Hatchablility of Broiler Breeder Eggs Sanitized with a Combination of Ultraviolet Light and Hydrogen Peroxide*

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1 Internatinal Jurnal f Pultry Science 0 (4): 30-34, 0 ISSN Asian Netwrk fr Scientific Infrmatin, 0 Hatchablility f Briler Breeder Eggs Sanitized with a Cmbinatin f Ultravilet Light and Hydrgen Perxide* J.B. Wells, C.D. Cufal, H.M. Parker, A.S. Kiess, K.M. Yung and C.D. McDaniel ** Department f Pultry Science, Mississippi State University, Mississippi State, Mississippi, 3976, USA Department f Pultry Science, Texas A & M University, Cllege Statin, TX 77845, USA Abstract: Research has shwn that exterir eggshell Aerbic Plate Cunts (APC) are greatly decreased using a cmbinatin f Ultravilet Light (UV) and hydrgen perxide (HO). Hwever, it is unknwn hw this treatment prcess wuld impact hatchability. Therefre, the bjective f this experiment was t determine if spraying eggs with.5% HO fllwed by UV irradiatin fr 8 min t reduce eggshell APC wuld affect hatchability. Eggs frm 3 cmmercial huses (57 wk-ld briler breeders) were cllected ver d (n =,944 eggs). Half were treated with.5% HO and UV and the ther half served as untreated cntrls (8 eggs/ tray). At time f treatment, egg was randmly selected frm each f 08 trays (n = 54 per treatment) fr eggshell APC enumeratin n TSA. Remaining eggs were stred at 8.3 C. Prir t set, egg per tray frm d f cllectin was sampled fr APC enumeratin. The 6 remaining eggs/tray were weighed prir t incubatin and at 8 d t determine egg weight lss. At hatch (.5 d), chick weights were btained, mecnium samples were cllected frm 8 chicks per incubatr and samples were plated n TSA t determine the presence f intestinal micrrganisms. A 3 lg0cfu/egg reductin in eggshell APC was fund fr treated eggs when cmpared t cntrl eggs. At hatch, n differences in chick weight, egg weight lss, psitive mecnium samples, r hatchability were bserved between treatments. In cnclusin, UV irradiatin fr 8 min with.5% HO reduced eggshell APC n briler breeder eggs with n affect n hatchability. Key wrds: Hatchability, sanitizatin, bacteria, ultravilet light, hydrgen perxide INTRODUCTION Micrbial cntaminatin lcated n the surface f the eggshell may have a negative effect n hatching eggs. It has been shwn that this cntaminatin can easily penetrate the eggshell (Berrang et al., 999). Wilsn (997) als suggested that the cntaminatin fund n hatching eggs can lead t embrynic mrtality, weak chicks, pr grwth and even chick mrtality. Safe and effective sanitizatin methds have been widely researched fr many years. Ultravilet Light (UV) has been shwn t be an effective methd f sanitizatin fr the eggshell (Berrang et al., 999; Gerzen and Sctt, 995). Unfrtunately, in research cnducted by Berrang et al. (995) as well as Gerzen and Sctt (995), it was determined that even thugh UV light was effective at reducing bacterial cntaminatin n the eggshell surface f briler breeder eggs it was nt effective at increasing hatchability. Bayliss and Waites (98) reprted that the cmbinatin f UV light and HO can reduce bacterial cunts n nutrient agar slpes by mre than 4 lg0cfu In mre recent studies cnducted by Wells et al. (008), the cmbinatin f UV light and HO was administered t the surface f eggshells and like the results f Bayliss and Waites (98) micrbial cntaminatin n the eggshell was reduced by 4 lg0cfu/egg. Hwever, it is still undetermined if this treatment cmbinatin will alter the hatchability f briler breeder eggs. Therefre, the bjective f this prject was t determine if the applicatin f HO and UV light in cmbinatin t reduce eggshell cntaminatin will affect the hatchability f briler breeder eggs. MATERIALS AND METHODS Egg cllectin: A ttal f,944 eggs frm 57 wk-ld briler breeder hens were utilized in this study. Egg Sanitizatin using hydrgen perxide (HO) has been cllectin was cmpleted ver a d perid, with 864 researched t determine if it alters hatchability. eggs cllected n the first day at 0:00 am. The fllwing Research cnducted by Sander and Wilsn (999) day,080 eggs were cllected during the same time demnstrated that fgging with 3% HO was effective at frame, beginning at 0:00 am. All eggs were cllected reducing bacteria but it had n effect n hatchability. directly frm the egg belts f 3 huses lcated n a Padrn (995) als fund n affect n hatchability when single cmmercial breeder farm. All,944 eggs frm the eggs were dipped in 6% HO, even thugh 95% f the d f cllectin were divided equally int a cntrl grup bacteria were reduced n the eggshell surface. and a treated grup. Crrespnding Authr: C.D. McDaniel, Department f Pultry Science, Mississippi State University, Mississippi State, Mississippi,

2 Int. J. Pult. Sci., 0 (4): 30-34, 0 Fig. : Experimental UV light chamber cnfiguratin fr the treatment f hatching eggs n wire trays Egg treatment and handling: Each day after all eggs were cllected, the treated eggs were placed hrizntally n wire trays (36.8 cm x 7.4 cm with 8 eggs per tray) and hand misted until cmpletely cated with.5% HO. The eggs were misted with enugh HO n the surface t be entirely cvered withut dripping. Immediately after treating each tray f eggs with HO, the eggs were placed in a UV chamber fr 8 min. The UV chamber used in this experiment had a UV-C intensity f apprximately mw/cm measured at egg level. The chamber was designed with levels s that trays culd be treated simultaneusly. A ttal f 0 UV-C lamps (9.4 cm G30T8) were munted in the chamber as clse t the eggs as pssible (see Fig. ). Immediately after each tray f eggs was treated with UV light, egg frm each tray (54 per treatment) was cllected frm bth treated and cntrl grups and used fr Aerbic Plate Cunt (APC) enumeratin as described belw. Using clean glves fr each repetitin, the remaining 7 eggs per tray were then placed int clean paper flats fr strage. All f the eggs, including the cntrl eggs, were then placed int a cler at 8.3 C. The eggs cllected n the first day were stred fr d in the cler and the eggs cllected n the secnd day were nly stred fr d. All eggs were remved frm the cler n d 3 f the experiment. Prir t setting bth the treated and cntrl eggs in the incubatr, initial egg weights were recrded fr calculating incubatinal egg weight lss. This was perfrmed t determine if the treatment altered misture lss thrugh the eggshell. Frm cntrl and treated eggs that had been stred in the cler fr d, egg frm each tray (4 ttal eggs) per treatment were randmly selected fr APC enumeratin t determine the effect f strage n micrbial survival fllwing sanitizatin. Eggs were then set int 6 different incubatrs (3 per treatment). All eggs were incubated under standard incubatin cnditins fr chicken eggs. Each incubatr cntained either all cntrl r all treated eggs frm a single breeder huse with apprximately 300 eggs per incubatr. Cntrl and treated eggs were placed in different incubatrs t prevent micrbial crss cntaminatin between the grups. After 8 d f incubatin, eggs were remved frm each incubatr and reweighed t determine weight lss during the incubatin perid relative t set egg weight. After weighing was cmplete, all f the eggs were placed int hatching baskets (8 baskets/incubatr). At.5 d f incubatin, all hatched chicks were remved frm the incubatrs, cunted and weighed by treatment. A single chick frm each hatching basket was randmly selected during weighing and a mecnium sample was btained by frced fecal expulsin. After chicks were remved frm the hatching baskets, all remaining eggs were cllected and hatch residue analysis was perfrmed. Bacterial enumeratin prcedure: Each egg was individually placed int a sterile Whirl-pak bag (Nasc, Frt Atkinsn, WI) cntaining 50 ml f 0% sterilized peptne water. Eggs were then massaged fr min t remve micrrganisms lcated n the uter surface f the eggshell. After the massage, 0 ml f each rinse slutin was aseptically pipetted int a sterile culture tube. Preliminary research revealed that cntrl eggs were highly cntaminated (Wells et al., 008). Therefre, serial dilutins were perfrmed fr each cntrl egg sample. Hwever, n serial dilutins were perfrmed n the treated egg samples. Fr cntrl eggs, 0.5 ml f each egg rinse and diluted samples were spread plated in duplicate n Tryptic Sy Agar (TSA). Als, 0.5 ml f egg rinse frm the treated eggs was spread plated in duplicate nt TSA plates. All plates were incubated fr 48 h at 37 C, after which clny enumeratin was perfrmed. At hatch, a ttal f 08 chicks ( chick per hatch tray) were randmly selected and mecnium samples were btained. Mecnium was expressed frm the claca f each chick directly int a sterile Whirl-pak bag and 5 ml f sterile peptne water was added. This slutin f peptne and mecnium was mixed and 0.5 ml was spread plated in duplicate n TSA. These plates were incubated fr apprximately 48 h at 37 C and assessed fr psitive r negative micrbial grwth. Statistical analysis: All data frm this study were analyzed as a randmized cmplete blck design and means were separated using Fisher s prtected least significant difference (p<0.05). Each f the 3 breeder huses served as a blck. Als, a x factrial arrangement f treatments was used t analyze egg sanitizatin and length f egg strage (Steel and Trrie, 980). 3

3 Int. J. Pult. Sci., 0 (4): 30-34, 0 Fig. : Effect f hydrgen perxide (HO) and Ultravilet (UV) light sanitizatin n average eggshell APC cunts f briler breeder hatching eggs cllected ver a day perid. Each treatment cntained 54 replicatins (4 replicatins fr d and 30 replicatins fr d ) with egg per replicatin (08 eggs ttal). a-b Means with different letters are significantly different at p<0.00 Table : Means fr hatch residue analysis data fr briler breeder eggs Early Middle Late Infertile dead dead dead Pipped Cntam. Parameter (%) Cntrl Treated SEM p-value n = 3 incubatrs cntaining apprximately 300 eggs each per treatment. Cntam. = Cntaminated Table : Means f chick characteristics f briler breeder chicks at hatch Chick Egg Mecnium samples weight weight psitive fr Characteristic (g) lss (%) 3 bacteria (%) Cntrl Treated SEM p-value n = 3 incubatrs per treatment cntaining apprximately 50 chicks each per incubatr. n = 3 incubatrs cntaining apprximately 300 eggs each per treatment. 3 n = 3 incubatrs per treatment with 8 chicks sampled per incubatr Fig. 3: Effects f egg sanitizatin and strage n eggshell bacterial cunts fr eggs stred fr days. Fresh eggs were sampled immediately after treatment and cllectin (n = 54 replicatins per treatment; 4 replicatins fr d plus 30 replicatins fr d, egg per replicatin). Stred eggs were sampled nly frm eggs cllected n d and stred d (n = 4 reps per treatment with egg per replicatin). a-c Means with different letters are significantly different at p<0.05 Fig. 4: Percentage hatchability f ttal eggs set fr cntrl and treated eggs. Means are nt different at p>0.9; n = 3 incubatrs cntaining apprximately 300 eggs each per treatment RESULTS treated eggs that were stred fr d when cmpared Effects f HO and UV light sanitizatin n average t treated eggs sampled immediately fllwing APC cunts frm briler breeder hatching eggs are given in Fig.. APC frm eggs treated with the cmbinatin f HO and UV light were significantly lwer than the cntrl eggs. There was a.8 lg0cfu/egg reductin in APC n the treated eggs when cmpared t the cntrl eggs. An interactin between the sanitizatin treatment and egg strage time was bserved fr the APC enumeratin data. A significant micrbial reductin during egg strage was bserved in treated eggs but nt cntrl eggs (Fig. 3). A.5 lg0cfu/egg reductin was fund in treatment. There were n differences between treatments fr infertile eggs, early dead (-7 d), mid dead (8-4 d), late dead (5- d), r pipped embrynic mrtalities (Table ). There were als n treatment effects bserved fr chick weight, egg weight lss and mecnium samples psitive fr micrbial grwth (Table ). In additin, when cmparing hatchability f ttal eggs set and hatchability f fertilized eggs, n significant treatment effects were bserved (Fig. 4 and 5). 3

4 Int. J. Pult. Sci., 0 (4): 30-34, 0 Fig. 5: Percentage hatchability f ttal fertilized eggs fr cntrl and treated eggs. Means are nt different at p>0.7; n = 3 incubatrs cntaining apprximately 300 eggs each per treatment DISCUSSION As shwn by Wells et al. (008) using White Leghrn eggs, the cmbinatin f UV light and HO effectively reduced micrbial cntaminatin fund n the surface f briler breeder eggshells. Wells et al. (008) demnstrated a 3.3 lg0 CFU/egg reductin in APC when treating White Leghrn eggs with.5% HO and 8 min f UV light. Hwever, in the current study, there was nly a.8 lg0 CFU/egg reductin in APC with the use f this treatment. The lwer reductin in the current study can likely be attributed t the different surces f eggs between the tw studies. In Wells et al. (008), the White Leghrn eggs were frm caged hens which had.9 lg0 CFU/egg fewer APC than the briler breeder eggs used in this experiment, which were frm litter and slat-flred cmmercial huses equipped with mechanical nest cllectin systems. Therefre, the briler breeder eggs cntained higher initial micrbial cunts and likely greater amunts f rganic material that wuld als react with the HO. Hwever, bth afrementined studies suggest that the frmatin f hydrxyl radicals, which ccurs when HO is split by UV light, is effective at prducing a rapid kill f bacteria, as suggested by Bayliss and Waites (98). Eggs that were treated and then stred fr d in the cler als had significantly lwer bacterial cunts than freshly treated eggs. Pssibly the cmbinatin f UV and HO damaged the bacterial cell wall (Ku et al., 997), which in turn caused the bacteria t be unable t withstand clder temperatures. Thieringer et al. (998) suggested that the stress f lw temperatures can hinder stabilizatin f DNA and RNA secndary structures in bacteria. Phadtare et al. (999) als suggested that clder temperatures can cause DNA replicatin t be less efficient in bacteria. If the treatment f UV and HO had already caused thymine dimers t frm n DNA strands (Bachmann, 975), then it may be pssible that nce eggs were placed int the cler, stabilizatin was impssible fr the bacteria, which resulted in their death. The data frm this experiment als demnstrated that treatment did nt affect hatchability. In ther experiments using nly UV light t sanitize eggs, researchers als demnstrated n effects n hatchability. Berrang et al. (995) shwed that the expsure f briler breeder eggshells t cntinuus UV light at 54 nm ver the entire d f incubatin demnstrated n effect n hatchability. Hwever, when using hatching eggs that were treated with a cmmercial sanitizer, % frmalin, r water and then incubated in an incubatr equipped with a UV light/air filtering system there was a significant increase in embry viability (Sctt, 993). Hwever, in the current study, n significant increase in embry viability r hatchability was bserved. Cx et al. (000) suggested that as sn as eggs are laid in nest bxes they cme in cntact with bacteria. Williams et al. (968) demnstrated that Salmnella was able t penetrate the cuticle and enter the shell almst immediately after expsing the shell t bacteria. Hwever, the Salmnella used in that experiment was inculated nt the egg using a liquid. The liquid may have facilitated the entry f the bacteria int the egg thrugh the eggshell pres. In a cmmercial setting, eggs are rarely submersed in water. Therefre, the bacteria that these eggs came in cntact with in the briler breeder huse may nt have penetrated the cuticle and shell s easily, resulting in a lw challenge f internal cntaminatin in the cntrl grup. Additinally, the fertility in bth cntrl and treated eggs frm this experiment was extremely lw. The percentage f infertile eggs fr the cntrl and treated eggs was 5 and 8%, respectively. This excessive infertility may have hindered ur ability t detect a significant effect n hatchability. In cnclusin, eggshell surface micrbial cunts were significantly lwered withut negatively affecting hatchability. ACKNOWLEDGEMENTS This research was supprted by a grant supplied by the US Pultry and Egg Assciatin (Tucker, GA). REFERENCES Bachmann, R., 975. Sterilizatin by intense ultravilet radiatin. Brwn Bveri Rev., 6: Bayliss, C. and W.M. Waites, 98. Effect f simultaneus high intensity ultravilet irradiatin and hydrgen perxide n bacterial spres. J. Fd. Technl., 7: Berrang, M., N.A. Cx and J.S. Bailey, 995. Efficacy f ultravilet light fr eliminatin f Salmnella n briler hatching eggs. J. Appl. Pult. Res., 4: Berrang, M.E., N.A. Cx, J.F. Frank and R.J. Buhr, 999. Bacterial penetratin f the eggshell and shell membranes f the chicken hatching egg: A review. J. Appl. Pult. Res., 8:

5 Int. J. Pult. Sci., 0 (4): 30-34, 0 Cx, N., M.E. Berrang and J.A. Casn, 000. Salmnella Sctt, T., 993. The effect f ultravilet light and air penetratin f egg shells and prliferatin in briler filtering system n embry viability and hatching eggs: A review. Pult. Sci., 79: micrrganism lad n the egg shell. J. Appl. Pult. Gerzen, P. and T.A. Sctt, 995. Ultravilet light Res., : 9-5. sanitatin fr briler hatching eggs. Pult. Sci., 74: Steel, R.G.D. and J.H. Trrie, 980. Principles and 83. Prcedures f Statistics. A Bimetrical Apprach. Ku, F., S.C. Ricke and J.B. Carey, 997. Shell egg nd Edn., McGraw Hill, New Yrk. sanitatin: Ultravilet radiatin and egg rtatin t Thieringer, H.A., P.G. Jnes and M. Inuye, 998. Cld effectively reduce ppulatins f aerbes, yeasts shck and adaptatin. Biessays, 0: and mlds. J. Fd Prt., 60: Wells, J., C. Cufal, H. Parker and C. McDaniel, 008. Padrn, M., 995. Egg dipping in hydrgen perxide Disinfectin f eggshells using ultravilet light and slutin t eliminate Salmnella typhimurium frm hydrgen perxide independently and in eggshell membranes. Avian Dis., 39: cmbinatin. Pult. Sci., 87(Suppl. ): 43. Phadtare, S., J. Alsina and M. Inuye, 999. Cld-shck Williams, J., l. Dillard and G. Hall, 968. The penetratin respnse and cld-shck prtein. Current Opinin patterns f Salmnella typhimurium thrugh the in Micr., : uter structures f chicken eggs. Avian Dis., : Sander, J.E. and J.L. Wilsn, 999. Effect f hydrgen perxide disinfectin during incubatin f chicken Wilsn, H., 997. Hatching egg sanitatin. University f eggs n micrbial levels and prductivity. Avian Flrida, Institute f Fd and Agricultural Sciences Dis., 43: (UF/IFAS). *Apprved fr publicatin as Jurnal Article N. J-0000 f the Mississippi Agriculture and Frestry Experiment Statin, Mississippi State University. **T whm crrespndence and reprint request shuld be addressed. cmcdaniel@pultry.msstate.edu. Phne: (66) , FAX: (66)

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