Abstract: Background and objective: Kashmir is a part of Jammu and Kashmir State of India where a large chunk of day old chicks are procured from outside the state and these chicks are transported across a distance of hundreds of kilometers over a period of several days. The long distance transport without any access to feed not only subjects the birds to early life stress but also affects their gut morphological development. Therefore, a study was conducted to evaluate the effect of delayed feeding on stress levels, mortality, intestinal morphometry and histomorphology of chabro broiler chicken. Materials and Methods: A total of 400 day old chabro chicks were randomly divided into 5 groups, each group comprising of four replicates of 20 birds. Chicks allotted to group-1 (G1) were offered feed at hatchery itself whereas feeding in groups G2, G3, G4 and G5 were initiated at the farm after the delay of 12, 24, 48 and 72 h, respectively. Results: The results revealed that the heterophil count showed a steady increase from G1-G5 and significantly (p<0.05) higher count was found in G3, G4 and G5 compared to G1. However, the lymphocyte count showed a steady decrease as delay in feeding increased. H:L ratio followed a regular increase from G1-G5 and was significantly (p<0.05) higher in G3, G4 and G5 compared to G1. An overall mortality of 13% was recorded during the trial and all of it occurred during first 2 weeks. The highest overall mortality of 22.50±3.23% was recorded in group G5 followed by 17.50±6.61% in G4. The villus height of duodenum and jejunum was significantly (p<0.05) higher in G2, G3, G4 and G5 as compared to G1. Crypt depth and muscular is thickness of duodenum was significantly (p>0.05) lower in G4 and G5 compared to G1. Conclusion: The duration of post hatch feeding delay gradually increased the stress level and mortality of birds and also decreased the length of different segments of small intestine in birds. No adverse effect on histomorphology was observed at the end of trial. Feeding at hatchery itself or feeding during transportation of birds would be a viable strategy to overcome the negative effects of delayed feeding in chicken.
INTRODUCTION
In commercial operations, chicks hatch over a period of time and are only taken out of the incubator once the maximum number of the birds have hatched out1. Most of the birds may have to even wait up to 2 days in the hatchery and also in the transit to the farms before they are given access to food and water2. Sexing, vaccinations and transportation of chicks further increases their delay in access to feed3. Moreover, there is an inevitable spread of 30-48 h in a hatch for late versus early hatchers4. This delay in initial access to feed and water can have lasting negative effects on birds5 as the magnitude of growth a broiler chicken attains indicates that every day in the life of a broiler chicken is very important6. In order to maximize the genetic potential and profitability of fast growing broiler chicken, post hatch feeding delay access must be minimized as possible7. The detrimental effects of delayed feeding in neonatal chicks could be overcome by reducing the transport time to the poultry farm, so as to provide them early access to feed8. The negative effects include reduction in growth and increase in early mortality, primarily due to dehydration and shortage of available energy9. Further, it causes immunosuppression and retarded gastrointestinal development in birds10.
The time period from the hatch of first chick to the hatch of the last chick (known as hatch window) may range from 24-48 h or more11. Several factors have been reported to influence hatch window viz., age of breeder flock, egg characteristics, sex of the embryo and length and temperature of egg storage before incubation. It has been reported that chicks from old aged breeder hens hatch earlier than those from young breeder hens8. Likewise, Ulmer-Franco et al.12 suggested that chicks from smaller or lighter eggs may hatch earlier than chicks from larger or heavier eggs. Moreover, female chicks have been reported to hatch earlier than male chicks13. Early hatching of chicks from eggs stored for short periods of time compared to eggs stored for a week or more has been documented14. Similarly, storage temperature affect hatch window for eggs stored for longer than 3 days15.
Kashmir is a part of Jammu and Kashmir State of India. Out of 140 lac broiler chicken raised in Kashmir valley annually16, a large chunk of day old chicks are procured from outside the state and these chicks are transported across a distance of hundreds of kilometers over a period of several days. The long distance transport without any access to feed not only subjects the birds to early life stress but also affects their gut morphological development. In view of the negative impacts of post-hatch delay in feeding, a study was conducted to evaluate its effect on stress levels, mortality, intestinal morphometry and histomorphology of chabro broiler chicken.
MATERIALS AND METHODS
Experimental site: The study was conducted in the Teaching and Research Farm of Center for Research on Poultry, Division of Livestock Production and Management, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shuhama.
Birds and experimental design: The experiment was carried out on relatively slow growing meat type chabro chicks procured from a local hatchery. Chabro is a broiler bird with colored plumage developed by Central Poultry Development Organisation, Northern Region. It attains a body weight of 1400-1500 g at 7 weeks of age with FCR17 of 2.48. A total of 400 day old chabro chicks were procured. The chicks were randomly divided into 5 groups, each group comprising of four replicates of 20 birds. Chicks allotted to group-1 (G1) were offered feed at hatchery itself whereas feeding in groups G2, G3, G4 and G5 were initiated at the farm after the delay of 12, 24, 48 and 72 h, respectively. The chick boxes were transported from the hatchery to the poultry farm where the chicks were brooded and reared up to 6 weeks of age in deep litter system using saw dust as litter material. The chicks were housed replicate wise in separate pens providing a floor space of one square feet per chick. Feed (commercially available pre-starter and starter mash) and water was offered ad libitum. Standard management and healthcare (vaccination) protocol was followed.
Parameters recorded
Stress levels and mortality: Stress levels was estimated on the basis of heterophil:lymphocyte (H:L) ratio. A drop of blood was collected randomly from 10 birds from each treatment group at the time of initiation of feeding and a smear was formed on glass slide. The smear was air dried and fixed with methanol. Later it was stained using Wright Giemsa stain and subjected to Differential Leukocyte Count (DLC) from which H:L ratio was worked out. Individual pens were inspected daily and mortality if any was recorded. Moreover, individual pens were inspected daily and mortality if any was recorded.
Intestinal morphometry and histomorphology: The intestines of the slaughtered birds were collected and duodenum, jejunum and ileum lengths were measured (cm) using measuring scale. Tissue samples from these parts were preserved and subjected to histomorphology. The intestinal samples were processed by paraffin embedding technique for histo-morphology. The formalin fixed intestinal samples were handled for processing in capsules labelled with identification number and processed by routine technique. Briefly, the samples were washed under running tap water for 6-8 h, dehydrated in acetone, three changes of 20 min each, followed by clearing in benzene (100%), two changes of 20 min each. Paraffin impregnation was achieved by giving one change in benzene-paraffin (1:1) [melting point of paraffin 54°C] for 20 min followed by three changes of 1 h each in paraffin wax maintained at 56°C in oven. Casting of blocks was carried out using 2 L shaped moulds which facilitates manipulation of size as per the requirement. The tissue was placed in the blocks so as to facilitate sectioning across all layers. The blocks were labeled alongside for identification. The paraffin blocks were cut using rotary microtome. Approximately 60 sections of 5-6 μm thickness were cut per sample and every fifth section was retained for staining. Twelve sections per samples were stained with Harris’ haematoxylin and eosin method18. The slides were observed under the microscope equipped with the camera for photomicrography. The microscopic images recorded were analysed using video test-5 image analyzing software calibrated for the purpose.
Ethics statement: All procedures performed in this study involving birds were approved by Institutional Animal Ethics Committee.
Statistical analysis: The data obtained was statistically assessed by one-way ANOVA as per the standard methods of Snedecor and Cochran19 using the general linear model procedure of Statistical Package for the Social Sciences, Base 10.0, 1999 (SPSS Software products, Marketing Department, SPSS Inc. Chicago, USA). To test the significance of difference between means Duncan’s multiple range test20 was used and differences were considered significant at 5% level.
RESULTS
Stress level of chabro birds: Stress levels in terms of heterophil:lymphocyte (H:L) ratio is given in Table 1 and Fig. 1. The heterophil count showed a steady increase from G1-G5 and significantly (p<0.05) higher heterophils were found in G3, G4 and G5 compared to G1.
| Table 1: | Effect of delayed feeding on heterophil, lymhocyte count and ratio in chabro chicken |
| Means across the columns in a particular row bearing similar superscript did not differ significantly (p<0.05) | |
| Fig. 1(a-d): | Heterophils and lymphocytes in blood smears of chabro chicken [Wright Giemsa X1000 (OZx4)] |
| Table 2: | Effect of delayed feeding on mortality pattern of chabro chicken |
| Means across the columns in a particular row bearing similar superscript did not differ significantly (p<0.05) | |
| Table 3: | Effect of delayed feeding on intestinal morphometry (cm) of chabro chicken |
| Means across the columns in a particular row bearing similar superscript did not differ significantly (p<0.05) | |
| Table 4: | Effect of delayed feeding on villus height (μm) of chabro chicken |
| Means across the columns in a particular row bearing similar superscript did not differ significantly (p<0.05) | |
| Table 5: | Effect of delayed feeding on crypt depth (μm) of chabro chicken |
| Means across the columns in a particular row bearing similar superscript did not differ significantly (p<0.05) | |
| Table 6: | Effect of delayed feeding on muscularis thickness (μm) of chabro chicken |
| Means across the columns in a particular row bearing similar superscript did not differ significantly (p<0.05) | |
However, on the other hand the lymphocyte count showed a steady decrease as delay in feeding increased. The H:L ratio was found to be 0.39±0.02, 0.41±0.03, 0.71±0.04, 0.83±0.04 and 1.11±0.06 in groups G1, G2, G3, G4 and G5, respectively. It followed a regular increase from G1-G5 and was significantly (p<0.05) higher in G3, G4 and G5 compared to G1.
Effect on mortality: The results of mortality in various chabro birds subjected to different durations of post-delay hatching are shown in Table 2. An overall mortality of 13% was recorded during the trial and all of it occurred during first 2 weeks. A mortality of 7.75% in 1st week and 5.5% in 2nd week was recorded. The highest overall mortality of 22.50±3.23% was recorded in group G5 followed by 17.50±6.61% in G4. In all the groups mortality whatsoever occurred in 1st 2 weeks only and no mortality was recorded in any of the groups from 3 weeks onwards. Between the groups significantly higher mortality was recorded during the 1st week in group G5 (16.25±3.15%) followed by G4 (11.25±6.25%) and least in G1 (2.50±1.44%).
Intestinal morphometry and histomorphology of birds: Table 3 depicts the morphometric results of different parts of small intestine. Duodenum and jejunum length showed a steady decrease from G1-G5. Although G2 and G3 showed no significant difference, the groups G4 and G5 were having significantly lower duodenal and jejunal length respectively as compared to G1. Similarly, length of ileum also followed a steady decreasing trend from G1-G5. Though G2 did not show any significant difference from G1, however, G3, G4 and G5 had significantly (p<0.05) lower length of ileum as compared to G1.
The effect of delayed feeding on histomorphological changes in different intestinal segments of chabro birds is presented in Table 4-6 and Fig. 2.
| Fig. 2(a-d): | Small intestinal histomorphology of chabro chicken, (a) Treatment fed soon after hatch was pulled out, (b) 24 h delay in feeding, (c) 48 h delay in feeding and (d) 72 h delay in feeding {H.EX40 (OZx3)} |
The villus height of duodenum and jejunum was significantly (p<0.05) higher in G2, G3, G4 and G5 as compared to G1. However, the villus height of ileum did not show any significant (p>0.05) difference among various groups. Crypt depth and muscularis thickness of duodenum was significantly (p>0.05) lower in G4 and G5 compared to G1, however, the jujenal and ileal crypt depths and muscularis thickness showed no significant (p>0.05) difference among various treatment groups.
DISCUSSION
The heterophil lymphocyte ratio has been reported to be good indicator of different types of stress including the feed deprivation21. Increase in the H:L ratio in the present study as a result of progressive increase in the delay of feeding is suggestive of increased stress levels in the birds. Cengiz et al.22 also found that delayed feed access significantly increased the H:L ratio at day 6. Chicks that had been deprived of feed for 48 h post-hatch had less lymphocyte synthesis after 72 h, lower bursa weight at 21 days of age, retarded lymphoid development at 21 days of age and lowered disease resistance10. Feed deprivation during first 12 h reportedly has no effect on immune response of layer chicken23. Bursa of Fabricius (Bursa) is an immune organ unique to avian species that produces antibodies in response to pathogen invasion24. Furthermore, 48 h of post-hatch delayed feeding lowers the immune capacity of broilers up to 42 days of age by way of reduced humoral and cellular immune capacity5.
The present study revealed that delayed feeding resulted in higher mortality and it was significantly higher in groups with increased extent of delay in feeding. Delayed access to water and feed post-hatch dehydrates the chicks25 resulting in depressed immune response26, increased early mortality27. Very high mortality to the extent of 60% was also reported by Mbajiorgu et al.28 in the broiler chicken subjected to feeding delay of 36 h between 1-3 days of age. The severe shortage of energy in the birds subjected to feed and water deprivation for 48 h causing alterations in body composition has been suggested as a reason for high mortality in turkey poults29.
All the segments appeared to be shorter in the groups subjected to delayed feeding as compared to control and more the duration of delay shorter was the intestinal length. It has been demonstrated that the intake of exogenous feed can stimulate the growth and development of gastrointestinal tract in newly hatched chicks. It has been reported that when chicks were subjected to delayed feeding of 24-72 h, GIT growth was stunted30. Gonzales et al.31 also reported that the delayed feeding delays GIT development. Maiorka et al.32 also suggested the need to feed chicks immediately after hatch to ensure proper development of gastrointestinal tract33. Similarly, birds that had access to feed immediately after hatch have been reported to exhibit more rapid development of the intestine during immediate post-hatch period34. Other studies in turkey poults have also indicated that early access to feed is critical for the development of intestinal tract35.
In the present study, the histomorphology of different segments of small intestine did not reveal any harmful effect of delayed feeding on villus height, crypt depth or muscularis thickness except for some changes in the duodenum but those too did not follow any definitive trend. This is in contrast to the results of Maiorka et al.32 who reported that feed deprivation of chicks depressed the intestinal villi, enterocyte proliferation and development of mucosal layer. Ganjali et al.8 reported that villus height of duodenum and jejunum at 0, 2, 4, 8 and 12 days of age in broiler chicken decreased significantly. Similarly, Shinde et al.23 reported that delay in feeding resulted in reduced duodenal, jejunal and ileal villus height in layer chicken at 36 h and 7 days post hatch. However, no such results were found in the present study which might be due to the fact that the histomorphological study was performed on 6 week old chicks and it appears that the difference if any at histomorphological level might have got masked with age. To our surprise, the villus height of duodenum and jejunum increased in 6 week old birds in all the groups subjected to delayed feeding, thus further studies in this regard are warranted.
CONCLUSION
It could therefore be concluded that with increase in the duration of post hatch feeding delay, the stress levels increased gradually and so did the mortality. A progressive decrease in the length of different segments of small intestine was noticed with increase in the duration of post hatch feeding delay. However, no adverse effect on histomorphology of small intestine was observed at the end of 6 week trial. In view of negative effects of delayed feeding on broiler chicken, early feeding strategies which may include hatchery feeding or feeding during transportation of birds need to be developed.
ACKNOWLEDGMENT
Financial support from B.V RaoPoultry Research Foundation Pune to carry out this piece of work is thankfully acknowledged.