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Poult Sci 2006. 85:1513-1519
© 2006 Poultry Science Association
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ENVIRONMENT, WELL-BEING, AND BEHAVIOR

Effects of Formalin Feeding or Administering into the Crops of White Leghorn Cockerels on Hematological and Biochemical Parameters

A. Khan1, S. M. Hussain and M. Z. Khan

Department of Veterinary Pathology, University of Agriculture, Faisalabad-38040, Pakistan

1 Corresponding author: ahrar1122{at}yahoo.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Formalin administered (15 and 20 mL) into the crops of White Leghorn cockerels resulted in behavioral alterations, included depression, dullness, staggering, somnolence, anorexia, and decreased frequency of crowing. A significant decrease was observed in red blood cell counts, hemoglobin concentration, packed cell volume, and leukocyte counts in birds fed higher levels of formalin. Decreasing and increasing trends in concentrations of serum proteins and enzymes, respectively, were observed with increases in formalin doses. Significantly lower blood glucose and testosterone levels and significantly (P < 0.05) higher blood urea and creatinine concentrations were observed in birds fed higher formalin levels. No gross or microscopic lesions were observed in the birds fed different levels of formalin mixed in feed. These changes suggested that the formalin mixed in feed might have evaporated, and, hence, the amount ingested might have been too low to induce pathological changes. It was concluded from the present study that formalin mixed up to 5 mL/kg of feed, corresponding to 10 mL/ bird of 3% formalin in drinking water by crop tube had no adverse effects on the health of White Leghorn cockerels.

Key Words: White Leghorn cockerels • formalin • feed administration • crop administration • hematology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cereals, oil seeds, and other poultry-feed ingredients are exposed to the external environment during their processing and storage, and they are contaminated by a variety of ubiquitous microorganisms, including fungi, bacteria, and viruses. Most of these organisms are not pathogenic, but their increased number is a burden on the intestines. Occasionally, some pathogenic microorganisms contaminate poultry feed, or its ingredients, and may produce a variety of diseases in birds. Some Salmonella serotypes of zoonotic importance are especially hazardous in this respect (Jones et al., 1991). Different methods are exercised to decrease the population of these microorganisms in poultry feed, including heat treatment and the incorporation of different antimicrobial agents like formic acid (Izat et al., 1990), propionic acid (Hinton and Linton, 1988), and formalin (Brown, 1996).

Formalin is an aqueous solution (37%) of formaldehyde, which is an excellent antimicrobial agent, and its efficiency is not affected in the presence of organic matter. In the poultry industry, formaldehyde is used as a disinfectant for poultry houses (Dietz et al., 1980; Williams, 1980) and poultry litter (Veloso et al., 1974), and it is used for the fumigation of hatching eggs (Proudfoot and Stewart, 1970). Formaldehyde, a gaseous chemical, is released from formalin as fumes in hatchers to disinfect the environment and prevent the spread of infections (Peckham, 1980). Formalin is highly corrosive, has a pungent odor, and is a strong irritant to the mucous membranes. Due to these characteristics, incorporation of formalin in poultry feed can develop a toxic potential. It may become injurious to birds, particularly when improperly mixed or incorporated inadvertently at higher than recommended levels.

Peroral toxicity of formaldehyde has been reported in rats, mice, dogs (Johannsen et al., 1986; Restani and Galli, 1991), and calves (Preston et al., 1960). Formalin at the dose rate of < 10 mL/kg fed to broiler chicks decreased feed consumption and BW (Babar et al., 2001). Similar levels of formalin, when fed to Japanese quails (Coturnix coturnix japonica), decreased testicular weight and diameter of seminiferous tubules (Anwar et al., 2001). Moreover, decreased BW, egg production and weight, erythrocyte and leukocyte counts, hemoglobin concentrations, and hematocrits have also been reported at 10 and 20 mL of formalin/kg of feed in Japanese quail (Khan et al., 2005).

Being a volatile compound, formalin evaporates when mixed in feed; hence, the amount of formalin ingested by the birds is usually lower than that mixed in feed. For this reason, the dose-related toxic effects of different levels of formalin in birds could not be ascertained by mixing it in the feed in the previous studies (Khan et al., 2003). The present study was executed to determine the toxic effects of different levels of formalin in birds by mixing it in the feed and also by administering the corresponding amounts of formalin in crops using a crop tube. The results obtained from this study would be helpful in evaluation of the toxic effects of formalin in White Leghorn (WLH) cockerels and also variations in the toxic effects when formalin is administered in 2 different media (feed and water).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
This experiment was conducted considering all the national and institutional legislations regarding animal protection and welfare. Moreover, synopsis of the project was approved by the Advanced Studies and Research Board, University of Agriculture, Faisalabad, Pakistan.

Birds and Feed
A total of 120 WLH cockerels, aged 10 wk, having similar BW and apparently free from any clinical ailments, were procured from a local farm. All birds were kept in wire cages under ambient temperature (23 to 26°C). Fresh water was provided ad libitum. The commercial layer grower mash, having 16.5% CP, was offered in the measured quantity, and the feed left unconsumed was weighed and subtracted to calculate daily feed consumed by each bird.

Experimental Procedure
After 3 d of acclimatization, birds were randomly divided into 8 equal groups (A to H). Birds in groups A, B, and C were given formalin (37% formaldehyde) mixed homogeneously in the feed at dose rates of 2.5, 5, and 10 mL/kg, respectively, on a daily basis. The birds in groups D, E, F, and G were given a 3% aqueous solution of formalin at dose rates of 5, 10, 15, and 20 mL per bird per day by crop intubation. These levels in the drinking water corresponded to 2.5, 5, 7.5, and 10 mL of formalin per kg of feed. The amount of formalin given in crops through intubation in different groups was adjusted according to the variations in feed intake by the corresponding formalinfed group during the experiment. Group H was given distilled water and kept as the control group. The duration of the experiment was 8 wk.

Clinical Signs and Behavioral Changes
Birds in each group were monitored subjectively twice daily for clinical signs, such as dullness, depression, staggering, somnolence, and anorexia. Alertness was monitored by knocking at the doors of the cages. Attraction toward feed was noted at the time of feeding. Crowing was recorded by visual observation.

Hematological and Biochemical Studies
Blood samples were collected from 6 birds at random from each group on d 28 and 56 of the experiment for hematological parameters, including erythrocyte counts (Natt and Herrick, 1952), hemoglobin concentrations (Benjamin, 1978), hematocrit (Benjamin, 1978), and total leukocyte counts (Natt and Herrick, 1952).

Total serum proteins were determined following the Biuret method of Reinhold (Oser, 1976). Serum albumin was determined by the bromocresol green dye-binding technique (Varley et al., 1980). Globulin concentration was obtained by subtracting albumin from total serum proteins.

Glucose concentration was measured following the method described by Quam et al. (1975). The diacetyl monoxime method, as described by Wybenga et al. (1971), was followed to estimate the concentration of serum urea. Serum creatinine concentration was determined by the alkaline picrate method (Bonses and Tausskay, 1945). Aspartate transaminase and alanine transaminase activities were measured by a colorimetric method (Reitman and Frankel, 1957). Serum alkaline phosphatase (AP) activity was estimated according to the method of Kind and King (1954). Testosterone was determined by using a colorimetric kit (catalog no. MB 20049, Biocheck Inc. Foster City, CA), according to instructions of the manufacturer.

Wattle and Comb Area
At the end of experiment, all the birds were killed humanely according to institutional animal welfare and protection legislation. The area of wattle and comb was measured using a planimeter (Takeda, Osaka, Japan).

Statistical Analysis
Mean values (±SE) of each parameter for various groups were computed. To ascertain the magnitude of variation of each parameter among groups, the data were subjected to the ANOVA under a randomized design. Duncan’s multiple range test was applied for multiple mean comparisons, when necessary, using the Mstat computer statistical program (version 4.00/EM, Department of Crop and Soil Sciences, Michigan State Univ., East Lansing). The level of significance was P ≤0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical Signs and Behavioral Changes
Clinical signs (e.g., severe dullness, depression, staggering, somnolence, and anorexia) were observed in birds of groups F and G (Table 1Go). An interest in feed was shown by the birds given 2.5 and 5 mL of formalin/kg of feed or 5 or 10 mL of formalin/bird through intubation; however, birds in group C (10 mL of formalin/kg of feed) showed less interest in feed compared with the control group (H), whereas the birds in groups F and G showed a minimum interest in feed. Alertness was absent in birds of groups F and G, with no response to disturbance most of the time. The birds in the control and low-formalinfed groups started crowing in wk 1 and 2 of the experiment, whereas birds in groups F and G (high dose of formalin) started crowing in wk 4 of the experiment. The intensity and frequency of crowing was much less in birds of group F and G compared with other experimental groups (Table 1Go).


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  		Table 1. Clinical signs and behavioral changes in different experimental groups of White Leghorn cockerels fed different levels of formalin1,2
 
Feed Intake
In wk 1, feed intake was significantly lower in birds of groups F and G compared with the control (group H). In wk 2 and 3, groups E, F, and G showed significantly lower feed intake than group H (Table 2Go). In wk 4 and 5, groups C, D, E, F, and G had significantly lower values of feed intake than the control. In wk 6, groups E, F, and G consumed significantly less feed than group H. In wk 7 and 8, groups F and G also consumed significantly less feed than group H. The lowest feed intake was observed in group G, followed by group F, throughout the experiment (Table 2Go).


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  		Table 2. Feed intake (g/bird per d) of White Leghorn cockerels fed different levels of formalin1
 
Hematological Parameters
Erythrocyte counts in wk 4 decreased significantly in all groups except A, B, and D compared with the control. In wk 8, all the groups except A and D showed significantly lower red blood cell counts than group H. Hemoglobin and hematocrit levels in wk 4 and 8 were significantly lower in all groups compared with the control. Leukocyte counts in wk 4 were significantly lower in all groups except A, whereas in wk 8, all the groups had significantly lower values compared with group H (Table 3Go).


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  		Table 3. Effect of formalin feeding through feed and intubation on various hematological values in White Leghorn cockerels1
 
Biochemical Parameters
Serum total proteins in wk 4 were significantly lower in birds of groups F and G, whereas in wk 8, only group G differed significantly from the control. Serum albumin levels of all the groups were nonsignificantly different from the control in wk 4 and 8. Serum globulin levels were significantly lower in groups F and G on wk 4, but they became nonsignificant in all groups on wk 8 compared with group H (Table 4Go).


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  		Table 4. Effect of formalin feeding through feed and intubation on certain biochemical parameters in White Leghorn cockerels1
 
Serum glucose levels in groups F and G were significantly lower in wk 4, whereas in wk 8, only group G showed significantly lower values than the control. Urea levels were significantly higher in groups F and G in wk 4, whereas in week 8, urea levels of groups C, F, and G became significantly higher than the control group. Creatinine levels in wk 4 were similar among all the groups, whereas in wk 8, it became significantly higher in groups C, E, F, and G compared with group H (Table 4Go).

Serum Enzymes
Serum alanine aminotransferase (ALT) levels in wk 4 and 8 significantly increased in all groups compared with the control. The maximum value was recorded in group G. Serum aspartate aminotransferase (AST) levels in wk 4 decreased significantly in group G as compared with all other groups except group E. At 8 wk, AST levels in all of the groups except group A had significantly lower values than group H. Alkaline phosphatase in wk 4 and 8 decreased significantly in all of the groups compared with the control (Table 5Go).


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  		Table 5. Effect of formalin feeding through feed and intubation on various enzymes (IU/L) in White Leghorn cockerels1
 
Wattle and Comb Area and Testosterone Concentration
Wattle area in groups A and B was significantly higher, whereas all other groups differed nonsignificantly from the control group. Comb area in group G was significantly lower than that of group H. Testosterone levels in wk 4 and 8 were nonsignificantly different in groups A and D, whereas all other groups had significantly lower values than the control. The lowest values were in group G, followed by F (Table 6Go).


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  		Table 6. Effect of formalin feeding through feed and intubation on wattle area along with serum testosterone levels in White Leghorn cockerels1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Formalin is a toxic substance and is an irritant to mucous membranes. It is corrosive and carcinogenic (Til et al., 1988; Kamata et al., 1997). Due to these characteristics, the incorporation of formalin in poultry feed may be detrimental to chicks. In the present study, the presence of clinical signs of dullness, depression, staggering, somnolence, and anorexia in birds of groups C, F, and G was indicative of dose-related harmful effects of formalin in WLH cockerels. Similar dose-related effects of formalin have been reported in broiler chicks (Babar et al., 2001) and male Japanese quail (Anwar et al., 2001). The level of dietary formalin (10 mL/kg of feed) used in these studies corresponds to the 20 mL/bird (3% aqueous solution) given directly by crop tube in the present study. The presence of clinical signs in birds given formalin (3% aqueous solution) at a level of 15 mL/bird by crop tube suggested that the corresponding formalin level of 7.5 mL/kg of feed might also have deleterious effects on birds. Peckham (1980) and Saunder (1996) reported harmful effects of formaldehyde vapors in chicks, such as distress, gasping, and photophobia. Bhatt and Panchal (1992) described decreased responsiveness in rats following oral administration of formalin in drinking water. A similar observation of decreased responsiveness in birds following oral administration of formalin was made in the present study.

A significant decrease in red blood cell counts, hemoglobin concentration, and packed cell volume in birds of groups given formalin at doses of 15 and 20 mL/bird by crop tube (groups F and G) and 10 mL/kg of feed (group C) is suggestive of deleterious effects of formalin on hematological parameters. A decreasing trend in erythrogram in birds given higher levels of formalin suggests that formalin might have an inhibitory effect on the synthesis of these cells in bone marrow. Tobe et al. (1989) reported similar depressant effects of high doses of formalin on different blood parameters in rats. However, those authors regarded the changes as clinically unimportant, because they were not accompanied by histopathological changes.

A decrease in serum proteins observed in birds fed different levels of formalin with an increase in formalin level (Table 3Go) indicates that formalin might have suppressed the synthesis of plasma proteins. Til et al. (1988) also observed a decrease in serum proteins in rats given formalin in drinking water at levels of 5, 25, and 125 mg/ kg of BW. Tobe et al. (1989) also reported a decrease in serum proteins in rats given formalin in drinking water at doses of 0.02, 0.1, and 0.5%. Deniz et al. (1993) reported a decrease in serum proteins in cattle fed a diet containing soybean meal treated with formaldehyde at 0.1, 0.6, 0.9, and 1.2 g/100 g of CP for 120 d.

Serum proteins are mainly synthesized in the liver, and a decrease in their level is indicative of a disturbance in the function of this organ (Benjamin, 1978). A decrease in serum proteins in WLH cockerels fed higher levels of formalin might have occurred due to its hepatotoxic effect (Beall and Ulsamer, 1984). Decreased glucose concentration in high formalin dose groups could also be related to the hepatotoxic effects of formalin that might have an inhibitory effect on glucogenesis in the liver.

Significantly higher levels of blood urea and serum creatinine observed in birds given higher levels of formalin (Table 4Go) could be ascribed to decreased fluid intake and urine production and, consequently, the retention of urea and creatinine in the blood, ultimately resulting in increased concentration of these parameters in the blood. Thus, an elevated concentration of urea and creatinine in the blood could be associated with impaired renal function at high formalin doses. Til et al. (1988, 1989) and Tobe et al. (1989) reported that rats given higher levels of formaldehyde in drinking water showed increased levels of urea and creatinine in the blood. Another study conducted in Friesian heifers given casein or soybean meal treated with formalin revealed an increased plasma concentration of urea (Oddham et al., 1982).

A significant dose-dependent increase in concentrations of ALT, AST, and AP, as compared with the control in the present study, was indicative of liver damage of variable degrees. The ALT is a cytoplasmic enzyme, and AST is a cytoplasmic and mitochondrial enzyme in hepatic cells, whereas AP is also synthesized by the liver. An increased activity of these enzymes is a very sensitive index of hepatic damage (Benjamin, 1978). Irrespective of the magnitude of alteration in serum enzymes, the changes reflect adverse effects of formalin on the function of hepatic cells. Murphy et al. (1964) reported that livers of rats exposed to formaldehyde inhalation had significantly greater AP activity than the control. Contrary to these observations, Til et al. (1988, 1989) and Tobe et al. (1989) reported a decrease in serum activities of enzymes including AP, AST, and ALT in rats given formalin in drinking water at higher doses of 1.8, 21, and 109 mg/kg of BW.

A significant difference in comb and wattle surface areas of the birds in various experimental groups fed different levels of formalin suggests that formalin affects the development of these organs. A significantly lower serum concentration of testosterone observed in birds given 3% formalin at a dose rate of 20 mL/bird by crop tube suggests that formalin at this dose level has deleterious effects on testes development and, hence, testosterone synthesis. No depressant effect of a corresponding level of formalin (10 mL/kg of feed) given to birds in feed may be because of the evaporation of formalin from the feed before it was ingested by the birds. At higher formalin doses, testes development becomes depressed, which could be a direct effect of reduced feed intake and BW (Khan et al., 2003).

In conclusion, the present study revealed that formalin mixed in feed at levels of 2.5, 5, and 10 mL/kg of feed has less deleterious effects compared with high levels of formalin given by crop intubation. However, the presence of clinical signs and adverse effects on different hemato-logical and serum biochemical parameters in birds administered formalin (3%) at levels of 15 and 20 mL/bird by crop tube and the absence of such deleterious effects in birds given corresponding formalin levels mixed in feed suggests that formalin, being a volatile substance, might have evaporated from the feed before being ingested by the birds.

Received for publication August 9, 2005. Accepted for publication April 11, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anwar, M. I., M. Z. Khan, G. Muhammad, A. Bachaya, and A. M. Babar. 2001. Effects of dietary formalin on the health and testicular pathology of male Japanese quails (Coturnix coturnix japonica). Vet. Hum. Toxicol. 43:330–333.[ISI][Medline]

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Beall, J. R., and A. G. Ulsamer. 1984. Formaldehyde and hepatotoxicity: A review. J. Toxicol. Environ. Health 13:1–21.[ISI][Medline]

Benjamin, M. M. 1978. Outline of Veterinary Clinical Pathology.3rd ed. The Iowa State Univ. Press, Ames.

Bhatt, H. V., and G. M. Panchal. 1992. Behavioural changes in rats due to chronic oral and systemic formaldehyde. Indian J. Physiol. Pharmacol. 36:270–272.[Medline]

Bonses, R. W., and H. H. Tausskay. 1945. The colorimetric determination of creatinine by the Jaffe reaction. J. Biol. Chem. 158:581–591.[Free Full Text]

Brown, R. H. 1996. FDA approved use of formaldehyde in poultry feed. Feedstuffs 15:40.

Deniz, S., B. Coskun, F. Inal, E. Seker, and K. Isik. 1993. The effect of formaldehyde treated soyabean meal on weight gain, feed efficiency and some blood and rumen fluid metabolites in calves. Hayvancilik Arastirma Dergisi 3:8–11.

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