrophy.

Hyperplasia is an embryonic period characterised by proliferation of muscle fiber number whereas hypertrophy is a post-hatch muscle growth which results in the enlargement of existing muscle fibers. Nutritional deprivation has a significant effect on the myoblast cells. Research was conducted to evaluate the effects of an immediate post-hatch feed restriction on breast muscle formation. The increased number of nuclei in muscle fibers correlates with increased synthesis of protein and muscle fiber size enlargement. Myoblast cells are extremely responsive to the mitogenic effects of their environment, including nutrition. A 42-day length of study conducted with feed restriction on the neonatal chickens showed a significant difference morphologically in the development and structure of the breast muscle between the feed restricted and unrestricted diet treatments. It also increased deposition of fat in the breast muscle of the birds with the 20% feed restriction.


Nutrient deprivation in the first few days after hatch may interfere with normal muscle protein development in broiler chicks. However, if you believe that flavor and juiciness follow the fat, there may be some benefit from early feed restriction.

What is the effect of KiFAY on IGF-1 and protein accretion in broilers?

What is the effect of KiFAY on IGF1 and protein accretion in broilers - Vinayak IngredientsA comparative study was performed to investigate the efficacy of KiFAY™ as a feed additive on performance parameters, thyroid, and pancreatic hormone levels in broilers. Ninety birds (Vencobb 400) were randomly divided into three groups viz., Control (no DL-methionine supplementation), Treatment 1 (containing added DL-methionine) and Treatment2 (containing KiFAY™ and without DL-methionine supplementation). The performance parameters (weekly body weight, body weight gain, feed intake, and feed consumption ratio) were recorded and calculated during the whole study of 4 weeks. Analysis of insulin and insulin-like growth factor (IGF1), triiodothyronine (T3), thyroxine (T4) and thyroid stimulating Hormone (TSH) were performed at the end of the study.

The results show that birds on supplementation of KiFAY™ performed significantly (p<0.001) better than other treatments. The weekly body weight, body weight gain, feed in-take and feed consumption ratio improved in KiFAY™ treated birds. The study shows an increase in insulin and IGF1 levels (p<0.001) in KiFAY™ than other treatments. Serum T3, T4 and TSH levels in the treatment2 were higher than other treatments (p<0.001). The KiFAY™ supplementation was able to improve performance with associated responses at a hormonal level in broilers.

Effect of Protein and Amino Acids on Fat Deposition in Poultry

Effect of Protein and Amino Acids on Fat Deposition in Poultry - Vinayak IngredientsThe abdominal fat tissue is very important in chickens due to its rapid growth as compared with other fat tissues. Most fatty acids are produced in the liver and stored as triglycerides in adipose tissues. Thus, the abdominal fat is a reliable parameter for estimating total body fat content as it directly correlates with the total lipid content in avian species. Nutritional factors play a key role in regulating body fat deposition. Therefore, this article discusses the effect of two such nutritional factors viz., protein and amino acids on the abdominal fat content and the mechanism of regulating abdominal fat deposition in poultry in a beneficial manner.

Protein is the most expensive component of poultry diets. The increase in the dietary protein content improves the daily weight gain, carcass yield, and meat quality by reducing body fat deposition and increasing protein content. A report shows that reducing dietary protein level during the starter, grower, and finisher phase, and compared with normal-protein diets as recommended by NRC, 1994 led to a significant increase in the abdominal fat content. An analogous study where increasing dietary protein level in the diets of broiler chickens in all three phases led to a significant reduction abdominal fat deposition compared with diets formulated according to NRC (1994) causing lean broiler chickens. Therefore, dietary protein content must play a direct or indirect role in the regulation of lipid metabolism. In 2002, it was found that reducing dietary protein content upregulates malic enzyme mRNA expression increases malic enzyme activity in the liver of broilers compared with the control, and vice versa. Further study also showed that increasing dietary protein content caused a significant reduction in hepatic enzyme mRNA expression in the livers of broiler chickens. Therefore, dietary protein level directly affects body fat deposition. Thus, it is important to suffice the protein requirements of birds to produce high-quality meat with low-fat deposition.

At present, only methionine, lysine, and arginine are known to beneficially regulate body fat deposition in poultry. Therefore, the addition of these amino acids in poultry diets should be ensured to prevent unnecessary fat deposition. Among these, methionine is the first limiting amino acid in poultry diet. It is essential amino acid as it directly affects on growth performance and helps in producing lean meat. A report shows that inclusion of L-methionine in poultry diet leads to a significant reduction in body fat content. The effect of dietary L-methionine in reducing the fat deposition may be associated with changes in lipolysis and lipogenesis. Lysine also has a prominent role in meat quality by increasing protein deposition, reducing the water-holding capacity, and enhancing muscle pH. The lysine supplementation in poultry diets significantly enhances lean meat production. A meat-type ducks fed with lysine-deficient diet gave significant high abdominal fat percentage while the inclusion of lysine eliminated this effect. Hence, addition of lysine in poultry diets promotes lean meat production by reducing carcass fatness via lipogenesis inhibition.

Another essential amino acid is the arginine which plays multiple roles in poultry production, implicated in reduction of carcass fat deposition. A study reports a significant reduction in the abdominal fat content in Japanese quails at 42 day of age 2.0% arginine supplementation at day zero of incubation. A corresponding study reported that providing 1.0% more arginine in addition to the NRC (1994) recommendations reduces the abdominal fat content by decreasing the activities of enzymes involved lipogenesis. In avian species, therefore, dietary L-arginine supplementation inhibits certain hepatic enzymes, which causes a reduction in the abdominal fat content by reducing the size of abdominal adipose cells.

Hence, the fat-reducing effects of protein and certain amino acids have not been fully clear. Thus this article makes an effort to elucidate our current understanding of the mechanism related to the effects of protein and amino acids that beneficially regulate abdominal fat deposition in poultry.

7 Main Nutrition Requirements in Egg Layers

7 Main Nutrition Requirements in Egg Layers - Vinayak IngredientsPoultry diets are a mixture of several feed stuffs such as soybean meal, cereal grains, fats, animal by-product meals, and vitamin and mineral premixes. Here are the few main nutrients which producer must not ignore when planning the feed diet.


The main source of energy for poultry is dietary carbohydrates. Corn, grain sorghum, wheat, and barley are important carbohydrates to poultry diets. These adversely affect the digestive processes of poultry when present in sufficient dietary concentrations. For example, pentosan and beta glucans of rye and barley respectively increase the viscosity of digesta and helps in nutrient absorption of poultry. Supplementation of rye or barley with dietary enzyme improves nutrient utilisation and growth of young poultry.


Dietary requirements for protein are actually requirements for the amino acids contained in the dietary protein. They are main constituents of structural and protective tissues, such as feathers, bone matrix, skin, and ligaments, including organs and muscles. The individual amino acids and short peptides after digestion-absorption may serve a variety of metabolic functions and precursor to biochemical pathways. Insufficient dietary protein leads to slow growth or less productivity.


Minerals are the inorganic part of feeds or tissues. Calcium and phosphorus are essential for the formation and maintenance of the skeleton and eggshell formation. Sodium, potassium, magnesium, and chloride function with phosphates and bicarbonate to maintain homeostasis of osmotic relationships and pH throughout the body. The forms of phosphorus, such as ATP and phospholipids if present in plants, can be digested by poultry; however, such digestible forms usually account for only 30 to 40 percent of the total phosphorus. The remaining phosphorus is present as phytate phosphorus and is poorly digested. Trace elements, including copper, iodine, iron, manganese, selenium, and zinc are required in small amounts in the diet. Trace elements function as part of larger organic molecules. Iron is a part of haemoglobin and cytochromes, and iodine is a part of thyroxine.


Vitamin C is synthesised by poultry and is, accordingly, not considered a required dietary nutrient. The dietary requirement for vitamin E is highly variable and depends on the concentration and type of fat in the diet, the concentration of selenium, and the presence of prooxidants and antioxidants. Vitamin K activity is exhibited by a number of naturally occurring and synthetic compounds with varying solubilities in fat and water.


Water must be regarded as an essential nutrient, although it is not possible to state precise requirements. The amount needed depends on environmental temperature and relative humidity, the composition of the diet, rate of growth or egg production, and efficiency of kidney resorption of water in individual birds.


The carotenoid pigments not only provide yellow-orange coloration of egg yolks and poultry fat but also contribute to coloration of the skin, feet, and beak. Alfalfa meal contains lutein which provides a yellow colour, whereas corn and corn gluten meal contain primarily zeaxanthin which impart as orange-red colour. Synthetic carotenoids are also used approved by the regulatory agencies used in poultry diets as the concentration of the desired pigments in natural feed stuff is not always constant.


Antimicrobial agents are nutritional feed additives/growth promoters and are not nutrients as they are essential to poultry. They are included in diets to improve growth, efficiency of feed utilisation and livability. They are added at relatively low concentrations (1 to 50 mg/kg), depending on the agent and stage of development of poultry.

Poultry diets are a mixture of several feed stuffs such as soybean meal, cereal grains, fats, animal by-product meals, and vitamin and mineral premixes. Here are the few main nutrients which producer must not ignore when planning the feed diet.

Bone Defects In Fast Growing Chicken

Bone Defects In Fast Growing Chicken - Vinayak IngredientsBirds pertaining rapid growth and heavy body weight, are usually associated with a week skeletal body. This has been implicated in musculoskeletal and cardiovascular disease in meat-type poultry. It does not always necessarily result in disease but many of the complications can be eliminated by slowing down the growth rate and research on this has produced contradictory results. Therefore it would be more correct to be called as metabolic disease, since most of these diseases are due to metabolic imbalances associated with rapid growth.

If the hypothesis that musculoskeletal deformity caused due to rapid growth is valid, then we must take into account how specific defects could be associated with rapid growth.

1) The defect may be due to increase in body weight.

2) The defect could occur because of undeveloped tissues (bones, ligaments, tendons, and muscles).  This is because as the strong tissue is produced, remodelling and bone alignment would require more duration than rapid growth.

3) The defect could be related to high amino acid supplement, enzyme, hormone, or oxygen requirement by specialised cells.

4) The defect may be due to metabolic by-products such as carbon dioxide and lactic acid that are increased by rapid growth.

5) Rapidly dividing cells could be more prone to toxic or metabolic injury.

Most of the skeletal deformities in birds result in birds that are not able to walk. Birds in these cases find difficult to get feed and water due to chronic pain and anxiety associated with aggression from other birds.

Skeletal deformities can be caused in a variety of ways. Nutritional deficiencies are one of the causes in skeletal disease in all birds. Birds that are growing fast have higher requirement of essential amino acid supplement and have more skeletal defects than in slower growing strains. Mechanically induced or trauma-associated problems are also much more frequent in fast-growing broilers. These problems may be caused due to immaturity and weight than rapid growth because tissue becomes stronger and more resilient with age. This age-related effect is particularly true of bone, tendon, and ligament. Toxins in feed or water can cause skeletal deformities. Toxin effects are not usually associated with rapid growth, although rapidly growing birds would consume more of the offending product. Genetic problems may also result in skeletal defects, but not related to growth.

To conclude, prevention of musculoskeletal disease in chickens must be the goal, and attempts should be made to find management and nutritional techniques to reduce bone defects such as better lighting programs appear to improve broiler mobility and better methods of catching and transferring birds.

IGF1 In Broilers

IGF1 In Broilers - Vinayak IngredientsInsulin like Growth Factor-1 (also called IGF-1 or somatomedin C), is a highly conserved molecule similar to the molecular structure of insulin. It is made up of 70 amino acids encoded by the IGF1 gene. IGF-1 has many effects on the body. It plays akey role in the control of skeletal characteristics, metabolism,and growth of adipose tissue and deposition of fat in chickens. IGF1 promotes cell division and cell growth in the body. It also plays a role in cellular repair in the brain, heart and muscles of the animal. The perturbation of IGF1 can cause many consequences to the animal. IGF-1 is a major mediator of effects of growth hormone (GH) produced in the pituitary gland, then released into the blood stream, later triggers the liver to produce IGF-1. Few studies have shown no direct correlation between GH levels and the growth rate in chickens; hence this has led to study IGFs as mediators of the functions of GH.

The action of mechanism is initiated by binding of IGF1 to its receptor called IGF1 receptor which is present on many cell types in many tissues. This mediates intracellular many cellular signal transductions at the molecular level.The mechanisms of involvement of these proteins in insulin/IGF signaling pathways are largely speculative and require further study. The IGF1 produced in the muscle offers main benefits to the gain of the muscle. They trigger different protein activities involved in the muscle protein synthesis. There are multiple factors associated with the production of IGF1, such as low levels of glucose or deficiency of protein can trigger a significant decline of the IGF1, vice versa.

Endocrinology in birds has always been an unfamiliar subject to the researchers, even though endocrinopathy in birds have high occurrence. Hormones such as the growth hormone, IGF, thyroid hormones and insulin, play important and diverse roles in animal growth. Very few information is available that explains the nutrient-IGF relationship in poultry industry. However,

IGF1 have been sensitive to the alteration in the nutrition in domestic fowl. Studies performed by two separate groups shows food deprivation for 5 day depresses circulating IGF1 concentration and upon re-feeding, concentration return to near initial concentration. Other studies in contrast reported that a complete return to normal IGF1 was observed following depriving from feeding suggesting that the extent of nutrient deprivation determines the rate at which IGF1 synthesis and secretion return to normal following periods of nutrient modification.The study led by Del vesco and its colleagues in 2013, has evaluated the effects of different dietary methionine levels on IGF1 and GH gene expression in liver and muscle tissues. The IGF1 and GH gene expression in muscle tissues was not affected by methionine supplementation. However, IGF1 gene expression in the liver was higher in broilers fed methionine diet. They further demonstrated the effect of heat stress and supplementation of methionine on the GH and IGF1gene expression in the liver and found that methionine supplementation increased IGF1 and GH expression. They observed that the highest GHR expression occurred at normal temperature and not at heat stress in supplementation of methionine in the diet. This suggests that the protein degradation is induced by the heat stress but supplementation of methionine triggers protein deposition because it increases the expression of gene related protein synthesis and reduces the expression of genes related to protein catabolism.

JaromirKadlec along with other workers has found IGF1 as a potential candidate gene responsible for various metabolic traits in chickens. They have identified single variable gene known as single nucleotide polymorphism (SNP) in total 132 birds using molecular techniques and have correlated the genotype frequencies with growth and fat deposition in chickens.The results depict identical IGF1 amino acid sequences among chickens, rats and human peptides.

In spite of the wealth of knowledge that has accumulated concerning IGF1 in past few decades, still many details of IGF1 in broilers remain to be clarified about the role of different pathways.