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Screening Sanitizing Agents and Methods of Application for Hatching Eggs III. Effect of Concentration and Exposure Time on Embryo Viability
01993Applied Poultly Science, Inc
TOM A. SCOTT' Agriculture Canada Research Station, Agassiz, British Colicmbia, VOM U O , Canada Phom atid FAX: (604) 796-2221 CHRISTINA SWETNAM and ROGER KINSMAN Agriculture Canada Research Station, Keiitville, Nova Scotia, B4N lJ.5, Caiiada ~
~
Primary Audience: Hatcheries, Veterinarians
1 T o whom
correspondence should be addressed
Downloaded from http://japr.oxfordjournals.org/ at United Arab Emirates University on June 18, 2015
SCREENING SANITIZING AGENTS AND METHODS OF APPLICATION FOR HATCHING EGGS 111. EFFECT OF CONCENTRATION AND EXPOSURE TIMEON EMBRYO VIABILITY
Research Report SCOTTet al.
13
the access of bacteria to the interior of the egg. DESCRIPTION OF PROBLEM One part of the defense of the shell is the cuticle, or proteineous coat, applied at time of lay, also referred to as the bloom. Handling and chemical exposure may alter the cuticle and result i n both negative and positive changes in porosity of the egg shell [7,10, 11, 12, 13, 14, 1.51. Vick and Brake [16] observed that removing the cuticle (and increasing moisture loss during incubation) improved hatchability of eggs from young, but not old, broiler breeder hens. There are two ways a chemical (sanitizer) may interact with the egg shell and reduce permeability: alteration of the proteineous cuticle, or alteration of the calcium carbonate matrix of the shell itself. Brake and Sheldon [2] found that solutions of quaternary ammonium interacted with the cuticle, increasing pernieability and resulting in an increase in hatchability of eggs laid by young, but not old, broiler breeder hens. Sheldon and Brake [ 171observed high microorganism destruction when 0.5% hydrogen peroxide was applied directly to plated cultures. However, concentrations of 5% (tenfold) were r e q u i r e d when t h e s a m e microorganisms where present on the egg shell. With a concentration of 5% hydrogen peroxide, Sheldon and Brake [17] demonstrated a 2% increase in hatchability compared to eggs treated with formaldehyde. These hatchability claims were made on relatively small numbers of eggs. Scott and Swetnam [3] have grave concerns about the use of hydrogen peroxide based on the MSDS (Material Safety Data Sheets) guidelines. Patterson et al. [l]observed that dipping, compared to foaming, hatching eggs in a chlorine dioxide solution resulted in decreased hatchability of heavily soiled duck eggs. Scott and Swetnam [4] observed that chlorine dioxide (Sanimist) was quickly neutralized by components of the egg shell and was inerfective in reducing microorganisms on the egg shell. Ozone gas was used to reduce microorganism loads of hatching eggs [18]. However, at the levels tested (3% ozone by weight for 2 hours), embryo toxicity occurred. In theory, ozone (having a molecular weight similar to oxygen and carbon dioxide) could transfer across the shell wall and destroy microorganisms which had penetrated the shell barrier. Unfortunately, this transfer across the shell
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Pattersonetal. [I], Brake and Sheldon [ 2 ] ) and Scott and Swetnam [3,4] provide a thorough review of the hazards involved in the use of formaldehyde in the work place. However, both Furuta and Maruyama [5] and Scott and Swetnam [3,4] were concerned about finding a sanitizer as effective against microorganisms as formaldehyde, and yet user and environmentally safe. There are many sanitizers available on the market; some have recommendations and claims for hatching egg sanitation, but others do not. Some of these compounds are as noxious and dangerous to use as formaldehyde [3], while still others are ineffective against bacteria when applied to the egg shell [4]. Cox et ai. [6] indicated that commercial hatcheries are switching from one chemical sanitizer or application method to another with little appreciable effect on Saltnonella contamination. Furthermore, Bruce and Drysdale [7] were not convinced that microorganism contamination of hatching eggs had any direct effect on hatchability. Therefore, it is imperative that the merits of a hatchery sanitizer or application procedure be reviewed closely. In the literature there are a number of reports which only include small numbers of eggs. This is a concern when reporting hatchability data, as Tullet [SI pointed out. He estimated that 10,000 eggs per treatment were required to demonstrate a 1% dilference in hatchability between treatments. There are several reports which support the idea that a dirty environinent and subsequently a dirty egg will decrease hatchability. Bruce and Drysdale [7] and Cox et ai. [6] implicated management as a source of poor hygiene. They cited the superior performance of "modell' grandparent compared to the lower performance of multiplier flocks. Tullct [8] compared floor ("dirty") eggs to nest run eggs and observed a 10 to 15% decrease in hatchability. Factors other than bacterial contaniination of these floor eggs were also indicated. Mowry et al. [SI observed that 50 day-old broiler chick performance was iniproved by sanitizing eggs prior to incubation. Several studies have measured a change in shell porosity and subsequent air exchange ( 0 2 in, C02 and HzO out) of the developing embryo. Porosity of a shell must also restrict
EMBRYO TXICOLOGY
14
twice. Ozone gas was applied at three concentrations for 3 hours. EGG DIPPING TREATMENTS Plastic 100 1 barrels were partly filled with 30 1 of tap water and allowed to equilibrate to room temperature over night. The following morning, the sanitizers to be tested were added and mixed thoroughly. Six flats of 30 eggs were submerged in the solutions for the required time (Table 1). The procedure was repeated until all eggs were treated. The eggs were allowed to drip dry and then placed in storage (18°C; 65%RH) for 72 hours before setting randomly in Robbins (Hatch-o-matic) incubators. OZONE TREATMENTS Hatching eggs were exposed to one of three concentrations of ozonegas (3.2,1.6, and 0.5p1) for a period of three hours at 10°C. As described previously for randomizing eggs, three flats of 30 eggs were placed into one of four 64 1 polyethylene containers. Ozone was produced by a Corona Discharge Ozone Generator (Triox Trucker model, Triox Co., Swindon, U.K.) and delivered to the containers through a three port manifold and 3.175 nun teflon tubing. Ozone concentration was monitored with a portable semiconductor thin film ozone detector (Ebra-Jitsugyo Co., Kawasaki, Japan) with a range of 0-5 p l . The sensor was placed through a hole in the top of one end of each chamber to the level of the eggs.
MATERIALS AND METHODS The physical screening and selection of a sanitizer was dependent on when the compound became available to the laboratory. Each sanitizer was tested at two different times over a six month period. To minimize variation in handling, environment, and equipment, a control group of eggs treated with 1% formalin was used in all comparisons. Hatching eggs were obtained from a commercial hatchery (ABS Hatchery of ACA Cooperatives, New Minas, Nova Scotia) on a weekly basis. All eggs during any given week were from a common flock of broiler breeder hens and were stored for less than 45 hours before being treated. On receipt, clean and sound eggs were selected and randomized into lots of 90 eggs per replicate. Four replications (360 eggs) for each of 12 treatment groups were tested to yield a total of 4320 cggdweek. In each comparison the control eggs were treated with 1%formalin for 1, 5, or 10 minutes, with four replications of 90 eggs per exposure time. The remaining nine treatments were divided into either 2, 3, or 4 concentration and/or exposure levels of three test sanitizers (Table 1). During a six month interval, assessment of concentration and/or exposure levels of each of the 23 sanitizers were tested
EGG HANDLING AND INCUBATION On day of setting, the pretreated eggs were removed from storage, placed on setting racks and weighed; a replicate was comprised of one tray of 90 eggs. The trays of eggs were identified and assigned randomly a position in the incubator. The eggs were allowed to equilibrate at room temperature for six hours before the incubators were switched on. Incubation temperatures were 37"C+OS"C and relative humidity was SO%. Eggs were turned hourly. At day 7, all eggs were candled by hand, The trays of eggs were removed, weighed, candled, and remaining viable embryos weighed and placcd back into the incubator. Eggs removed were broken out and visually classified as infertile, live, dead before 2 days (mem-
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wall also puts ozone into direct contact with the embryo. Ozone may be a serious health hazards for those working with it [3]. The level of ozone used in the study by Whistler and Sheldon [ 181 was extremely high, far exceeding amounts which have been recommended for food products [19]. This high level of exposure may explain the high embryo mortality which was reported in their study [IS]. Mauldin and Wilson [20] reviewed the basic considerations for a successful hatchery sanitation program. Good sanitation practices must begin at the point of lay and continue through the placement of chicks. Part of this sanitation practice often includes treatment of eggs prior to and during incubation. In the present study, 23 sanitizers wcre applied to hatching eggs. Gross embryo mortality was monitored. Sanitizers that demonstrate user and environmental safety, effectiveness against microorganism on the eggshell, and n o gross toxicity to the embryo, will be further tested in large scale hatchability studies.
Research Report 15
SCOTTet al.
PARAMETERS MEASURED The moisture loss from setting to transfer (18 days) for each lot of 90 eggs was recorded as a percentage of initial weight. Following classification of all broken out eggs at candling, and after the hatch was taken off, the number of healthy viable chicks from fertile eggs were used to determine percent hatchability.
STATISTICAL ANALYSIS The data within each hatch was expressed relative (as a percentage of) to formaldehyde control to minimize differences between hatches. The statistical model assessed the main effects of sanitizer and concentration and/or exposure time of each sanitizer. Significant differences between mean values were separated by Duncan's Multiple Range Test [21].
RESULTS AND DISCUSSION To simplify the presentation of data, only the relative response (formalin treated eggs equal 100%) in moisture loss (0 to 18 days incubation) and hatchability of fertile eggs are presented (Table 1). The actual values for eggs treated with formaldehyde are 10.1% moisture loss from 0 to 18 days incubation, and 81.5% hatchability of fertile eggs set. The effect of exposure time (I, 5, or 10 minutes) to 1% formalin on moisture loss or hatchability was not significant. Overall, fertility of eggs (greater than 60,000) set in this study was 92.6+4.0%.
TABLE 1. The relative (to formaldehyde control) loss in moisture (0 to 18 day incubation) and response of sanitizer treatments hatchability of fertile eggs and dose (Tl, T2, or T34l/)
0.70
1.40
1.90
102.6
ia
+
++
+
-
-
-
100.3
i abcd
+
+
+
+ + 0 . 8 0 1.60 3 . 1 0 97.9 j abc 100.0 i abcd lo-ldehyd. w .. equal t o . 1 formalin and eggs dipped for 1, 5 or 10 min. I'clutaraldehyde was applied full strength for 1 or 10 nin. I oron. qaa cor c:antratLon. -re held for 180 mi". h11 other treatment# involved eggs dipped in SOlUtiOn for 10 min. - or + Indicate a L.S ID. value of I 5 . 0 units in relative moimture lose or hatchability reaponse from the control (1\ indicate- a reeponse of between B 5 to 90% of the control. d Icmte. a value of between 100 and lost,
'
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brane, but no blood development), dead between 2 and 4 days (blood ring development and no visible embryo), or dead between 4 and 7 days (blood ring and recognizable embryo). On day 18, all eggs were removed from the incubators, weighed and transferred to hatching trays in a Robbins hatcher. The hatch was taken off on day 22. All cull chicks, dead chicks, pipped live, and pipped dead embryos were recorded. The remaining unhatched intact eggs were broken out and classified as infertile, early dead (7 days or less), middle dead (8 to 14 days), late dead (15 days or greater), or "other",when cause was obscured by bacterial growth or by egg breakage.
EMBRYO TXICOLOGY
16
I'
'I-
Quat 800. Moisture loss from eggs treated with quaternary ammonia was lower than moisture loss from formalin treated eggs. This is in disagreement with observations by Brake and Sheldon [2]. The sanitizers listed above, which contained quaternary ammonia, did not affect hatchability significantly relative to eggs treated with formaldehyde. The relative hatchability scores for Germex, Quam, Super Quam, Tryad, and Quat 800 were 100.1,102.3, 97.5,100.2, and 100.3, respectively. Even at the highest concentration of each of these five sanitizers, hatchability was not reduced significantly. The significant moisture loss response and the lack of significant response in hatchability score reemphasises the importance of incorporating large sample sizes when attempting to identify small differences between hatchability studies [SI. Other sanitizers which also influenced moisture loss significantly,but did not contain quaternary ammonia as an active ingredient, were Virkon, Coverage 256, and Egg Wash. However, all three of these sanitizers did share EDTA as a common ingredient. In particular, it was noted that eggs treated with Virkon had a "waxy" coat which became progressively more evident as the concentration of Virkon increased. The average relative moisture loss of eggs treated with Virkon, Coverage 256, and Egg Wash was 80.8, 84.4, and 83.2%, respectively. There was a definite dose response (in moisture loss) with increasing levels of Virkon. The response at levels T1 and T2 were not significant compared to the eggs treated with formaldehyde. However, at twice and four times the recommended levels of Virkon, the relative moisture loss was significantly lower. Relativeinoisture loss of eggs treated with Egg Wash or Coverage 256 was reduced significantly by all concentrations tested. Overall hatchability of eggs treated with Virkon, Egg Wash, and Coverage 256 was decreased relative to eggs treated with formaldehyde (73.9, 88.9, and 81.8%, respectively). In fact, at the very high concentration of Virkon, less than 10% of the eggs hatched. At the two lowest concentrations (T1 and T2) of Virkon, the hatchability of eggs was not significantly different from the eggs treated with formaldehyde. There was a dose response in hatchability to increasing levels of Virkon, Egg Wash, and Coverage 256. Embryo mortality was
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Dose response (i.e., concentration or exposure time) to each sanitizer is important to demonstrate toxicity to the embryo. The concentration or exposure time effects are expressed as T1 (approx. half the recommended level), T2 (recommended level), T3 (twice the recommended level) concentration and/or exposure time, and T4 for Virkon only (four times recommended concentration for 10 minutes). Gross toxicity to the embryo was classitied in this study (Table 1). Therefore, only large differences in hatchability were noted. Hatchability was presented as five percentile point increments above or below those values obtained for formalin treatedeggs (e.g., + +" indicates that the response is between 105 and 110% of the control; 'I- - -'I indicates that the response is between 85 and 90% of the control). If a dose response is significantlydifferent (by + or -) from the formalin treatment, the value must be greater than or equal to + + " or -". Moisture loss is an important criteria for assessing incubation conditions and egg shell porosity 141. In order to optimize hatchability, the moisture loss (measured as weight loss between the freshly laid egg and the point at which the egg is pipped) should be equal to 12% [SI. Moisture loss from setting to 18 days incubation over all treatments was equal to 9.7%. Moisture loss ranged from 4.6% when dipped in a solution of 4% Virkon, to 11.0% when eggs were dipped in solutions of Iodophor or hydrogen peroxide. Brake and Sheldon 12) reported that quaternary ammonia increased the permeability of egg shells to respiratory gases (and to moisture loss). However, only young broiler breeders benefited (increased hatchability) by these treatments, presumably due to an increased permeability of the egg shell in response to treatment. In addition, hatchability was determined with rather small numbers of eggs per treatment. In the present study, sanitizers that contained quaternary ammonium as an active ingredient were Germex, Quam, Super Quam, Tryad, and Quat 800. For the three concentrations (Tl, T2, and T3) tested for each sanitizer, the relative moisture loss for eggs treated with Germex, Quam, Super Quam, Tryad, and Quat 800 was 96.1, 93.8, 91.6, 93.8, and 90.4%, respectively. The concentrations tested produced a "dose1'response for Germex, Quam, and Super Quam, but not for Tryad or
Research Report 17
SCOTTetal.
The highest level of hatchability in this study was for eggs treated with D.O.C. Hatchability for these eggs was 103.4% relative to eggs treated with formalin (100.0%). D.O.C. is a phenol and has a relatively high hazardous ingredient rating [41. Hydrogen peroxide is currently being used as a (5% vol/vol) spray for sanitizing hatching eggs in commercial hatcheries based on recommendations by Sheldon a n d Brake [17]. In the present study, hatching eggs were dipped for 10 minutes in 0.70, 1.40, and 2.90 (voVvol) hydrogen peroxide solutions. There was no effect of hydrogen peroxide on moisture loss or hatchability relative to eggs treated wilh formaldehyde. Of interest was the chalky texture of the shell of eggs treated with hydrogen peroxide, indicative of a reaction between the compound and the components of the egg shell. Chemical reactions of this nature influence the effectiveness of a sanitizer on microorganisms, and may explain why Sheldon and Brake [17] required a ten-fold increase in hydrogen peroxide to achieve an equal microorganism kill on agar plates. Scott and Swetnam [3] have strong reservations about user safety when handling hydrogen peroxide.
CONCLUSIONS AND APPLICATIONS 1. Taking into account the difficulty of making comparisons between sanitizers containing different ingredients, different levels of ingredients and, in some situations, no recommendations for use on hatching eggs, it is apparent that there are several potential sanitizers available on the market which perform as well or better than formaldehyde. 2. In the present survey of sanitizers, further study (large scale hatchability trials) were
recommended for Germex, Iodophor, Lysovet, Iocide-14, Hydrogen Peroxide, Quam, 1-Stroke (Tektrol has similar ingredients), Virkon, Quat 900, and Quat 800. Similarly, trials will be conducted to examine the influence of UV-light as a potential for sanitizing hatching eggs and scrubbing circulating air during incubation.
REFERENCES AND NOTES 1. Patterson, P.II., S.C. Ricke, M. Suodr, and D.M. Schaeler, 1990. Hatching eggs sanitized with chlorine dioxide foam: Egg hatchability and bactericidal properties. Avian Dis. 34:1-6.
2. Brake, J. and B.W. Sheldon, 1990. Effect of a quaternary ammonium sanitizer for hatching e g s on their contamination, ermeability, water loss and hatchability. Poultry Sci. 69Q17-525.
3. Scoll, T.A. nod C. Swelnam, 1993a. Screeningsanitizing agents and methods of application for hatching
eggs. I. User and environmental friendliness. J. Appl. Poultiy Res. 2: 1 4 . 4. Scull, T.A. and C. Sweloam, 1993b. Screening sanitizing agents and methods of application for hatching 11. Effectiveness against a "cocktail"of microorganisms on the egg shell. J. Appl. Poultry Res. 2:7-11.
ew.
5. Furula, K. mid S. Maruynma, 1981. Bacterial contamination on eggs during incubation and hatching and of llufl-sof newly hatched chicks. Br. Poultry Sci., 22247254.
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evenly distributed between early (7 days or less) and late (greater than 7days) embryo mortality. This could indicate that optimum air exchange is as important for the early growing embryo as for the high metabolism late embryo. Bagley and Christensen [15] observed marked improvements in late turkey embryo survival when egg shell permeability was increased. However, this could also indicate that equally high early and late embryo mortality was due to penetration of some eggs by the sanitizer (early mortality), while subsequently sealing the pores and suffocating the embryos which survived (late mortality). Of these three compounds, only Virkon at the lower concentrations (T1 or T2) was indicated worthy of large scale hatchability studies. The maximum concentration of ozone gas tested in the present study was approximately 100 times less concentrated than that used by Whistler and Sheldon [18]. However, even at this relatively low concentration, we observed a numerical decrease (P > .OS) in hatchability relative to eggs treated with formaldehyde. Although ozone gas is viewed as a potential preservative and sanitizer in the food industry, there are too many restrictions relative to user and environmental safety to justify its use in large scale hatchability studies.
18 6. Cox, N.A., J.S.Bailey, J.M. Mauldiiq LC. Blankenship, and J . L Wilson, 1991. Research Note: Extent of Salrrlpnellae contamination in breeder hatcheries. Poultry Sci. 70416418. 7. Bruce, 1. and EM. Drysdale, 1991. Egg h giene: ,Poultry lcience Routesof infection. In: 2 m p o s i u m 22 (Tullet, S.G., Ed.) Butterwortheinemann, Toronto, pp 257-267. 8. Tullel, S.C., 1990. Science and the art of incubation. Poultry Sci. 691-15. 9. Mowry, D.J., D.J. Fagerberg, and C.L Qunrles, 1980. Effect of hatcher fogging on hatcher airborne bacteria and broiler performance. Poultry S i . 59:714-718.
level with increased eggshell permeability. Poultry Sci. 70:1412-1418. 16. Vkk, S.V. and J. Brake, 1986. Effect of incubation humidity on hatchabili with respect to egg weight and flock age. Poultry Sci. (Suppl):130 (abstract).
d
17. Sheldon, B.W. and J. Brake, 1991. Hydrogen peroxide as an alternative hatching egg disinfectant. Poultry Sci. 701092-1098. 18. Whisller, P.E and B.W. Sheldon, 1989. Biocidal activity of ozone versus formaldehyde against oultry pathogens inoculated in a prototype setter. Goultly Sci. 68:106&1073.
19. Rice, R.C., J.W. Farquhar, and L J . Ballyky, 1982. Review of the applicationsof ozone for increasingstorage times of erishable foods. Ozone: Science and Engineering, 4:14$-163. 20. Mauldin, J.M. and J.L. Wilson, 1990.Twelve components of good hatcherysanitation. Misset-World Poultry, DeclJan, 1991, pp. 29-31,35,37. 21. SAS Inslilute, 1986. Statistical Analysis System, 1986 ed., SAS Inst, Inc., Cay, NC. 22. The authors thank the Canada/Nova Scotia AgriFood Development Agreement and the Canada/New Brunswick Agri-Food Development Agreement for their combined financial support of this project through the Atlantic Poultry Research Institute (A.P.R.I.). The assistance and support from Agriculture Canada and A.P.RI. employees at the Kentville Research Station is also acknowledged.
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10. Christensen, V.L and F.M. McCorkle, 1982. Turkey egg wei ht losses and embryonic mortality during incubation. foultry Sci. 61:1209-1213. 11.Syurks, N.H.C. and ILG. Board, 1984. Cuticle shell y i t y and water u take through hen’seggshells. British oultly Sci. 25:267-!76.’ 12. Peebles, E D . and J. Brake, 1986. The role of the cuticle in water vapour conductance by the eggshell of broiler breeders. Poultry Sci. 65:1034-1039. 13. Christensen, V.L and LC. Bagley, 1987. Water balance in incubating turkey eggs. Poultly Sci. 66: 18351840. 14. Wineland, M.J., 1990. Minimizing cootaniination of hatching eggs a must. Poultry Digest, Sept., pp. 36-37. 15. Bagley, LG. arid V.L. Christensen, 1991. Hatchability and physiology of turkey embryos incubated at sea
EMBRYO TXICOLOGY
TOM A. SCOTT' Agriculture Canada Research Station, Agassiz, British Colicmbia, VOM U O , Canada Phom atid FAX: (604) 796-2221 CHRISTINA SWETNAM and ROGER KINSMAN Agriculture Canada Research Station, Keiitville, Nova Scotia, B4N lJ.5, Caiiada ~
~
Primary Audience: Hatcheries, Veterinarians
1 T o whom
correspondence should be addressed
Downloaded from http://japr.oxfordjournals.org/ at United Arab Emirates University on June 18, 2015
SCREENING SANITIZING AGENTS AND METHODS OF APPLICATION FOR HATCHING EGGS 111. EFFECT OF CONCENTRATION AND EXPOSURE TIMEON EMBRYO VIABILITY
Research Report SCOTTet al.
13
the access of bacteria to the interior of the egg. DESCRIPTION OF PROBLEM One part of the defense of the shell is the cuticle, or proteineous coat, applied at time of lay, also referred to as the bloom. Handling and chemical exposure may alter the cuticle and result i n both negative and positive changes in porosity of the egg shell [7,10, 11, 12, 13, 14, 1.51. Vick and Brake [16] observed that removing the cuticle (and increasing moisture loss during incubation) improved hatchability of eggs from young, but not old, broiler breeder hens. There are two ways a chemical (sanitizer) may interact with the egg shell and reduce permeability: alteration of the proteineous cuticle, or alteration of the calcium carbonate matrix of the shell itself. Brake and Sheldon [2] found that solutions of quaternary ammonium interacted with the cuticle, increasing pernieability and resulting in an increase in hatchability of eggs laid by young, but not old, broiler breeder hens. Sheldon and Brake [ 171observed high microorganism destruction when 0.5% hydrogen peroxide was applied directly to plated cultures. However, concentrations of 5% (tenfold) were r e q u i r e d when t h e s a m e microorganisms where present on the egg shell. With a concentration of 5% hydrogen peroxide, Sheldon and Brake [17] demonstrated a 2% increase in hatchability compared to eggs treated with formaldehyde. These hatchability claims were made on relatively small numbers of eggs. Scott and Swetnam [3] have grave concerns about the use of hydrogen peroxide based on the MSDS (Material Safety Data Sheets) guidelines. Patterson et al. [l]observed that dipping, compared to foaming, hatching eggs in a chlorine dioxide solution resulted in decreased hatchability of heavily soiled duck eggs. Scott and Swetnam [4] observed that chlorine dioxide (Sanimist) was quickly neutralized by components of the egg shell and was inerfective in reducing microorganisms on the egg shell. Ozone gas was used to reduce microorganism loads of hatching eggs [18]. However, at the levels tested (3% ozone by weight for 2 hours), embryo toxicity occurred. In theory, ozone (having a molecular weight similar to oxygen and carbon dioxide) could transfer across the shell wall and destroy microorganisms which had penetrated the shell barrier. Unfortunately, this transfer across the shell
Downloaded from http://japr.oxfordjournals.org/ at United Arab Emirates University on June 18, 2015
Pattersonetal. [I], Brake and Sheldon [ 2 ] ) and Scott and Swetnam [3,4] provide a thorough review of the hazards involved in the use of formaldehyde in the work place. However, both Furuta and Maruyama [5] and Scott and Swetnam [3,4] were concerned about finding a sanitizer as effective against microorganisms as formaldehyde, and yet user and environmentally safe. There are many sanitizers available on the market; some have recommendations and claims for hatching egg sanitation, but others do not. Some of these compounds are as noxious and dangerous to use as formaldehyde [3], while still others are ineffective against bacteria when applied to the egg shell [4]. Cox et ai. [6] indicated that commercial hatcheries are switching from one chemical sanitizer or application method to another with little appreciable effect on Saltnonella contamination. Furthermore, Bruce and Drysdale [7] were not convinced that microorganism contamination of hatching eggs had any direct effect on hatchability. Therefore, it is imperative that the merits of a hatchery sanitizer or application procedure be reviewed closely. In the literature there are a number of reports which only include small numbers of eggs. This is a concern when reporting hatchability data, as Tullet [SI pointed out. He estimated that 10,000 eggs per treatment were required to demonstrate a 1% dilference in hatchability between treatments. There are several reports which support the idea that a dirty environinent and subsequently a dirty egg will decrease hatchability. Bruce and Drysdale [7] and Cox et ai. [6] implicated management as a source of poor hygiene. They cited the superior performance of "modell' grandparent compared to the lower performance of multiplier flocks. Tullct [8] compared floor ("dirty") eggs to nest run eggs and observed a 10 to 15% decrease in hatchability. Factors other than bacterial contaniination of these floor eggs were also indicated. Mowry et al. [SI observed that 50 day-old broiler chick performance was iniproved by sanitizing eggs prior to incubation. Several studies have measured a change in shell porosity and subsequent air exchange ( 0 2 in, C02 and HzO out) of the developing embryo. Porosity of a shell must also restrict
EMBRYO TXICOLOGY
14
twice. Ozone gas was applied at three concentrations for 3 hours. EGG DIPPING TREATMENTS Plastic 100 1 barrels were partly filled with 30 1 of tap water and allowed to equilibrate to room temperature over night. The following morning, the sanitizers to be tested were added and mixed thoroughly. Six flats of 30 eggs were submerged in the solutions for the required time (Table 1). The procedure was repeated until all eggs were treated. The eggs were allowed to drip dry and then placed in storage (18°C; 65%RH) for 72 hours before setting randomly in Robbins (Hatch-o-matic) incubators. OZONE TREATMENTS Hatching eggs were exposed to one of three concentrations of ozonegas (3.2,1.6, and 0.5p1) for a period of three hours at 10°C. As described previously for randomizing eggs, three flats of 30 eggs were placed into one of four 64 1 polyethylene containers. Ozone was produced by a Corona Discharge Ozone Generator (Triox Trucker model, Triox Co., Swindon, U.K.) and delivered to the containers through a three port manifold and 3.175 nun teflon tubing. Ozone concentration was monitored with a portable semiconductor thin film ozone detector (Ebra-Jitsugyo Co., Kawasaki, Japan) with a range of 0-5 p l . The sensor was placed through a hole in the top of one end of each chamber to the level of the eggs.
MATERIALS AND METHODS The physical screening and selection of a sanitizer was dependent on when the compound became available to the laboratory. Each sanitizer was tested at two different times over a six month period. To minimize variation in handling, environment, and equipment, a control group of eggs treated with 1% formalin was used in all comparisons. Hatching eggs were obtained from a commercial hatchery (ABS Hatchery of ACA Cooperatives, New Minas, Nova Scotia) on a weekly basis. All eggs during any given week were from a common flock of broiler breeder hens and were stored for less than 45 hours before being treated. On receipt, clean and sound eggs were selected and randomized into lots of 90 eggs per replicate. Four replications (360 eggs) for each of 12 treatment groups were tested to yield a total of 4320 cggdweek. In each comparison the control eggs were treated with 1%formalin for 1, 5, or 10 minutes, with four replications of 90 eggs per exposure time. The remaining nine treatments were divided into either 2, 3, or 4 concentration and/or exposure levels of three test sanitizers (Table 1). During a six month interval, assessment of concentration and/or exposure levels of each of the 23 sanitizers were tested
EGG HANDLING AND INCUBATION On day of setting, the pretreated eggs were removed from storage, placed on setting racks and weighed; a replicate was comprised of one tray of 90 eggs. The trays of eggs were identified and assigned randomly a position in the incubator. The eggs were allowed to equilibrate at room temperature for six hours before the incubators were switched on. Incubation temperatures were 37"C+OS"C and relative humidity was SO%. Eggs were turned hourly. At day 7, all eggs were candled by hand, The trays of eggs were removed, weighed, candled, and remaining viable embryos weighed and placcd back into the incubator. Eggs removed were broken out and visually classified as infertile, live, dead before 2 days (mem-
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wall also puts ozone into direct contact with the embryo. Ozone may be a serious health hazards for those working with it [3]. The level of ozone used in the study by Whistler and Sheldon [ 181 was extremely high, far exceeding amounts which have been recommended for food products [19]. This high level of exposure may explain the high embryo mortality which was reported in their study [IS]. Mauldin and Wilson [20] reviewed the basic considerations for a successful hatchery sanitation program. Good sanitation practices must begin at the point of lay and continue through the placement of chicks. Part of this sanitation practice often includes treatment of eggs prior to and during incubation. In the present study, 23 sanitizers wcre applied to hatching eggs. Gross embryo mortality was monitored. Sanitizers that demonstrate user and environmental safety, effectiveness against microorganism on the eggshell, and n o gross toxicity to the embryo, will be further tested in large scale hatchability studies.
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SCOTTet al.
PARAMETERS MEASURED The moisture loss from setting to transfer (18 days) for each lot of 90 eggs was recorded as a percentage of initial weight. Following classification of all broken out eggs at candling, and after the hatch was taken off, the number of healthy viable chicks from fertile eggs were used to determine percent hatchability.
STATISTICAL ANALYSIS The data within each hatch was expressed relative (as a percentage of) to formaldehyde control to minimize differences between hatches. The statistical model assessed the main effects of sanitizer and concentration and/or exposure time of each sanitizer. Significant differences between mean values were separated by Duncan's Multiple Range Test [21].
RESULTS AND DISCUSSION To simplify the presentation of data, only the relative response (formalin treated eggs equal 100%) in moisture loss (0 to 18 days incubation) and hatchability of fertile eggs are presented (Table 1). The actual values for eggs treated with formaldehyde are 10.1% moisture loss from 0 to 18 days incubation, and 81.5% hatchability of fertile eggs set. The effect of exposure time (I, 5, or 10 minutes) to 1% formalin on moisture loss or hatchability was not significant. Overall, fertility of eggs (greater than 60,000) set in this study was 92.6+4.0%.
TABLE 1. The relative (to formaldehyde control) loss in moisture (0 to 18 day incubation) and response of sanitizer treatments hatchability of fertile eggs and dose (Tl, T2, or T34l/)
0.70
1.40
1.90
102.6
ia
+
++
+
-
-
-
100.3
i abcd
+
+
+
+ + 0 . 8 0 1.60 3 . 1 0 97.9 j abc 100.0 i abcd lo-ldehyd. w .. equal t o . 1 formalin and eggs dipped for 1, 5 or 10 min. I'clutaraldehyde was applied full strength for 1 or 10 nin. I oron. qaa cor c:antratLon. -re held for 180 mi". h11 other treatment# involved eggs dipped in SOlUtiOn for 10 min. - or + Indicate a L.S ID. value of I 5 . 0 units in relative moimture lose or hatchability reaponse from the control (1\ indicate- a reeponse of between B 5 to 90% of the control. d Icmte. a value of between 100 and lost,
'
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brane, but no blood development), dead between 2 and 4 days (blood ring development and no visible embryo), or dead between 4 and 7 days (blood ring and recognizable embryo). On day 18, all eggs were removed from the incubators, weighed and transferred to hatching trays in a Robbins hatcher. The hatch was taken off on day 22. All cull chicks, dead chicks, pipped live, and pipped dead embryos were recorded. The remaining unhatched intact eggs were broken out and classified as infertile, early dead (7 days or less), middle dead (8 to 14 days), late dead (15 days or greater), or "other",when cause was obscured by bacterial growth or by egg breakage.
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I'
'I-
Quat 800. Moisture loss from eggs treated with quaternary ammonia was lower than moisture loss from formalin treated eggs. This is in disagreement with observations by Brake and Sheldon [2]. The sanitizers listed above, which contained quaternary ammonia, did not affect hatchability significantly relative to eggs treated with formaldehyde. The relative hatchability scores for Germex, Quam, Super Quam, Tryad, and Quat 800 were 100.1,102.3, 97.5,100.2, and 100.3, respectively. Even at the highest concentration of each of these five sanitizers, hatchability was not reduced significantly. The significant moisture loss response and the lack of significant response in hatchability score reemphasises the importance of incorporating large sample sizes when attempting to identify small differences between hatchability studies [SI. Other sanitizers which also influenced moisture loss significantly,but did not contain quaternary ammonia as an active ingredient, were Virkon, Coverage 256, and Egg Wash. However, all three of these sanitizers did share EDTA as a common ingredient. In particular, it was noted that eggs treated with Virkon had a "waxy" coat which became progressively more evident as the concentration of Virkon increased. The average relative moisture loss of eggs treated with Virkon, Coverage 256, and Egg Wash was 80.8, 84.4, and 83.2%, respectively. There was a definite dose response (in moisture loss) with increasing levels of Virkon. The response at levels T1 and T2 were not significant compared to the eggs treated with formaldehyde. However, at twice and four times the recommended levels of Virkon, the relative moisture loss was significantly lower. Relativeinoisture loss of eggs treated with Egg Wash or Coverage 256 was reduced significantly by all concentrations tested. Overall hatchability of eggs treated with Virkon, Egg Wash, and Coverage 256 was decreased relative to eggs treated with formaldehyde (73.9, 88.9, and 81.8%, respectively). In fact, at the very high concentration of Virkon, less than 10% of the eggs hatched. At the two lowest concentrations (T1 and T2) of Virkon, the hatchability of eggs was not significantly different from the eggs treated with formaldehyde. There was a dose response in hatchability to increasing levels of Virkon, Egg Wash, and Coverage 256. Embryo mortality was
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Dose response (i.e., concentration or exposure time) to each sanitizer is important to demonstrate toxicity to the embryo. The concentration or exposure time effects are expressed as T1 (approx. half the recommended level), T2 (recommended level), T3 (twice the recommended level) concentration and/or exposure time, and T4 for Virkon only (four times recommended concentration for 10 minutes). Gross toxicity to the embryo was classitied in this study (Table 1). Therefore, only large differences in hatchability were noted. Hatchability was presented as five percentile point increments above or below those values obtained for formalin treatedeggs (e.g., + +" indicates that the response is between 105 and 110% of the control; 'I- - -'I indicates that the response is between 85 and 90% of the control). If a dose response is significantlydifferent (by + or -) from the formalin treatment, the value must be greater than or equal to + + " or -". Moisture loss is an important criteria for assessing incubation conditions and egg shell porosity 141. In order to optimize hatchability, the moisture loss (measured as weight loss between the freshly laid egg and the point at which the egg is pipped) should be equal to 12% [SI. Moisture loss from setting to 18 days incubation over all treatments was equal to 9.7%. Moisture loss ranged from 4.6% when dipped in a solution of 4% Virkon, to 11.0% when eggs were dipped in solutions of Iodophor or hydrogen peroxide. Brake and Sheldon 12) reported that quaternary ammonia increased the permeability of egg shells to respiratory gases (and to moisture loss). However, only young broiler breeders benefited (increased hatchability) by these treatments, presumably due to an increased permeability of the egg shell in response to treatment. In addition, hatchability was determined with rather small numbers of eggs per treatment. In the present study, sanitizers that contained quaternary ammonium as an active ingredient were Germex, Quam, Super Quam, Tryad, and Quat 800. For the three concentrations (Tl, T2, and T3) tested for each sanitizer, the relative moisture loss for eggs treated with Germex, Quam, Super Quam, Tryad, and Quat 800 was 96.1, 93.8, 91.6, 93.8, and 90.4%, respectively. The concentrations tested produced a "dose1'response for Germex, Quam, and Super Quam, but not for Tryad or
Research Report 17
SCOTTetal.
The highest level of hatchability in this study was for eggs treated with D.O.C. Hatchability for these eggs was 103.4% relative to eggs treated with formalin (100.0%). D.O.C. is a phenol and has a relatively high hazardous ingredient rating [41. Hydrogen peroxide is currently being used as a (5% vol/vol) spray for sanitizing hatching eggs in commercial hatcheries based on recommendations by Sheldon a n d Brake [17]. In the present study, hatching eggs were dipped for 10 minutes in 0.70, 1.40, and 2.90 (voVvol) hydrogen peroxide solutions. There was no effect of hydrogen peroxide on moisture loss or hatchability relative to eggs treated wilh formaldehyde. Of interest was the chalky texture of the shell of eggs treated with hydrogen peroxide, indicative of a reaction between the compound and the components of the egg shell. Chemical reactions of this nature influence the effectiveness of a sanitizer on microorganisms, and may explain why Sheldon and Brake [17] required a ten-fold increase in hydrogen peroxide to achieve an equal microorganism kill on agar plates. Scott and Swetnam [3] have strong reservations about user safety when handling hydrogen peroxide.
CONCLUSIONS AND APPLICATIONS 1. Taking into account the difficulty of making comparisons between sanitizers containing different ingredients, different levels of ingredients and, in some situations, no recommendations for use on hatching eggs, it is apparent that there are several potential sanitizers available on the market which perform as well or better than formaldehyde. 2. In the present survey of sanitizers, further study (large scale hatchability trials) were
recommended for Germex, Iodophor, Lysovet, Iocide-14, Hydrogen Peroxide, Quam, 1-Stroke (Tektrol has similar ingredients), Virkon, Quat 900, and Quat 800. Similarly, trials will be conducted to examine the influence of UV-light as a potential for sanitizing hatching eggs and scrubbing circulating air during incubation.
REFERENCES AND NOTES 1. Patterson, P.II., S.C. Ricke, M. Suodr, and D.M. Schaeler, 1990. Hatching eggs sanitized with chlorine dioxide foam: Egg hatchability and bactericidal properties. Avian Dis. 34:1-6.
2. Brake, J. and B.W. Sheldon, 1990. Effect of a quaternary ammonium sanitizer for hatching e g s on their contamination, ermeability, water loss and hatchability. Poultry Sci. 69Q17-525.
3. Scoll, T.A. nod C. Swelnam, 1993a. Screeningsanitizing agents and methods of application for hatching
eggs. I. User and environmental friendliness. J. Appl. Poultiy Res. 2: 1 4 . 4. Scull, T.A. and C. Sweloam, 1993b. Screening sanitizing agents and methods of application for hatching 11. Effectiveness against a "cocktail"of microorganisms on the egg shell. J. Appl. Poultry Res. 2:7-11.
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5. Furula, K. mid S. Maruynma, 1981. Bacterial contamination on eggs during incubation and hatching and of llufl-sof newly hatched chicks. Br. Poultry Sci., 22247254.
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evenly distributed between early (7 days or less) and late (greater than 7days) embryo mortality. This could indicate that optimum air exchange is as important for the early growing embryo as for the high metabolism late embryo. Bagley and Christensen [15] observed marked improvements in late turkey embryo survival when egg shell permeability was increased. However, this could also indicate that equally high early and late embryo mortality was due to penetration of some eggs by the sanitizer (early mortality), while subsequently sealing the pores and suffocating the embryos which survived (late mortality). Of these three compounds, only Virkon at the lower concentrations (T1 or T2) was indicated worthy of large scale hatchability studies. The maximum concentration of ozone gas tested in the present study was approximately 100 times less concentrated than that used by Whistler and Sheldon [18]. However, even at this relatively low concentration, we observed a numerical decrease (P > .OS) in hatchability relative to eggs treated with formaldehyde. Although ozone gas is viewed as a potential preservative and sanitizer in the food industry, there are too many restrictions relative to user and environmental safety to justify its use in large scale hatchability studies.
18 6. Cox, N.A., J.S.Bailey, J.M. Mauldiiq LC. Blankenship, and J . L Wilson, 1991. Research Note: Extent of Salrrlpnellae contamination in breeder hatcheries. Poultry Sci. 70416418. 7. Bruce, 1. and EM. Drysdale, 1991. Egg h giene: ,Poultry lcience Routesof infection. In: 2 m p o s i u m 22 (Tullet, S.G., Ed.) Butterwortheinemann, Toronto, pp 257-267. 8. Tullel, S.C., 1990. Science and the art of incubation. Poultry Sci. 691-15. 9. Mowry, D.J., D.J. Fagerberg, and C.L Qunrles, 1980. Effect of hatcher fogging on hatcher airborne bacteria and broiler performance. Poultry S i . 59:714-718.
level with increased eggshell permeability. Poultry Sci. 70:1412-1418. 16. Vkk, S.V. and J. Brake, 1986. Effect of incubation humidity on hatchabili with respect to egg weight and flock age. Poultry Sci. (Suppl):130 (abstract).
d
17. Sheldon, B.W. and J. Brake, 1991. Hydrogen peroxide as an alternative hatching egg disinfectant. Poultry Sci. 701092-1098. 18. Whisller, P.E and B.W. Sheldon, 1989. Biocidal activity of ozone versus formaldehyde against oultry pathogens inoculated in a prototype setter. Goultly Sci. 68:106&1073.
19. Rice, R.C., J.W. Farquhar, and L J . Ballyky, 1982. Review of the applicationsof ozone for increasingstorage times of erishable foods. Ozone: Science and Engineering, 4:14$-163. 20. Mauldin, J.M. and J.L. Wilson, 1990.Twelve components of good hatcherysanitation. Misset-World Poultry, DeclJan, 1991, pp. 29-31,35,37. 21. SAS Inslilute, 1986. Statistical Analysis System, 1986 ed., SAS Inst, Inc., Cay, NC. 22. The authors thank the Canada/Nova Scotia AgriFood Development Agreement and the Canada/New Brunswick Agri-Food Development Agreement for their combined financial support of this project through the Atlantic Poultry Research Institute (A.P.R.I.). The assistance and support from Agriculture Canada and A.P.RI. employees at the Kentville Research Station is also acknowledged.
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10. Christensen, V.L and F.M. McCorkle, 1982. Turkey egg wei ht losses and embryonic mortality during incubation. foultry Sci. 61:1209-1213. 11.Syurks, N.H.C. and ILG. Board, 1984. Cuticle shell y i t y and water u take through hen’seggshells. British oultly Sci. 25:267-!76.’ 12. Peebles, E D . and J. Brake, 1986. The role of the cuticle in water vapour conductance by the eggshell of broiler breeders. Poultry Sci. 65:1034-1039. 13. Christensen, V.L and LC. Bagley, 1987. Water balance in incubating turkey eggs. Poultly Sci. 66: 18351840. 14. Wineland, M.J., 1990. Minimizing cootaniination of hatching eggs a must. Poultry Digest, Sept., pp. 36-37. 15. Bagley, LG. arid V.L. Christensen, 1991. Hatchability and physiology of turkey embryos incubated at sea
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