In Ovo Technology Applications in Poultry
In ovo (In Latin- in egg) supplementation of nutrients or the vaccine is a unique technique which is basically aimed to supply the nutrients or the vaccine at early stages of embryonic development of the chick. The technique is intended to assure better growth rate, better feed conversion efficiency, higher bird weights and relatively superior immunity in poultry. Sharma and Burmester first used in ovo technique where they vaccinated the chicks with Marek’s vaccine during early embryonic stages and observed significant increase in immune protection against the disease. Now the technology is commercialized. The five basic compartments in an incubating egg i.e. Air cell, Allantoic sac, Amniotic sac, the Embryo itself and the Yolk sac used as a site for injection in in ovo technique and the most preferred period to use it is during 17th to 19th day of incubation. The in ovo technology is now utilized for supplementation of nutrients to the chicks before hatch to enhance growth as well as immunity where different nutrients like carbohydrates, amino acids, minerals and vitamins are under investigation singly or in combination. The major advantages of technology are precise delivery of vaccines, reduced labour cost, improvement in final body weights and immunity. In conclusion, lots of development is expected in in ovo technology in coming future as it has potential to save economical losses in poultry due to diseases as well as increased productivity and sustainability.
In ovo (In Latin- in egg) supplementation of nutrients or the vaccine is a unique technique which is basically aimed to supply the nutrients or the vaccine at early stages of embryonic development of the chick. The first administration of in ovodelivery of exogenous materialwas reported in the 1980s for vaccination againstMarek’s disease by Sharma and Burmester. Later on, a wide research was carried out worldwide to inject smaller quantities of vaccines, drugs and nutrients in ovoto validate its effect in later stages of life. The basic aim of using in ovo technique in poultry is to assure better growth rate, better feed conversion efficiency, higher bird weights and relatively superior immunity.
In ovo technology One of the useful tools for precision poultry farming and production is the in ovo technology for transforming the situations within the egg through the application of vaccines, nutrients and other bioactives. With this technique, one can deposit a definite quantity of cautiously chosen material into a particular location inside an incubating egg. In ovo technology is targeted for the most decisive time in the chick’s development, that is, the perinatal period. The perinatal period extends from the last days of the egg’s incubation to the initial few days post-hatching. At this time, the embryo needs to adjust to a change in food, which is from the fat-rich to the carbohydraterich, as well as contact with environmental microbes. In commercial settings, the hatching window is a necessary evil and so unavoidable. Because of these various factors, in ovo method has been developed to facilitate manipulation of the chicken embryo before hatching. In principle, this technology is basically dependent on the delivery of matters by mechanical means straight inside the incubating egg First, in ovo inoculation was used for the vaccination purpose against Marek’s disease (MD) on the 18th day of incubation and observed better immunization (Sharma and Burmester, 1982). In ovo, immunization is mainly used for MD, Gumboro, Ranikhet and Avian Influenza. However, in ovo technology as a tool of prenatal nutrition is still an area of exploration, and its application is being investigated to deliver carbohydrates, peptides, vitamins, prebiotics, probiotics, synbiotics, etc. Such precise manipulation of the embryo may develop the vigor and resilience of the hatched chicks, which contributes to their further posthatching growth.
Conceptualization of in ovo technique
In the decade of 80s it was observed that, there is mortality in birds due to Marek’s disease in poultry in spite of vaccination, as adequate time period was not available to develop protective immunity post vaccination. To overcome the issue, Sharma and Burmesterfirst used in ovo technique where they vaccinated the chicks with Marek’s vaccine during early embryonic stages and observed significant increase in immune protection against the disease The technology was patented and later made available commercially which significantly decreased mortality due to disease and ultimately enhanced the production performance of the poultry. Now, the in ovo commercial vaccines are also available against Infectious bursal disease virus (IBDV) and Pox virus in chicken.
Sites of injection
During the final stage of incubation, there are five basic compartments in an egg i.e. Air cell, Allantoic sac, Amniotic sac, the Embryo itself and the Yolk sac. These compartment(s) are basically used as a site for injection in in ovo technique. Usually the embryonic development is at its peak during the 17th to 19th day of incubation and this period provides window for in ovo injection. During this period the compartments change fast and each compartment is responsible for different support to embryonic development. Therefore, the site and time for in ovo injection is very essential to be recognized as the placement of vaccines and/or other compounds into those compartments may allow or limit their absorption by the embryo.
In ovo technology and nutrition
The in ovo technology is now utilized for supplementation of nutrients to the chicks before hatch to enhance growth as well as immunity. A major intention of pre-hatch feeding is to equip the embryo with the nutrients necessary to continue intestinal development post-hatch at or close to the same rate as pre-hatch. Supplementing the embryo with exogenous nutrients would allow the gastrointestinal tract to develop the structures and functionality to properly digest and absorb nutrients immediately when exogenous nutritional supplementation is provided after hatch. These nutrients, along with the yolk sac reserves, can contribute not only to maintaining the systems and metabolism already established but also to continuing growth, development, and proper nutritional status. The nutrients that are under investigation for in ovo administration are carbohydrates, amino acids, minerals and vitamins. Some studies have shown comparatively larger digestive organs by amino acids; greater surface area of villi, higher goblet cell density on jejunal villi and higher mucin gene expression by carbohydrates administration, stimulation of intestinal development of hatching eggs; increase post-hatch growth and humoral immune response by Vit. E; beneficial effect on antibody, macrophage response, immunomodulation and cellular immunity by amino acids, vitamins and fatty acids. Due to these reasons now a day more and more studies are aimed to investigate the incorporation of various nutrients in ovo.
In ovo feeding
In ovo feeding is a technique of providing essential nutrients in the egg itself. Early nutrients access has a fundamental function in the initial stage of growth and utilization of nutrients by chicks. Rapid development, feed utilization efficiency, getting early to marketable bodyweight (Bakyaraj et al., 2012), increased perinatal development (Ferket and Qureshi, 1992) can be achieved with in ovo method of feeding. The supplementation of macro and micronutrients and probiotics also causes a valuable outcome on hatchability as well as disease resistance of hatchlings (Uni and Ferket, 2003).
Different routes of in ovo feeding
In ovo feeding of nutrients is done via intraembryonic and extraembryonic routes. The intra-embryonic route involves direct inoculation of nutrients into embryonic tissue. On the other hand, in extraembryonic routes, nutrients are inoculated into different extraembryonic contents, viz. yolk sac, albumen, amnion, allantois and chorion and air cell, from where nutrients are absorbed by various mechanisms. According to the time of inoculation, it is defined as early and later stage from e12. The development of egg-laying birds after hatching is improved through feeding a subject before hatching. The subject should be fed with nutrients or an enteric modifier. The nutrient composition preferably should have a minimum of one amino acid, protein, peptide, or carbohydrate. A favourite enteric modulator is hydroxymethyl butyrate. Feeding may be done by giving the nutrients and/or enteric modulator in the amnion where these are orally consumed by the subject (Uni et al., 2003). Therefore, the nutrients can be better supplemented to the developing embryo via the amniotic sac at the later phase of growth, i.e. 18 days of incubation to give a nonstop supply of vital nutrients during the first few days (3- 4 days) of the after-hatch period, so facilitating enteric development and metabolism. This is distinct from other methods of in ovo nutrient administration, for which reason, it was patented with the United States patent US 6592878 B2 (2003) by Uni and Ferket (2003). The three crucial determinants for in ovo feeding include route of injection, the timing of injection and dose of nutrients (Ohta and Kidd, 2001). Presently 18 to 24 G needles or specialized Inovoject systems are used for injecting the nutrients. After the injection of the egg with the sterile needle under the controlled environment, the site of injection is closed with the paraffin film and the egg is placed into an incubator. The ideal time to administer nutrients in an in ovo feeding programme is 456 hours from fertilization that is roughly e 19 through the amniotic route (Kornasio et al., 2011).
Nutrients for in ovo feeding
The main purpose of in ovo feeding is to make the embryo able for faster post-hatch growth. Supplying the embryo with exogenous nutrients would allow the GIT to build up the structures and functionality to correctly digest and absorb nutrients instantaneously when an exogenous nutritional supplement is provided after hatch (Uni and Ferket, 2003). These exogenous nutrients, together with the yolk sac reserves, can contribute not only to maintaining the systems and metabolism already established but also to continuing growth, development, and proper nutritional condition (Noy and Skalan, 1998). Feeding of carbohydrates, proteins, and amino acids, vitamins or other modulators through in ovo injection have been evaluated (Bakyaraj et al., 2012; Araújo et al., 2019; Han et al., 2019; Xu et al., 2019). Carbohydrates commonly act as a glucose source and are essential for the process of hatching and hatchling development (Uni et al., 2003). With eggs inoculated with carbohydrates such as, maltose, sucrose and dextrose, it was observed that the extra source of energy improved the development of goblet cells and augmented the surface area of villi in the intestine. Al-Murrani (1982) first experimented with the amino acid supplement to the yolk sac at the 7 day of incubation. Outcomes showed that the embryo did not utilize the amino acids till the late-stage embryonic development to gain. Both Filho et al. (2018) and Xu et al. (2019) mentioned that the supplementation of amino acids incited the development of GIT and hatchability. Nutrients that can be used in ovo feeding include carbohydrates, all amino acids, fatty acids, vitamins, and other modulators.
In ovo feeding and gut development
Early access to feed does a key job in the development of the gut (GIT). The critical thing is that the digestive tract grows faster than any other body system in the earlier life stage of chicks. The method of in ovo feeding permits the deliverance of a range of supplements straight into chicken embryos; facilitate the early founding of a healthy gut microbiome prior to it is exposed to any pathogenic bacteria. In the final phase of incubation, nutrients, which are ingested with the amniotic fluid, arrive at the intestine of the embryo (Romanoff, 1960; Moran, 2007). Through this time, the intestine of the embryo experiences molecular, cellular and morphological changes, rapid proliferation and differentiation of enterocytes, enhanced capacity of absorption and nutrient uptake, and higher gene expression concerned in the process of digestion and absorption in the epithelial enterocytes (Geyra et al., 2001; Uni et al., 2003). mRNA of the digestive enzymes sucrase-isomaltase (SI) and aminopeptidase N (APN) is abundant in the intestine of chick embryo on e19 (Uni et al., 2003). Those of peptide transporter (PepT1), as well as sodium-glucose transporter (SGLT1), enhance significantly from e18 to e21 (Chen et al., 2005; Gilbert et al., 2007), and coding of genes for various transporters of amino acids demonstrate elevated levels of expression on e18 (Filho et al., 2018). Nutrients of in ovo feed supplied by in ovo feeding are then exposed to the tissues of the gut after the embryo orally ingests the amniotic contents before the emergence of air cells (Moran, 2007). This also makes the nutritional condition of the hatchlings better by means of speeding up the development of the gut for superior competence of digestion and nutrient absorption.
In ovo feeding and immune response
In ovo feeding may also stimulate immunocompetence during early stages by enhancing the protective function of enteric mucosa. Hatchlings are highly vulnerable to the colonization of gut pathogens because of the nominal competitive exclusion by symbiotic microflora, which colonizes the mucin layer of the mucosa of the gut. The primary defence against gut infection is the mucus gel layer of the intestinal epithelium. Kidd (2004) suggested that the nutrients (fat, amino acids or watersoluble vitamins and trace metals) use at a quantity higher than the requirements confers immune benefits to poultry. Providing natural nutrients by in ovo feeding maintains and hastens the development of the gut, giving the bird a perfect beginning point towards enhanced immune response to enteric antigens. Uni et al. (2003) noticed that in ovo feeding augmented surface area of villus at hatch and 3 day posthatch by approximately 27% and 21%, respectively. Besides, the fraction of goblet cells containing acidic mucin was enhanced by 50% over controls at 36 h after in ovo feeding, which was related to increase mucin mRNA expression. Therefore, in ovo feeding perhaps will be helpful for improving the colonization resistance of enteric pathogenic organisms of newborn and young chicks. Bhanja and Mandal (2005) reported that in ovo injection of vitamin E (0.25-0.50 IU) may be sufficient to augment post-hatch development as well as humoral immune response; while linoleic acid may enhance cellmediated immunity in broiler chickens and increases lipid in brain tissue as well as the status of antioxidants in hatched chicks. Researchers have definitely recognized that in ovo feeding is helpful for early immune response as well as growth. Greater enlightenment of the needs of main immune-modulatory nutrients will be helpful for the nutritionists to formulate in ovo feeds to improve immunity and the overall health of birds. The aptness of products for in ovo application in poultry nutrition and production necessitates further investigation.
In ovo stimulation and embryonic microbiome
The concept of in ovo stimulation has come from that of perinatal programming. The concept of perinatal programming was originated from human studies, which gave the hypothesis that a stimulus (or adverse factor) given to a foetus during the early stage of its development could lead to permanent phenotypic alteration (Calkins and Devaskar, 2011). The key notion of the in ovo stimulation is centered on stimulating the colonization of the embryonic gastrointestinal tract with its native microbiota that will help in establishing the optimal microbiota already existing during the incubation of the egg. It is shown that delivering 0.2 mL volume of dissolved bioactive stimulus right on day 12 of incubation of egg can kick off an entire flow of incidents on various phenotypic concentrations, from modulation of gene expression to growth performance. Scientists, K. Gulewicz and M. Bednarczyk, have developed and patented this procedure (Siwek et al., 2018). The majority of the microbes are used as probiotics in animal feeding and belonging to various species of bacteria like Lactobacillus, Bacillus, Bifidobacterium and Enterococcus, yeasts like Saccharomyces cerevisae and Saccharomycces bourlardii as well as fungi like Aspergillus oryzae and Candida pintolepsii can also be meant for the same rationale. In ovo stimulation is especially significant in the industrial production of chicken with a poor microbial profile leading to enteric microbial infestation and infectious disease outbreaks, which become even more customary after the removal of growth-promoting antibiotics from various global markets.
Advantages-
- Precise delivery of vaccines • Reduced labour cost • Reduced bird handling stress • Advanced development of intestinal mucosa • Increased body weights at hatch and better hatchability • Improves post hatch immune status • Increased breast muscle size at hatch and final body weight of broilers .
Future scope of in ovo technology
Though there are some constraints in effective use of in ovo technology like economical viability at large volume, higher maintenance cost, requirement of skilled manpower, chances of low hatchability due to any error etc., still there is lot of scope for its development as the technology has already established its usefulness till date. In future the chickens can be more benefitted through this technology by use of combinations of nutrients than alone. Even scope is there to incorporate nutrients in vaccine diluents for in ovo administration. Though the current machinery is costly and suitable for large scale production only, the compact, portable, low cost machine to fit the need for small scale operations can be developed for overall benefit of the poultry industry.
Future studies
There are still many limitations of in ovo feeding. To remove these limitations following problems are required to be solved through further studies: 1. Needs of appropriate nutritional products for in ovo administration 2. Needs of standardization of nutrients composition, doses of nutrients, concentration and viscosity of nutrient solutions, volumes of solutions, timing during pre-hatch period, routes, sites of injection, type of needles, sealing of holes, etc. to obtain different or all desirable post-hatch effects 3. Needs to remove doubt in a decrease in hatchability as a result of in ovo feeding of some nutrients
Although in ovo feeding method has produced many positive nutritional responses at the perinatal stage of chicks, such advantages, whether this method is sustainable for growth and production in poultry is still not well established. There are still lots of debates among scientists on the long-term benefits of in ovo feeding. It is obvious that there are inconsistent outcomes of research trials on in ovo feeding. So, this is necessary to develop a standard.
Compiled & Shared by- Team, LITD (Livestock Institute of Training & Development)
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