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The Role of The Cuticle in Water Vapor Conductance by the Eggshell of Broiler Breeders1,2
EDUCATION AND PRODUCTION The Role of The Cuticle in Water Vapor Conductance by the Eggshell of Broiler Breeders1'2 E. D. PEEBLES and J. BRAKE 3 Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7608 (Received for publication September 26, 1985) ABSTRACT The effects of cuticle removal on the water vapor conductance (G) by eggshells from broiler breeders were determined in five experiments. The efficacy of washing eggs in sodium hypochlorite (NaCIO) as a means to remove cuticle was demonstrated in Experiment 1. Birds housed in slat litter pens were used in Experiment 2, 4, and 5, while individually caged birds were used in Experiment 3. Conductance measurements before and after cuticle removal were made in desiccators in Experiments 2, 3, and 4, and G measurements, with and without the cuticle, were made in incubators in Experiment 5. Conductance before and after cuticle removal was measured at various time periods between Weeks 33 and 66. Water vapor conductance was increased to a highly significant degree after cuticle removal at Weeks 54, 58, and 61 in Experiment 2. Conductance was significantly higher after cuticle removal at Weeks 42, 46, 50, and 54 and significantly lower after cuticle removal at Weeks 38 and 64 in Experiment 3. Conductance was significantly higher after cuticle removal at Weeks 33, 37, 45, 49, and 61 and significantly lower after cuticle removal at Week 65 in Experiment 4. The greatest changes in conductance with cuticle removal (AG) were + 1.80 at Week 33 and —2.40 at Week 65 in Experiment 4. In Experiment 5, G was significantly higher with the cuticle removed than it was with the cuticle intact at Weeks 48, 54, and 66. It was concluded that the cuticle may either impede or enhance water vapor diffusion as a function of a bird's age. The influence of the cuticle is, in turn, affected by the humidity of the air surrounding the egg. (Key words: broiler breeders, conductance, cuticle) 1986 Poultry Science 65:1034-1039 INTRODUCTION
The avian eggshell is both a conduit through which diffusive water vapor, oxygen, and carbon dioxide are permitted to pass and a barrier to the free diffusion of these same substances (Rahn et al, 1979; Rahn, 1981). In order to hatch, an egg must lose 12 to 15% of its weight primarily as water during incubation and maintain proper vital gas exchange (Rahn et al, 1979; Tazawa, 1980). The major role of resistance to this exchange has been attributed to the CaC0 3 portion of the shell with lesser contributions from the shell membranes and cuticle (Tullett, 1978; Rahn et al, 1979). A majority of the scientific literature assigns an insignificant contribution by the cuticle to vital
'Paper No. 10117 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. 2 The use of trade names in this paper does not constitute endorsement of products mentioned, nor criticism of similar products not mentioned. 3 To whom correspondence should be addressed.
gas and water vapor diffusion (Wangensteen et al, 1970/71; Paganelli et al, 1978; Rahn et al, 1979; Kayar et al, 1981; Board, 1982; Tranter et al, 1983; Sparks and Board, 1984). However, there are other reports in the literature that regard the cuticle as an influence on gas exchange. The cuticle has been described as a physical barrier to water vapor loss (Simons, 1971; Board and Halls, 1973; Meir et al, 1984a) and vital gas exchange (Fromm, 1963; Romanoff, 1943), but earlier workers (Bryant and Sharp, 1934; Marshall and Cruickshank, 1938) have reported that the cuticle may actually enhance evaporative water loss. The majority of these studies were made at a single point in time with respect to bird age, and no attempts were made to look at different bird ages. Because eggshell quality and hatchability of broiler breeder eggs are different between ages (McDaniel et al, 1979), it was postulated that the cuticle itself may contribute to this dynamic system and exhibit changes over a production cycle that would explain the discrepancies in the literature. The cuticle has been reported to
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CUTICLE AND CONDUCTANCE
contribute to water conservation differently at different humidities (Board and Halls, 1973). This variability would imply that the method of G measurement before and after cuticle removal (i.e., desiccator vs. incubator) is an important factor in determining the role of the cuticle as a diffusion barrier. The objectives of the present study were to assess the contribution of the cuticle to G at various flock ages and to determine the effects of measuring G with and without the cuticle under humid and dry conditions. MATERIALS AND METHODS
General. Arbor Acres female broiler breeders were utilized in five separate experiments. Birds were reared with standard husbandry practices for floor-reared, feed-restricted pullets (Brake et al, 1984), housed and photostimulated (14L:10D) at 20 weeks of age and maintained in either slat litter pens or individual cages. All birds were fed a basal corn-soy mash ration (Brake et al, 1984) with amounts of feed adjusted to maintain the body weights recommended by Arbor Acres (1982). Egg weight was determined on each egg on the day of oviposition and G (mg H 2 0 / d a y / Torr) was determined in a desiccator by the method of Ar et al. (1974) at a constant temperature of 25 ± 1 C on the succeeding 4 days. Egg weight loss was adjusted to a 24-hr basis and a standard barometric pressure of 760 Torr (mm Hg). Eggs were washed in an egg washer containing either water or 2500 mg sodium hypochlorite (NaClO; SHS-900, Oxford Chemicals, Inc., Atlanta, GA) per liter of solution at 40 C for 5 min. The eggs were rinsed with tap water and allowed to dry at 20 to 25 C after washing. The H 2 0 G was again determined on the same eggs by the method of Ar etal (1974). Experiment 1. Eggs were collected from broiler breeders in slat litter pens at 49 weeks of age. A total of 30 nest-laid eggs were collected at random and then divided into control, water washed, and NaClO solution-washed groups with 10 eggs/group. Experiment 2. Eggs were collected from broiler breeders in slat litter pens at 54, 58, and 61 weeks of age during September and October. A total of 36 nest-laid eggs were collected at random at each sample time. Experiment 3. Eggs were collected from individually caged broiler breeders at 34, 38,
42, 46, 50, 54, 58, 62, and 64 weeks of age during the period from December through July. A total of 46 eggs were sampled at random at each time. Experiment 4. Birds from the same growing flock as those of Experiment 3 were placed in slat litter pens in a house adjacent to the flock used for Experiment 3. A total of 36 nest-laid eggs were collected at random from these birds at 4-week intervals between 33 and 65 weeks of age. Experiment 5. Eggs were collected at random at 48, 54, and 66 weeks of age from the same birds as those in Experiment 4. In this experiment, however, G for each egg was measured in a Jamesway 252B incubator at 37.5 C dry bulb and 28.3 C wet bulb over a 17-day period (Butler Mfg. Co., Ft. Atkinson, WI) according to the methods of Paganelli (1980) and Meir et al (1984b). A total of 70 eggs in each of the cuticle-intact and cuticleremoved groups were used at each sample period. Statistical Analyses. Data relevant to the comparison of G before and after cuticle removal were arranged in a randomized block design with individual eggs used as a block and were subjected to one-way analysis of variance. Data concerning the change in G due to cuticle removal (AG) were subjected to one-way analysis of variance to assess effects due to time (week). Where appropriate, differences among means were partitioned using Duncan's new multiple range test (SAS Institute, 1982). Slopes of the changes in AG in Experiment 2 and 3 were calculated by linear regression. Statements of statistical significance are based on P<.05 unless otherwise indicated. RESULTS
Experiment 1. Water vapor conductance increased in eggs after being washed in water and NaClO solution, whereas G decreased in the unwashed eggs. These changes were significant for the unwashed and NaClO-washed groups. The change in G due to cuticle removal was significantly higher in the washed than in the unwashed eggs. The change in G due to cuticle removal was numerically higher in NaClO than in water-washed eggs (Table 1). This method appeared to be effective in removing the cuticle, based on the results of Experiment 1 showing AG to be significantly higher in the NaClO-washed eggs than in unwashed controls.
PEEBLES AND BRAKE
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TABLE 1. Water vapor conductance (G) of broiler breeder hatching eggs before and after washing (Experiment 1) G1 T y p e of wash
Before
After
AG
C o n t r o l , unwashed Water NaCIO 2
8.51A 8.33A 7.79B
8.00B 8.63A 8.27A
-.51b + .30a + .48a
AR
' M e a n s within a treatment for G different superscripts differ significantly a 'b Means within a column for AG different superscripts differ significantly
which possess (P<.05). which possess (P=S.0005).
1 Expressed as mg H, O/day/Torr either before or after cuticle removal. 2
Sodium hypochlorite.
Experiment 2. C o n d u c t a n c e was increased to a very highly significant degree after cuticle removal at Weeks 54, 5 8 , and 6 1 . At Week 5 4 , AG was significantly higher t h a n at either Weeks 58 or 6 1 , while AG at Weeks 58 and 61 were n o t significantly different (Table 2 ) . Experiment 3. C o n d u c t a n c e was significantly higher after cuticle removal at Weeks 4 2 , 4 6 , 50, and 54. At Weeks 38 and 6 4 , G was significantly higher before cuticle removal t h a n it was after cuticle removal. This result is indicated by t h e negative AG values at these 2 weeks. T h e change in G d u e t o cuticle removal ranged from + 1.79 at Week 4 2 t o - 1 . 2 9 at Week 6 4 (Table 3).
Experiment 4. C o n d u c t a n c e was significantly higher after cuticle removal at Weeks 3 3 , 37, 4 5 , 4 9 , and 6 1 . C o n d u c t a n c e was significantly higher before cuticle removal t h a n it was after cuticle removal a t Week 6 5 . T h e change in G d u e t o cuticle removal ranged from + 1.80 at Week 37 t o - 2 . 4 0 at Week 65 (Table 4). Experiment 5. C o n d u c t a n c e in an i n c u b a t o r was significantly higher for eggs with cuticle removed at Weeks 4 8 , 54, and 66 (Table 5). DISCUSSION Water vapor c o n d u c t a n c e was generally increased b y cuticle removal. In a t o t a l of 2 4 tests, G increased significantly with cuticle removal 15 times. This suggests cuticle removal t o b e a m e a n s t o increase G. However, G was significantly higher before t h a n after cuticle removal at Weeks 38 and 6 4 in E x p e r i m e n t 2 a n d a t Week 65 in E x p e r i m e n t 3. In Experim e n t 1, t h e G of unwashed control eggs was found t o be t h e lower o n t h e second meas u r e m e n t . This, in c o m b i n a t i o n with altered cuticle m o r p h o l o g y , m a y a c c o u n t for part of t h e decrease in G with cuticle removal at these times in E x p e r i m e n t s 2 and 3. T h e slopes of t h e changes in AG w i t h increasing bird age in E x p e r i m e n t s 2 and 3 were similar (—.031 and
TABLE 3. Water vapor conductance (G) of broiler hatching eggs from individually caged hens before and after cuticle removal (Experiment 3) G1
TABLE 2. Water vapor conductance (G) of broiler hatching eggs before and after cuticle removal (Experiment 2) G
Age
1
Before
After
AG
12.05 B 13.87 B 13.24 B
13.84A 14.45 A 13.91 A
+1.79 a +.58 b +.67 b
(wk)
54 58 61
A,BMeans within a week for G which possess different superscripts differ significantly (P<.0001). a ' b Means within a column for AG which possess different superscripts differ significantly (P<.05). 'Expressed as mg H 2 0/day/Torr either before or after cuticle removal.
Age
Before
After
AG
11.31A 11.50A 11.05B 11.54B 11.78B 11.30B 12.57A 12.31A 10.80A
11.36A 11.26B 12.85A 12.34A 12.28A 12.52A 12.53A 12.39A 9.63B
+.03de -.24e +1.79a +.93bc +.45cd + 1.22D -.04de +.07de -1.29f
(wk)
34 38 42 46 50 54 58 62 64
A ' B Means within a week for G which possess different superscripts differ significantly (P<.01). a—f Means within a column for AG which possess different superscripts differ significantly (P<.05). 'Expressed as mg H 2 0/day/Torr either before or after cuticle removal.
CUTICLE AND CONDUCTANCE TABLE 4. Water vapor conductance (G) of broiler hatching eggs from slat and floor pen hens and after cuticle removal (Experiment 4) G1 Age
Before
After
AG
10.47 B 9.73 B 11.40 A 10.58 B 10.96 B 11.40A 12.24A 11.48 B 12.HA
11.48 A 11.54 A 12.38 A 11.80 A U.82A 12.08 A 12.73 A 12.46 A 9.40 B
+1.01 b + 1.80a +.98 b +1.23 a b +.87 b +.67 b +.50 b +.98 b -2.40c
(wk)
' Means within a week for G which possess different superscripts differ significantly (P<.05). a ' ' c Means within a column for AG which possess different superscripts differ significantly (P<.05).
1 Expressed as mg H2 O/day/Torr either before or after cuticle removal.
—.073, respectively). This indicated that the resistance of the cuticle to water loss tended to decrease with breeder age. This may be a function of cuticle morphology per se, or an interaction of cuticle with changing shell morphology. Because the influence of the cuticle on water vapor diffusion changes over the production cycle, cuticle removal may increase G only at certain times during production. These changes, particularly at the end of production, may explain some of the discrepancies reported in the literature as to the functionality of the cuticle as a barrier. Ball et al. (1975) found hen age (22, 50, 59, and 68 weeks of age) to have no effect on amount of cuticle on SCWL eggs. However, the structure or morphology of the cuticle is known to differ among avian species (Board, 1982) and can change with length of egg storage (Simons, 1971; Ballet al., 1975). It may, therefore, follow that morphology, as well as amount of cuticle in broiler breeders, may change over the production cycle and thereby change its influence on water vapor diffusion. At the end of production, a cuticle that is thinner or different in morphology may actually promote G, allowing an egg to have a high G than one with the cuticle removed (Bryant and Sharp, 1934; Marshall and Cruickshank, 1938). Board and Scott (1980) suggest that fissures or cracks in the cuticle link
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the lumens of pore canals to the exterior of the egg and provide pathways for diffusion of gases. It is postulated that fissured cuticles may contribute to the increased G of eggs late in production. Egg weight is known to increase with age in broiler breeders; as seen in the data of the present study and as egg weight and shell surface area increase, there is a proportional increase in G (Tullet, 1978). Our data are in general agreement with those of Tullett (1978); however, despite increased surface area, decreases in G before cuticle removal were evident at some weeks. Changes in cuticle morphology may be partly responsible, because declines in G with bird age were often accompanied with an increase in AG. The decrease in G at Week 64 in Experiment 3 was unexpected, based on other unpublished data from these eggs that showed that shell pore concentration was higher, whereas shell thickness was the same in comparison to eggs at Week 62. In addition, AG was negative at this time in the present study. Because G is proportional to total functional pore area and inversely proportional to shell thickness (Ar et al., 1974), smaller pore diameter is suggested as an explanation for the above result. It was noteworthy that AG varied in magnitude between Experiments 3 and 4 at certain times and that the floor birds of Experiment 4 usually exhibited a lower initial G. The birds in these experiments were from the same flock but were housed differently. The different means of confinement may have allowed the
TABLE 5. Water vapor conductance (G1) of broiler hatching eggs with and without the cuticle in an incubator (Experiment 5) Cuticle Age
Intact
Removed
17.76 B 19.11 B 22.18 B
19.39 A 21.00 A 26.92A
(wk) 48 54 66
' Means within a week for G which possess different superscripts differ significantly (P>C.01). 'Expressed as mg H 2 0/day/Torr either with or without the cuticle. Eggs kept in incubator maintained at 37.5 C dry bulb and 28.3 C wet bulb for 17 days.
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PEEBLES AND BRAKE
floor hens t o recycle additional n u t r i e n t s from t h e litter, such as calcium, so as t o alter shell and cuticle m o r p h o l o g y . Board and Halls ( 1 9 7 3 ) r e p o r t t h a t t h e cuticle c o n t r i b u t e s t o water conservation only w h e n eggs are stored at low humidities, and using a desiccated env i r o n m e n t in which t o measure G, Meir et al. ( 1 9 8 4 a ) showed significant differences in G before and after cuticle removal. However, Sparks and Board ( 1 9 8 4 ) were u n a b l e to show an appreciable difference in G before and after cuticle removal using t h e same m e t h o d . In E x p e r i m e n t 4, significant differences in G, before and after cuticle removal, were observed in a desiccated environment. Similarly, in E x p e r i m e n t 5 in which t h e same birds were used, significant differences in G b e t w e e n eggs with t h e cuticle intact and with t h e cuticle removed were observed in incubators having high humidities. These results are consistent with t h e observations b y Christensen and Bagley ( 1 9 8 4 ) w i t h t u r k e y eggs. T h e influence of t h e cuticle o n G, however, differed b e t w e e n t h e t w o m e t h o d s of m e a s u r e m e n t at similar times in t h e p r o d u c t i o n cycle. With t h e use of desiccators, decreases in G with cuticle removal occurred late (Weeks 6 4 a n d 6 5 ) , b u t this was n o t t r u e if an i n c u b a t o r was used. H u m i d i t y obviously has an effect on t h e character of t h e cuticle, changing its resistance to water loss from t h e egg. These effects, however, again appear to be influenced b y bird age. Higher levels of G in E x p e r i m e n t 5, compared w i t h those in E x p e r i m e n t 4 , m a y have been a result of measuring G in an i n c u b a t o r (37.5 C) r a t h e r t h a n a desiccator (25 C). According to t h e modification of F i c k ' s first law of diffusion b y Wangensteen et al. ( 1 9 7 0 / 7 1 ) , an alteration in t h e diffusion coefficient of w a t e r vapor in air would affect G (Ar et al, 1 9 7 4 ) . B o l t o n ( 1 9 5 7 ) similarly observed higher weight losses from eggs k e p t in i n c u b a t o r s vs. those k e p t in desiccators at lower t e m p e r a t u r e s . In conclusion, t h e cuticle m a y act as a barrier t o w a t e r vapor diffusion or act t o e n h a n c e water vapor diffusion, and these properties m a y be affected b y t h e age of t h e b i r d . T h e h u m i d i t y of t h e air a r o u n d t h e egg m a y likewise be an i m p o r t a n t factor in predicting t h e effects of cuticle removal o n G and o n e m b r y o n i c d e v e l o p m e n t during incubation. ACKNOWLEDGMENTS. T h e a u t h o r s express appreciation to J i m m y H i n n a n t and J a m e s Bronson for animal care and
t o Grace B r o c k m a n for e x p e r t technical assistance.
REFERENCES Ar, A., C. V. Paganelli, R. B. Reeves, D. G. Greene, and H. Rahn, 1974. The avian egg: Water vapor conductance, shell thickness and functional pore area. Condor 76:153-158. Arbor Acres, 1982. Broiler breeder male and female feeding and management guide. Arbor Acres Farm, Inc. Glastonbury, CT. Ball, R. F., V. Logan, and J. F. Hill, 1975. Factors affecting the cuticle of the egg as measured by intensity of staining. Poultry Sci. 54:1479—1484. Board, R. G., 1982. Properties of avian eggshells and their adaptive value. Biol. Rev. 57:1—28. Board, R. G., and N. A. Halls, 1973. The cuticle: a barrier to liquid and particle penetration of the shell of the hen's egg. Br. Poult. Sci. 14:69-97. Board, R. G., and V. D. Scott, 1980. Porosity of the avian eggshell. Am. Zool. 20:339-349. Bolton, W., 1957. The effect of the age of the hen on the porosity of the egg. J. Agric. Sci. 49:384— 386. Brake, J., J. F. Ort, T. A. Carter, and W. R. Campbell, 1984. Effect of the insect growth regulator CGA-72662 (Larvadex®) on broiler breeder production, hatchability, and subsequent chick performance. Poultry Sci. 63:910—916. Bryant, R. L., and P. F. Sharp, 1934. Effect of washing on the keeping quality of hen's eggs. J. Agric. Res. 4 8 : 6 7 - 8 9 . Christensen, V. L., and R. A. Bagley, 1984. Vital gas exchange and hatchability of turkey eggs at high altitude. Poultry Sci. 63:1350-1356. Fromm, D., 1963. Permeability of the hen's eggshell. Poultry Sci. (Abstr.) 42:1271. Kayar, S. R., G. K. Snyder, G. F. Birchard, and C. P. Black, 1981. Oxygen permeability of the shell and membranes of chicken eggs during development. Respir. Physiol. 4 6 : 2 0 9 - 2 2 1 . Marshall, W., and D. B. Cruickshank, 1938. The function of the cuticle in relation to the porosity of eggs. J. Agric. Sci. 28:24-42. McDaniel, G. R., D. A. Roland, Sr., and M. A. Coleman, 1979. The effect of eggshell quality on hatchability and embryonic mortality. Poultry Sci. 5 8 : 1 0 - 1 3 . Meir, M., A. Ar, and A. Nir, 1984a. Preincubation dipping of turkey eggs. Does it affect eggshell conductance? Poultry Sci. 63:2475-2478. Meir, M., A. Nir, and A. Ar, 1984b. Increasing hatchability of turkey eggs by matching incubator humidity to shell conductance of individual eggs. Poultry Sci. 63:1489-1496. Paganelli, C. V., 1980. The physics of gas exchange across the avian eggshell. Am. Zool. 20:329—338. Paganelli, C. V., R. A. Ackerman, and H. Rahn, 1978. The avian egg: in vivo conductances to oxygen, carbon dioxide, and water vapor in late development. Pages 212—218 in Respiratory Function in Birds, Adult and Embryonic. J. Piiper, ed. Springer-Verlag, Berlin, Germany. Rahn, H., 1981. Gas exchange of avian eggs with special reference to turkey eggs. Poultry Sci. 60:1971-1980. Rahn, H., A. Ar, and C. V. Paganelli, 1979. How bird
CUTICLE AND CONDUCTANCE eggs breathe. Sci. Am. 240:46-55. Romanoff, A. L., 1943. Study of various factors affecting permeability of birds' eggshell. Food Res. 5:212-223. SAS Institute, Inc., 1982. SAS User's Guide: Basics. SAS Inst., Cary, NC 27511. Simons, P.C.M., 1971. Ultrastructure of the hen eggshell and its physiological interpretation. Centre for Agricultural Publishing and Documentation, Wageningen, Netherlands. Sparks, N.H.C, and R. G. Board, 1984. Cuticle, shell porosity and water uptake through hens' eggshells. Br. Poult. Sci. 25:267-276. Tazawa, H., 1980. Oxygen and C0 2 exchange and
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acid-base regulation in the avian embryo. Am. Zool. 20:395-404. Tranter, H. S., N.H.C. Sparks, and R. G. Board, 1983. Changes in structure of the limiting membrane and in oxygen permeability of the chicken egg integument during incubation. Br. Poult. Sci. 24:5 37-547. Tullett, S. G., 1978. Pore size versus pore number in avian eggshells. Pages 217—226 in Respiratory Function in Birds, Adult and Embryonic. J. Piiper, ed. Springer, Berlin, Germany. Wangensteen, O. D., D. Wilson, and H. Rahn, 1970/71. Diffusion of gases across the shell of the hen's egg. Respir. Physiol. 11:16—30.
The avian eggshell is both a conduit through which diffusive water vapor, oxygen, and carbon dioxide are permitted to pass and a barrier to the free diffusion of these same substances (Rahn et al, 1979; Rahn, 1981). In order to hatch, an egg must lose 12 to 15% of its weight primarily as water during incubation and maintain proper vital gas exchange (Rahn et al, 1979; Tazawa, 1980). The major role of resistance to this exchange has been attributed to the CaC0 3 portion of the shell with lesser contributions from the shell membranes and cuticle (Tullett, 1978; Rahn et al, 1979). A majority of the scientific literature assigns an insignificant contribution by the cuticle to vital
'Paper No. 10117 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. 2 The use of trade names in this paper does not constitute endorsement of products mentioned, nor criticism of similar products not mentioned. 3 To whom correspondence should be addressed.
gas and water vapor diffusion (Wangensteen et al, 1970/71; Paganelli et al, 1978; Rahn et al, 1979; Kayar et al, 1981; Board, 1982; Tranter et al, 1983; Sparks and Board, 1984). However, there are other reports in the literature that regard the cuticle as an influence on gas exchange. The cuticle has been described as a physical barrier to water vapor loss (Simons, 1971; Board and Halls, 1973; Meir et al, 1984a) and vital gas exchange (Fromm, 1963; Romanoff, 1943), but earlier workers (Bryant and Sharp, 1934; Marshall and Cruickshank, 1938) have reported that the cuticle may actually enhance evaporative water loss. The majority of these studies were made at a single point in time with respect to bird age, and no attempts were made to look at different bird ages. Because eggshell quality and hatchability of broiler breeder eggs are different between ages (McDaniel et al, 1979), it was postulated that the cuticle itself may contribute to this dynamic system and exhibit changes over a production cycle that would explain the discrepancies in the literature. The cuticle has been reported to
1034
1035
CUTICLE AND CONDUCTANCE
contribute to water conservation differently at different humidities (Board and Halls, 1973). This variability would imply that the method of G measurement before and after cuticle removal (i.e., desiccator vs. incubator) is an important factor in determining the role of the cuticle as a diffusion barrier. The objectives of the present study were to assess the contribution of the cuticle to G at various flock ages and to determine the effects of measuring G with and without the cuticle under humid and dry conditions. MATERIALS AND METHODS
General. Arbor Acres female broiler breeders were utilized in five separate experiments. Birds were reared with standard husbandry practices for floor-reared, feed-restricted pullets (Brake et al, 1984), housed and photostimulated (14L:10D) at 20 weeks of age and maintained in either slat litter pens or individual cages. All birds were fed a basal corn-soy mash ration (Brake et al, 1984) with amounts of feed adjusted to maintain the body weights recommended by Arbor Acres (1982). Egg weight was determined on each egg on the day of oviposition and G (mg H 2 0 / d a y / Torr) was determined in a desiccator by the method of Ar et al. (1974) at a constant temperature of 25 ± 1 C on the succeeding 4 days. Egg weight loss was adjusted to a 24-hr basis and a standard barometric pressure of 760 Torr (mm Hg). Eggs were washed in an egg washer containing either water or 2500 mg sodium hypochlorite (NaClO; SHS-900, Oxford Chemicals, Inc., Atlanta, GA) per liter of solution at 40 C for 5 min. The eggs were rinsed with tap water and allowed to dry at 20 to 25 C after washing. The H 2 0 G was again determined on the same eggs by the method of Ar etal (1974). Experiment 1. Eggs were collected from broiler breeders in slat litter pens at 49 weeks of age. A total of 30 nest-laid eggs were collected at random and then divided into control, water washed, and NaClO solution-washed groups with 10 eggs/group. Experiment 2. Eggs were collected from broiler breeders in slat litter pens at 54, 58, and 61 weeks of age during September and October. A total of 36 nest-laid eggs were collected at random at each sample time. Experiment 3. Eggs were collected from individually caged broiler breeders at 34, 38,
42, 46, 50, 54, 58, 62, and 64 weeks of age during the period from December through July. A total of 46 eggs were sampled at random at each time. Experiment 4. Birds from the same growing flock as those of Experiment 3 were placed in slat litter pens in a house adjacent to the flock used for Experiment 3. A total of 36 nest-laid eggs were collected at random from these birds at 4-week intervals between 33 and 65 weeks of age. Experiment 5. Eggs were collected at random at 48, 54, and 66 weeks of age from the same birds as those in Experiment 4. In this experiment, however, G for each egg was measured in a Jamesway 252B incubator at 37.5 C dry bulb and 28.3 C wet bulb over a 17-day period (Butler Mfg. Co., Ft. Atkinson, WI) according to the methods of Paganelli (1980) and Meir et al (1984b). A total of 70 eggs in each of the cuticle-intact and cuticleremoved groups were used at each sample period. Statistical Analyses. Data relevant to the comparison of G before and after cuticle removal were arranged in a randomized block design with individual eggs used as a block and were subjected to one-way analysis of variance. Data concerning the change in G due to cuticle removal (AG) were subjected to one-way analysis of variance to assess effects due to time (week). Where appropriate, differences among means were partitioned using Duncan's new multiple range test (SAS Institute, 1982). Slopes of the changes in AG in Experiment 2 and 3 were calculated by linear regression. Statements of statistical significance are based on P<.05 unless otherwise indicated. RESULTS
Experiment 1. Water vapor conductance increased in eggs after being washed in water and NaClO solution, whereas G decreased in the unwashed eggs. These changes were significant for the unwashed and NaClO-washed groups. The change in G due to cuticle removal was significantly higher in the washed than in the unwashed eggs. The change in G due to cuticle removal was numerically higher in NaClO than in water-washed eggs (Table 1). This method appeared to be effective in removing the cuticle, based on the results of Experiment 1 showing AG to be significantly higher in the NaClO-washed eggs than in unwashed controls.
PEEBLES AND BRAKE
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TABLE 1. Water vapor conductance (G) of broiler breeder hatching eggs before and after washing (Experiment 1) G1 T y p e of wash
Before
After
AG
C o n t r o l , unwashed Water NaCIO 2
8.51A 8.33A 7.79B
8.00B 8.63A 8.27A
-.51b + .30a + .48a
AR
' M e a n s within a treatment for G different superscripts differ significantly a 'b Means within a column for AG different superscripts differ significantly
which possess (P<.05). which possess (P=S.0005).
1 Expressed as mg H, O/day/Torr either before or after cuticle removal. 2
Sodium hypochlorite.
Experiment 2. C o n d u c t a n c e was increased to a very highly significant degree after cuticle removal at Weeks 54, 5 8 , and 6 1 . At Week 5 4 , AG was significantly higher t h a n at either Weeks 58 or 6 1 , while AG at Weeks 58 and 61 were n o t significantly different (Table 2 ) . Experiment 3. C o n d u c t a n c e was significantly higher after cuticle removal at Weeks 4 2 , 4 6 , 50, and 54. At Weeks 38 and 6 4 , G was significantly higher before cuticle removal t h a n it was after cuticle removal. This result is indicated by t h e negative AG values at these 2 weeks. T h e change in G d u e t o cuticle removal ranged from + 1.79 at Week 4 2 t o - 1 . 2 9 at Week 6 4 (Table 3).
Experiment 4. C o n d u c t a n c e was significantly higher after cuticle removal at Weeks 3 3 , 37, 4 5 , 4 9 , and 6 1 . C o n d u c t a n c e was significantly higher before cuticle removal t h a n it was after cuticle removal a t Week 6 5 . T h e change in G d u e t o cuticle removal ranged from + 1.80 at Week 37 t o - 2 . 4 0 at Week 65 (Table 4). Experiment 5. C o n d u c t a n c e in an i n c u b a t o r was significantly higher for eggs with cuticle removed at Weeks 4 8 , 54, and 66 (Table 5). DISCUSSION Water vapor c o n d u c t a n c e was generally increased b y cuticle removal. In a t o t a l of 2 4 tests, G increased significantly with cuticle removal 15 times. This suggests cuticle removal t o b e a m e a n s t o increase G. However, G was significantly higher before t h a n after cuticle removal at Weeks 38 and 6 4 in E x p e r i m e n t 2 a n d a t Week 65 in E x p e r i m e n t 3. In Experim e n t 1, t h e G of unwashed control eggs was found t o be t h e lower o n t h e second meas u r e m e n t . This, in c o m b i n a t i o n with altered cuticle m o r p h o l o g y , m a y a c c o u n t for part of t h e decrease in G with cuticle removal at these times in E x p e r i m e n t s 2 and 3. T h e slopes of t h e changes in AG w i t h increasing bird age in E x p e r i m e n t s 2 and 3 were similar (—.031 and
TABLE 3. Water vapor conductance (G) of broiler hatching eggs from individually caged hens before and after cuticle removal (Experiment 3) G1
TABLE 2. Water vapor conductance (G) of broiler hatching eggs before and after cuticle removal (Experiment 2) G
Age
1
Before
After
AG
12.05 B 13.87 B 13.24 B
13.84A 14.45 A 13.91 A
+1.79 a +.58 b +.67 b
(wk)
54 58 61
A,BMeans within a week for G which possess different superscripts differ significantly (P<.0001). a ' b Means within a column for AG which possess different superscripts differ significantly (P<.05). 'Expressed as mg H 2 0/day/Torr either before or after cuticle removal.
Age
Before
After
AG
11.31A 11.50A 11.05B 11.54B 11.78B 11.30B 12.57A 12.31A 10.80A
11.36A 11.26B 12.85A 12.34A 12.28A 12.52A 12.53A 12.39A 9.63B
+.03de -.24e +1.79a +.93bc +.45cd + 1.22D -.04de +.07de -1.29f
(wk)
34 38 42 46 50 54 58 62 64
A ' B Means within a week for G which possess different superscripts differ significantly (P<.01). a—f Means within a column for AG which possess different superscripts differ significantly (P<.05). 'Expressed as mg H 2 0/day/Torr either before or after cuticle removal.
CUTICLE AND CONDUCTANCE TABLE 4. Water vapor conductance (G) of broiler hatching eggs from slat and floor pen hens and after cuticle removal (Experiment 4) G1 Age
Before
After
AG
10.47 B 9.73 B 11.40 A 10.58 B 10.96 B 11.40A 12.24A 11.48 B 12.HA
11.48 A 11.54 A 12.38 A 11.80 A U.82A 12.08 A 12.73 A 12.46 A 9.40 B
+1.01 b + 1.80a +.98 b +1.23 a b +.87 b +.67 b +.50 b +.98 b -2.40c
(wk)
' Means within a week for G which possess different superscripts differ significantly (P<.05). a ' ' c Means within a column for AG which possess different superscripts differ significantly (P<.05).
1 Expressed as mg H2 O/day/Torr either before or after cuticle removal.
—.073, respectively). This indicated that the resistance of the cuticle to water loss tended to decrease with breeder age. This may be a function of cuticle morphology per se, or an interaction of cuticle with changing shell morphology. Because the influence of the cuticle on water vapor diffusion changes over the production cycle, cuticle removal may increase G only at certain times during production. These changes, particularly at the end of production, may explain some of the discrepancies reported in the literature as to the functionality of the cuticle as a barrier. Ball et al. (1975) found hen age (22, 50, 59, and 68 weeks of age) to have no effect on amount of cuticle on SCWL eggs. However, the structure or morphology of the cuticle is known to differ among avian species (Board, 1982) and can change with length of egg storage (Simons, 1971; Ballet al., 1975). It may, therefore, follow that morphology, as well as amount of cuticle in broiler breeders, may change over the production cycle and thereby change its influence on water vapor diffusion. At the end of production, a cuticle that is thinner or different in morphology may actually promote G, allowing an egg to have a high G than one with the cuticle removed (Bryant and Sharp, 1934; Marshall and Cruickshank, 1938). Board and Scott (1980) suggest that fissures or cracks in the cuticle link
1037
the lumens of pore canals to the exterior of the egg and provide pathways for diffusion of gases. It is postulated that fissured cuticles may contribute to the increased G of eggs late in production. Egg weight is known to increase with age in broiler breeders; as seen in the data of the present study and as egg weight and shell surface area increase, there is a proportional increase in G (Tullet, 1978). Our data are in general agreement with those of Tullett (1978); however, despite increased surface area, decreases in G before cuticle removal were evident at some weeks. Changes in cuticle morphology may be partly responsible, because declines in G with bird age were often accompanied with an increase in AG. The decrease in G at Week 64 in Experiment 3 was unexpected, based on other unpublished data from these eggs that showed that shell pore concentration was higher, whereas shell thickness was the same in comparison to eggs at Week 62. In addition, AG was negative at this time in the present study. Because G is proportional to total functional pore area and inversely proportional to shell thickness (Ar et al., 1974), smaller pore diameter is suggested as an explanation for the above result. It was noteworthy that AG varied in magnitude between Experiments 3 and 4 at certain times and that the floor birds of Experiment 4 usually exhibited a lower initial G. The birds in these experiments were from the same flock but were housed differently. The different means of confinement may have allowed the
TABLE 5. Water vapor conductance (G1) of broiler hatching eggs with and without the cuticle in an incubator (Experiment 5) Cuticle Age
Intact
Removed
17.76 B 19.11 B 22.18 B
19.39 A 21.00 A 26.92A
(wk) 48 54 66
' Means within a week for G which possess different superscripts differ significantly (P>C.01). 'Expressed as mg H 2 0/day/Torr either with or without the cuticle. Eggs kept in incubator maintained at 37.5 C dry bulb and 28.3 C wet bulb for 17 days.
1038
PEEBLES AND BRAKE
floor hens t o recycle additional n u t r i e n t s from t h e litter, such as calcium, so as t o alter shell and cuticle m o r p h o l o g y . Board and Halls ( 1 9 7 3 ) r e p o r t t h a t t h e cuticle c o n t r i b u t e s t o water conservation only w h e n eggs are stored at low humidities, and using a desiccated env i r o n m e n t in which t o measure G, Meir et al. ( 1 9 8 4 a ) showed significant differences in G before and after cuticle removal. However, Sparks and Board ( 1 9 8 4 ) were u n a b l e to show an appreciable difference in G before and after cuticle removal using t h e same m e t h o d . In E x p e r i m e n t 4, significant differences in G, before and after cuticle removal, were observed in a desiccated environment. Similarly, in E x p e r i m e n t 5 in which t h e same birds were used, significant differences in G b e t w e e n eggs with t h e cuticle intact and with t h e cuticle removed were observed in incubators having high humidities. These results are consistent with t h e observations b y Christensen and Bagley ( 1 9 8 4 ) w i t h t u r k e y eggs. T h e influence of t h e cuticle o n G, however, differed b e t w e e n t h e t w o m e t h o d s of m e a s u r e m e n t at similar times in t h e p r o d u c t i o n cycle. With t h e use of desiccators, decreases in G with cuticle removal occurred late (Weeks 6 4 a n d 6 5 ) , b u t this was n o t t r u e if an i n c u b a t o r was used. H u m i d i t y obviously has an effect on t h e character of t h e cuticle, changing its resistance to water loss from t h e egg. These effects, however, again appear to be influenced b y bird age. Higher levels of G in E x p e r i m e n t 5, compared w i t h those in E x p e r i m e n t 4 , m a y have been a result of measuring G in an i n c u b a t o r (37.5 C) r a t h e r t h a n a desiccator (25 C). According to t h e modification of F i c k ' s first law of diffusion b y Wangensteen et al. ( 1 9 7 0 / 7 1 ) , an alteration in t h e diffusion coefficient of w a t e r vapor in air would affect G (Ar et al, 1 9 7 4 ) . B o l t o n ( 1 9 5 7 ) similarly observed higher weight losses from eggs k e p t in i n c u b a t o r s vs. those k e p t in desiccators at lower t e m p e r a t u r e s . In conclusion, t h e cuticle m a y act as a barrier t o w a t e r vapor diffusion or act t o e n h a n c e water vapor diffusion, and these properties m a y be affected b y t h e age of t h e b i r d . T h e h u m i d i t y of t h e air a r o u n d t h e egg m a y likewise be an i m p o r t a n t factor in predicting t h e effects of cuticle removal o n G and o n e m b r y o n i c d e v e l o p m e n t during incubation. ACKNOWLEDGMENTS. T h e a u t h o r s express appreciation to J i m m y H i n n a n t and J a m e s Bronson for animal care and
t o Grace B r o c k m a n for e x p e r t technical assistance.
REFERENCES Ar, A., C. V. Paganelli, R. B. Reeves, D. G. Greene, and H. Rahn, 1974. The avian egg: Water vapor conductance, shell thickness and functional pore area. Condor 76:153-158. Arbor Acres, 1982. Broiler breeder male and female feeding and management guide. Arbor Acres Farm, Inc. Glastonbury, CT. Ball, R. F., V. Logan, and J. F. Hill, 1975. Factors affecting the cuticle of the egg as measured by intensity of staining. Poultry Sci. 54:1479—1484. Board, R. G., 1982. Properties of avian eggshells and their adaptive value. Biol. Rev. 57:1—28. Board, R. G., and N. A. Halls, 1973. The cuticle: a barrier to liquid and particle penetration of the shell of the hen's egg. Br. Poult. Sci. 14:69-97. Board, R. G., and V. D. Scott, 1980. Porosity of the avian eggshell. Am. Zool. 20:339-349. Bolton, W., 1957. The effect of the age of the hen on the porosity of the egg. J. Agric. Sci. 49:384— 386. Brake, J., J. F. Ort, T. A. Carter, and W. R. Campbell, 1984. Effect of the insect growth regulator CGA-72662 (Larvadex®) on broiler breeder production, hatchability, and subsequent chick performance. Poultry Sci. 63:910—916. Bryant, R. L., and P. F. Sharp, 1934. Effect of washing on the keeping quality of hen's eggs. J. Agric. Res. 4 8 : 6 7 - 8 9 . Christensen, V. L., and R. A. Bagley, 1984. Vital gas exchange and hatchability of turkey eggs at high altitude. Poultry Sci. 63:1350-1356. Fromm, D., 1963. Permeability of the hen's eggshell. Poultry Sci. (Abstr.) 42:1271. Kayar, S. R., G. K. Snyder, G. F. Birchard, and C. P. Black, 1981. Oxygen permeability of the shell and membranes of chicken eggs during development. Respir. Physiol. 4 6 : 2 0 9 - 2 2 1 . Marshall, W., and D. B. Cruickshank, 1938. The function of the cuticle in relation to the porosity of eggs. J. Agric. Sci. 28:24-42. McDaniel, G. R., D. A. Roland, Sr., and M. A. Coleman, 1979. The effect of eggshell quality on hatchability and embryonic mortality. Poultry Sci. 5 8 : 1 0 - 1 3 . Meir, M., A. Ar, and A. Nir, 1984a. Preincubation dipping of turkey eggs. Does it affect eggshell conductance? Poultry Sci. 63:2475-2478. Meir, M., A. Nir, and A. Ar, 1984b. Increasing hatchability of turkey eggs by matching incubator humidity to shell conductance of individual eggs. Poultry Sci. 63:1489-1496. Paganelli, C. V., 1980. The physics of gas exchange across the avian eggshell. Am. Zool. 20:329—338. Paganelli, C. V., R. A. Ackerman, and H. Rahn, 1978. The avian egg: in vivo conductances to oxygen, carbon dioxide, and water vapor in late development. Pages 212—218 in Respiratory Function in Birds, Adult and Embryonic. J. Piiper, ed. Springer-Verlag, Berlin, Germany. Rahn, H., 1981. Gas exchange of avian eggs with special reference to turkey eggs. Poultry Sci. 60:1971-1980. Rahn, H., A. Ar, and C. V. Paganelli, 1979. How bird
CUTICLE AND CONDUCTANCE eggs breathe. Sci. Am. 240:46-55. Romanoff, A. L., 1943. Study of various factors affecting permeability of birds' eggshell. Food Res. 5:212-223. SAS Institute, Inc., 1982. SAS User's Guide: Basics. SAS Inst., Cary, NC 27511. Simons, P.C.M., 1971. Ultrastructure of the hen eggshell and its physiological interpretation. Centre for Agricultural Publishing and Documentation, Wageningen, Netherlands. Sparks, N.H.C, and R. G. Board, 1984. Cuticle, shell porosity and water uptake through hens' eggshells. Br. Poult. Sci. 25:267-276. Tazawa, H., 1980. Oxygen and C0 2 exchange and
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acid-base regulation in the avian embryo. Am. Zool. 20:395-404. Tranter, H. S., N.H.C. Sparks, and R. G. Board, 1983. Changes in structure of the limiting membrane and in oxygen permeability of the chicken egg integument during incubation. Br. Poult. Sci. 24:5 37-547. Tullett, S. G., 1978. Pore size versus pore number in avian eggshells. Pages 217—226 in Respiratory Function in Birds, Adult and Embryonic. J. Piiper, ed. Springer, Berlin, Germany. Wangensteen, O. D., D. Wilson, and H. Rahn, 1970/71. Diffusion of gases across the shell of the hen's egg. Respir. Physiol. 11:16—30.