- Email: [email protected]
Water Quality III: The Effect of Water Nitrate and Bacteria on Broiler Breeder Performance
01W Applied Poultry Science. Inc
WATERQUALITY 111: THEEFFECT OF WATER NITRATE AND BACTERIA ON BROILER BREEDER PERFORMANCE
Primarv Audience: Flock SuDervisors. Researchers
creased resistance to respiratory and gastroDESCRIPTION OF PROBLEM intestinal infections [3]. The effect of intensive agricultural practices on the quality of our water supplies has led to concerns about bacterial and nitrogen poisoning. Water nitrate has been reported to decrease broiler growth rate [l]. In growing chicks and poults, chronic nitrate poisoning has resulted in anorexia, hypovitaminosis A, incoordination, and lowered weight gains [2]. In mammals, chronic lowlevel nitrates have been associated with de1
To whom correspondence should be addressed
While the detrimental effect of nitrate-nitrogen levels above 20 m g L on turkey and chick growth has been well documented [2,4], the effect of low-level ( < 2 0 m@) nitratenitrogen and other compounding contaminants on long-term broiler breeder or laying hen performance is not known. Adams et al. [5] found no change in hen day egg production (HDP) or egg quality among White Leghorn hens drinking up to 300 mg/L nitrate or
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
J. M. GRIZZLE', T.A. ARMBRUST, M. A. BRYAN, and A. M. SAXTON D e p m e n t ofAnimal Science, The Universityof Tennessee, Knoxville, TN 37760 Phone: (423) 9747226 F M : (423) 974-7448
Research Report 57
GRIZZLE et al.
tions, as is the case in the mammal [3], and that it could also affect egg production and fertility. This paper describes the third in a series of studies designed to look at the compounding effects of water contaminants on the production characteristics of domestic poultry. This experiment addressed the effect of low-level chronic exposure to water nitrates plus E. coli on the reproductive performance of broiler breeder hens.
MATERIALS AND METHODS ANIMALS W Ohundred forty Arbor Acres broiler breeder hens were raised from day-old chicks on restricted daily intake diets per breeder recommendation. At 20 wk of age, all birds were randomly assigned to 1.5 x 3.0 m floor pens, 10 hens plus 1rooster per pen. Each pen was equipped with two hanging tube feeders and a 10-compartment nest box. Birds received a standard breeder layer diet, which was formulated per breeder recommendations (Table 1). Supplemental light was proTABLE 1. Composition of breeder layer diet INGREDIENT
%O
Yellow corn
67.48
Soybean oil meal (48%)
11.53
Dehydrated alfalfa meal (17%)
6.99
Fish meal - Menhaden
5.00
Ground limestone
7.31
Vitamin-Tracemineral mixA
1.00
Dicalcium phosphate (22% Ca;18.5% P)
0.27
Salt
0.33
DL-Methionine
0.09
Crude protein (%) Metabolizableenergy (Cal/Kg)
15.00 2800.00
Methionine + Cystine (%)
0.62
L-Lysine (%)
0.80
Calcium (%)
3.30
Total phosphorus (%)
0.50
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
50 mgL nitrite ( N 0 2 ) over a 28-day period. However, the length of the production cycle in breeder or layer buds offers greater opportunity for long-term chronic effects of water contaminates. Unfortunately there is confusion in the literature when describing nitrogen contamination of feed or water supplies. Nitrate refers to the amount of NO3 in the water, while nitrate-nitrogen (NO3-N) refers to the nitrogen (22.58%) contribution to nitrate. These terms have been used interchangeably, and it is not always clear whether the author(s) meant nitrate or nitrate-nitrogen. The National Academy of Sciences has set the safe upper limit of nitrate-nitrogen in human drinking water at 10 mg/L [6]. Bacterial contamhation of poultry water supplies is a significant problem, particularly if open trough or bell-type drinkers are used. The presence of coliform bacteria in water supplies is usually an indication of fecal contamination, which may be the result of poor well construction or maintenance [A. Under certain conditions, some strains of E.coli are capable of respiratory invasion, with subsequent blood and pericardial sac involvement [SI. This infection can be established if respiratory disease agents such as pathogenic pleuropneumonia-like organisms (Mycoplasma), Infectious Bronchitis Virus, or Newcastle disease virus are present, as these agents enable the bacteria to enter the respiratory tract via an aerosol route [8]. Airsacculitis, pericarditis, and perihepatitis appear more extensive in the presence of a mixed infection, Mycoplasma and E. coli, rather than a single agent [9,10]. This condition is more prevalent in young birds than old, during periods of high stress, e.g., during the establishment of dominance in young birds (crowding), and during extremes in weather, particularly cold [11, 121. Chlorinationof water supplies has been shown to improve gumboro and airsacculitis conditions [13]. The effect of water nitrate and E. coli interaction on the breeder animal is relatively unknown. The size and maturity of the animal should make it less susceptible to nitrate toxicity than the younger, more immunologically incompetent bird. It is certainlylogical to think that chronic nitrate-nitrogen exposure would lower the resistance of breeder hens to respiratory diseases and secondary E. coli infec-
WATER QUALITY AND BR. BREEDERS
vided for a total of 16 hr/day. The experiment began in late December when hens reached 26 wk of age and 15% hen day egg production (HDP). The experiment continued until October, when hens reached 66 wk of age and 40 wk egg production. All roosters were removed after 20 wk of egg production, and were replaced with younger 26-wk-old birds of the same strain. Replacement birds had been raised on the same restricted intake diet as the original flock and had received 16 hr of tight/day from 20 wk of age. W o hens and no roosters died during the experiment.
A minimum of 50 eggs/pen were set, and eggs were kept separate by pen. At 15,25,and 40wk of egg production (41,51, and 66 wk of age), 10 hens per treatment were killed by cervical dislocation. The ovary was removed, and the size of the largest (Fl), third largest (F3), and fifth largest (F5) preovulatory follicle was measured. Follicles greater than 10 mm, 6-8 mm, 4-6 mm, 1-3 mm, and those atretic were counted. The designation F1, F2,M,etc., describes the size and hierarchal order of ovulation as described by Warren and Scott [17]. Atretic follicles were identified as those in a collapsed and hemorrhagic condition [181. Data were analyzed by the mixed model procedure of SAS [19,20].
WATER TREATMENTS Hens were assigned to one of eight water treatments consisting of 0, 10,20, or 40 mg/L added sodium nitrate and 0 or 100 CFU/mL E. coli (ATTC #11775 from urine). Municipal water contained 1.90 mg/L nitrate-nitrogen by analysis [14]. Final calculated nitrate-nitrogen levels were 1.90, 3.55, 5.19, or 10.38 mg/L. E.coli treatment level was chosen per a Tennessee poultry farm survey that found coliform contamination of well water to 100 CFU/mL [15]. Each treatment was replicated3 times for a total of 30 hens per treatment. Water was supplied to each pen via a 20-L plastic bucket positioned outside of the pen and connected by plastic tubing to hard cup drinkers, three drinkers per pen. Drinkers were shielded from pen contamination by a wooden shelf positioned 6 in. above the drinkers. Water buckets were emptied and filled with fresh water treatments daily. Bacteria were grown as overnight cultures and were counted by spectrophotometric methods before inoculation. Water samples were collected once per month and cultured on Violet Red Bile Agar with Mug (Difco, Detroit, MI), an agar selective for coliform bacteria. The API 20E system (BioMerieux Vitek, Hazelwood, MO) further verified bacterial identification. DATA COLLECTION Data were collected on HDP and monthly fertility and fertile hatchability [16] of eggs until hens reached 40 wk of egg production. Eggs were set on the morning of the 4th Monday of every month from January through September after a preceding 10-daycollection period (14-day period during the last 60 days of the experiment, August and September).
RESULTS AND DISCUSSION Nitrate-nitrogen or bacteria alone did not affect (P2.05) overall HDP from weeks 5 to 40. However, a significant interaction occurred between nitrate-nitrogen and bacteria (Table 2). Least square means comparison showed that nitrate-nitrogen at 10.38 mg/L in the presence of added bacteria depressed HDP during the 40-wk production period. However, when not combined with bacteria, nitrate-nitrogen to 10.38 mg/L did not affect HDP. More careful examination of the data shows that the highest HDP occurred in the 1.90 mg/L nitrate-nitrogen hens consuming E. coli, and the lowest rate of egg production in those consuming 10.38 mg/L nitratenitrogen and E. coli, which explains the difference observed. There is room to question whether this data truly indicates a casual relationship between egg production and the nitrate-bacteria interaction, especially since the negative control (1.90 mg/L nitratenitrogedno E. coli) rate of egg production was the second-lowest of all treatments and very close to the lowest value observed (10.38 mg/L nitrate-nitrogen and E. coli). These findings are consistent with those of A d a m et al. [5], who were not able to show a reduction in egg production during a 28-day period due to 300 mg/L water nitrate. However, Bentley et al. [21] reported that 300 mg/L water nitrate reduced early egg production in laying hens. As would be expected, a significant ( P s .OS) main effect due to reproductive age @-day period) was found. Average HDP for the 2nd, 3rd, 4th, 5th, and 6th 28-day periods of egg production (February, March, April,
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
58
Research Report 59
GRIZZLE et al.
BACIERIA
HEN-DAY EGG
NITRAm-NITROGEN
PRODUCTION^
(%)
60.16f2.48ab
1.90 mg/L
None
3.55 m g L
61.63f2.54ab
5.19 mg/L
62.56f 2.03ab
10.38 mdL
64.28 f 2.03ab
1.90 mg/L
65.262 2.03a
3.5.5 mg/L
61.4.5?2.03ab 64.11+2.03ab 59.43+2.03b
*Mean f standard error a’bLeastsquare means within the same column with different superscripts are significantly different (P1.05).
May, or June, respectively) were 66.62, 66.14, 66.44, 68.85, and 63.25%. HDPs for these periods were all significantly higher ( P I .OS) than those for the 7th, 8th, and 9th 28-day periods (July, August, and September; 58.26, 58.81, and 50.52%, respectively). Since these data were averaged across all treatments, the lowered HDP during the last three time periods of the experiment was considered an effect of reproductive age and high summer temperatures. Least square means compari-
sons showed differences among treatments during the summer months (Pr.05). However, it was not clear whether the differences were attributable to the additive effect of temperature stress on the three-way interaction among nitrate-nitrogen, bacteria, and age. Neither water-borne nitrate-nitrogen nor bacteria affected (Pr.05) follicle size or number (Table 3). Follicle number decreased ( P I .OS) with age as would be expected. There was no change in the size of the F1 and F3
None
35.45
27.78
16.47
0.76
4.36
10.19
24.66
12.15
E. d i
34.57
27.52
16.61
0.67
3.30
10.80
22.50
13.29
0.38
0.66
0.70
0.16
0.49
0.73
1.51
1.01
Pooled SEMA
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
5.19 mg/L 10.38 m d L
WATER QUALITY AND BR. BREEDERS
follicles as the age of the hen increased. The fifth largest preovulatory follicle (F5) decreased in size, and was smaller (P5.OS) after 25 wk of egg production and numerically smaller (P 1.OS) after 40 wk of egg production. Likewise, there were fewer follicles greater than 10 mm after 25 and 40 wk of egg production (PS.05). Among the small follicles (Le., those less than 9 mm), the number of 4-6 mm follicles decreased, and those atretic increased over the experimental period (Ps.05). Gilbert et al. [18] suggested that the number of follicles in each size class regulates rate of lay. As the hen ages, fewer follicles advance into the larger size classes due to increased atresia. Our data indicates that atresia targets 4-6 mm follicles. This is in contrast to Gilbert et al. [18], who suggested that 7-8 mm follicleswere most susceptible to atresia. The decrease in the F5 follicle during the reproductive cycle of these hens probably indicates that fewer follicles are advancing toward ovulation, and corresponds to, or is the cause of, the declining rate of egg production. The size of the F1 and the F3 follicles changed very little during 40 wk of egg production, indicating that an optimal size must be reached before ovulation can occur. Every 28 days, a minimum 150 eggs per treatment (50 eggs/pen) were set for evaluation of fertility and fertile hatchability. Overall fertility or fertile hatchability was not affected (Pr.05) by E. coli or nitrate-nitrogen alone. Fertility and fertile hatchability decreased as the reproductive age of the hen increased, and
was lowest after 30 wk egg production ( P S .05; Table 4). This was also the hottest month of the experiment (July) for mean house high and low temperatures. The decrease observed during this month seemed attributable more to high temperatures than to the age of the hen, particularly since fertility increased as temperatures cooled in late summer and early fall. Fertile hatchability also declined as the age of hen increased, to 69.86% after 16 wk of egg production (Table 4). During May, after roosters were replaced, fertile hatchability increased slightly but then declined to 62.01% in July and was lower (PS.05) than in any other time period. During the fall, fertile hatchability once again increased as temperatures cooled. Least square means comparisons of treatments during the summer months showed differences in fertility and fertile hatchability ( P I .OS), but as with egg production, the results may have been compounded by the temperature effect rather than the treatment effect. Analysis of least square means did not show an interactive effect of nitrate-nitrogen and bacteria on fertility (Table 5). Although this result points to the younger males used to spike the flock, it also demonstrates that prolonged exposure to low-level nitrate-nitrogen and E. coli as used in this study did not affect the hens' ovum viability. In contrast, fertile hatchability was affected by water nitrate treatment (Table 5). Among hens drinking only nitrate-nitrogen-contaminated water, fertile hatchability was significantly
I
MONTHSET
,
WEEKEGG PRODUCIION~
AVERAGE HOUSE TEMPERATURE LOW "C
HIGH "C
FERTILITY
FERTILE HATCHABILITY
%
76
Janualy
4
7.2
16.8
95.45rt 1Ma
76.9 21.98b
Februaty
8
6.9
16.7
95.91f l . M a
76.47k1.98'
12
5.2
17.3
95.36k l.04ab
73.78f1.98"
1 March April
16
10.3
20.0
94.90%1.04ab
69.865 1.98'
Mav
20
16.9
28.8
96.13k 1.07a
74.83L2.03b
July
30
24.9
34.9
92.822 1.09b
62.01k2.md
34
22.5
32.2
94.56&1.0?'
85.8352.03a
38
19.4
29.9
95.74kl.OP
83.04f2.03a
IAugust
September
*June data lost to hatchery fire
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
60
Research Report GRIZZLE et al.
61
TABLE 5. Interactive effect of water nitrate and bacteria on percent fertility and fertile hatchability of eggs
a*bL.eastsquare means in the same column with different superscripts are significantly diffcrent (PS.05).
less ( P r .05) for those consuming 10.38 mg/L nitrate-nitrogen compared to control hens consuming only municipal water with 1.90 mg/L nitrate-nitrogen. Interestingly, there was no change in fertile hatchability among hens drinking E. coli and any level of nitrate-nitrogen. The reduction in fertile hatchability among hens consuming only nitrate-nitrogen-contaminated water may have resulted from impaired or limited vitamin A availability to the developing embryo despite dietary vitamin A levels (15,400 IU/kg diet) that were more than adequate. Adams ef al. [5] reported a significant ( P S .05) %-day decline in liver vitamin A and /3-carotene among poults drinking 200 mgL nitrite, but not among laying hens. The short duration of their experiment, the difference in species, or the tolerance of nitrite and increased liver stores of vitamin A characteristic of adults may explain their results. Early studies by Tomov [22] reported leg weakness and plaques on the esophageal mucosa indicative of vitamin A deficiency in hens fed high levels of nitrate and nitrites (100 to 200 mgL sodium nitrite or 1000or 2000 mgL potassium nitrate). Eggs were reported to contain blood spots. The question of why hens consuming only nitrate-nitrogen and not those consuming both nitrate and bacteria experienced reduced hatchability is intriguing. The bacteria in the water supply may have removed the nitrate by metabolizing it. However, these bacteria are capable of nitrate reduction, and the resulting nitrite is more detrimental to animal health than the original nitrate [23]. Whether detri-
mental amounts of nitrite formed at the low levels of nitrate-nitrogen used in this study is questionable. A more plausible explanation may come from Adams et al. [5],who reported that nearly half of the water nitrite was lost from trough waterers when the environmental temperature was 21-24°C. Therefore it is possible that even if bacterial reduction of nitrate to nitrite occurred, much of the resulting nitrite may have been lost during the last 4 months of the experiment when house temperatures were over 21°C (Table 6). Coliform bacterial counts from water samples taken from the buckets were much higher during the summer months, but did not exceed 250 CFU/mL at any sampling period (data not shown). Whether 250 coliform colonies/mL. TABLE 6. Average daily water consumption per hen for a 3@-day period from June 12 to July 17
NITRATE NITROGEN
None
619.18k17.01
Erali
631.54 k 16.94
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
I
JAPR WATER QUALITY AND BR. BREEDERS
represents sufficient numbers to remove significant amounts of nitrate-nitrogen from the water supply, or whether this is high enough to compromise the birds' performance, is not known. As reported by Good [4], only 50 coliform colonies/ml of water is permitted in potable water. Neither bacteria nor nitratenitrogen affected water consumption during a 30-day sampling period from June 17 to July 12 (P2.05;Table 6). Average maximum daily temperature was 28.7"Cduring this time. We had anticipated that birds consuming 10.38 mg/L nitrate-nitrogen would consume more water because of the sodium contribu-
tion. However, there was in fact no difference (Pr.05) in water consumption, perhaps because of the increased temperature. This experiment confirms that broiler breeder performance is affected by water nitrates at 10.38 mglL. The compound effect of water nitrate-nitrogen and E. coli compromised egg production, an effect perhaps attributable to immune suppression and opportunistic disease organisms. Fertile hatchability was reduced by nitrate-nitrogen, which may cause reduced vitamin A availability to the embryo. Further research is planned in this area.
CONCLUSIONS AND APPLICATIONS 1. Broiler breeder egg production was not changed by water nitrate or bacteria alone. However, the combination of E. coli and 10.38 mg/L nitrate-nitrogen reduced HDP measured over an entire production cycle. 2. Neither water nitrate nor bacteria affected fertility of hatching eggs. 3. The treatments of 10.38 mglL nitrate-nitrogen reduced fertile hatchability of eggs; E. coli either alone or in combination with nitrate-nitrogen did not affect fertile hatchability. 4. Reduction in fertile hatchability associated with high levels of water nitrate-nitrogen may be mediated by augmenting vitamin A available for embryonic development.
REFERENCES AND NOTES 1.Barton, T.L, LH. Hileman, and T.S.Nelson, 1986. A survey of water quality on Arkansas broiler farms and its effect on performance. Mimeo, 36 pp. Proc. 21st Natl. Meeting Poultry Health and Condemnations, Univ. Arkansas, Fayetteville, AFL 2. Marrelt, LE and M.L. Sunde, 1968. The use of turkey poults and chickens as test animals for nitrate and nitrite toxicity. Poultry Sci. 68(2):511-519. 3. Wood, P A , 1980. The molecular pathology of chronic nitrate intoxication in domestic animals: A hypothesis. Vet. Human Toxic. 2226-27. 4. Good, B., 1985.Water ualityaffectspoultryproduction. Poultry Dig. 44(517):1~&109. 5. Adam, AW.,RJ. Emerick, and C.W. Carlson, 1966. Effects of nitrate and nitrite in the drinkin water on chicks, poults, and laying hens. Poultry Sci. 4!:12151222. 6. National Academy of Sciences, National Research Council, Assembly of Life Sciences, 1981. The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds. Natl. Acad. Sci., Washington, DC. 7. Carter, T.A.and RE Sneed, 1986. Drinking water for poultry. Poultry Science and Technology Guide No. 42, North Carolina State Univ., Raleigh, NC. 8. Gross,W.B., 1962.Blood cultures, blood counts, and temperature records in an experimentally produced "air ' ' rpliinfection sac disease" and uncomplicated Eschenchla of chickens. Poultry Sci. 41:691-700. 9. Hamdy, AH. and CJ. Blanchard, 1969. Effect of lincomycin and spectinomycin water medication on
chickens experimenta1l.y infected with and Eschenchla d. Poultry Sci. 48:17031708. 10. Gross, W.B., 1958. The role of E. & in the cause of chronic res irato diseases and certain respiratory diseases. Am. Vet.%es. 19:448452.
f
11.Gross, W.B. and G. Colmano, 1969. The effect of social isolation on resistance to some infectious diseases. Poultry Sci. 48514-520. 12.Yoder, H.W., Jr., 1991. Mycoplasmosis. Pages 196235 in: Diseases of Poultry. B.W. Calnek, H.J. Barnes, C.W.Beard,W.M. Reid,andH.W. Yoder, eds. IowaState Univ. Press, Ames, IA. 13. Cam, L E , D.W. Murphy, and C.J. Wabeck, 1988. Chlorination of drinkin water for broilers. Livestock environment 111. Pages $79-285 in: Proc. 3rd Internatl. Livestock Environment Symp., Toronto, Canada. ASAE Publ. 1-88.St. Joseph, MI. 14. American Public Health Assn., 1975. Standard Methods for the Examination of Water and Wastewater. 14th Edition., Amer. Public Health Assn., Washington, DC. 15. Burcham, T.N.,H.C. Goan, P.H. Denlon, andC.L Ahrens, 1991. Quality of ground water on Tennessee oultry farms. Mimeo, 4 p Proc. 2nd Tenn. Water kesources Conf., Knoxville,
h.
16. Fertility was determined by candling all eggs at 7 days incubation; those showing embryonic development and the establishment of vascular development were determined to be fertile. Eggs showing development were returned to the incubator, transferred on the evening of
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
62
Research Report GRIZZLE et al. the 18th day of incubation, and removed on the morning of the 21st day of incubation. All eggs that did not show development and those deemed dead were broken out and examined for the presence of embryonic development. These were examined by visual inspection, and not with a stereo microscope. Any egg showing obvious s i p s of development, Le., those with enlarged germinal disk, visible area pellucida and primitive streak, blood islets, or obvious differentiation were declared fertile but dead, and records were changed according1 Fertility was calculated as the number of fertile eggs Gertile and alive + fertile but dead) at 7 days/number of eggs set. Fertile hatchabilitywas calculated as the percent chicks hatched from fertile eggs. 17. Warren, D.C. and H.M. Scott, 1935. The time factor in egg formation. Poultry Sci. 14:195-207.
(m
19. SAS Inslitute, Ine, 1996. The mixed procedure. Pages 531-656 in: SAWSTAT Software: Changes and Enhancements through release 6.11. SAS Institute, Inc., Cary, NC. 20. The design of the experiment was a three-factor split plot, with bacteria assigned the main plot, nitrate level the sub-plot, and reproductive age the sub-sub plot. Data from each individual pen were entered as replicate treatments. values of the eight nitrate-nitrogen&. The mixed model procedure correctly estimates error terms used to test effects in the model. For the model used in this experiment, incorrect standard errors and painvise tests among means would occur if a standard futed effects
analysis were used (i.e., SAS general linear models procedure). Least square means were separated by Fisher’s Least Significant difference test (a = 0.05). Power calculations showed that with an anticipated mean square error of 20, approximately30 birds per treatment would be required to detect a 4 percentage point difference in e production at a = 0.05 with 95.0% confidence. Theregre the number of hensltreatment used in the experiment was considered adequate. Weeks 1-4 eggproduction data were omitted from the data deck to remove any potential bias due to the inconsistent manner in which the flock came into production. This was considered to be partly due to subzero weather during this time; even though the house was heated, 4 of 24 pens had not reached 40% hen-day egg production (from a startingpoint of 15%) by the secondweek of data collection. 21. Bentley, AB., Q.B. Kinder, G.B. Garner, and J.E. Savage, 1965. Effect of nitrate in water on performance of laying hens. Poultry Sci. 44.1351. 22. Tomov, A, 1965. Effect of prolonged intake of nitrates and nitrites on hens and the quality of their eggs. Vet. Med. Nauki (Sofia) 2:31>321. 23. Bruning-Fann, C.S. and J.B. Kaneene, 1993. The effects of nitrate, nitrite, and N-nitroso compounds on animal health. Vet. Hum. Toxicol. 35(3):237-250.
ACKNOWLEDGEMENT The authors thank Arbor Acres Farm, Inc., Blairsville, GA for providing all birds used in this study.
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
18. Gilbert, AB., M.M. Perry, D. Wadington, and M A Hardie, 1983. Role of atresia in establishing the follicular hierarchy in the ovary of the domestic hen domesticus). J. Reprod. Fert. 69221-227.
63
WATERQUALITY 111: THEEFFECT OF WATER NITRATE AND BACTERIA ON BROILER BREEDER PERFORMANCE
Primarv Audience: Flock SuDervisors. Researchers
creased resistance to respiratory and gastroDESCRIPTION OF PROBLEM intestinal infections [3]. The effect of intensive agricultural practices on the quality of our water supplies has led to concerns about bacterial and nitrogen poisoning. Water nitrate has been reported to decrease broiler growth rate [l]. In growing chicks and poults, chronic nitrate poisoning has resulted in anorexia, hypovitaminosis A, incoordination, and lowered weight gains [2]. In mammals, chronic lowlevel nitrates have been associated with de1
To whom correspondence should be addressed
While the detrimental effect of nitrate-nitrogen levels above 20 m g L on turkey and chick growth has been well documented [2,4], the effect of low-level ( < 2 0 m@) nitratenitrogen and other compounding contaminants on long-term broiler breeder or laying hen performance is not known. Adams et al. [5] found no change in hen day egg production (HDP) or egg quality among White Leghorn hens drinking up to 300 mg/L nitrate or
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
J. M. GRIZZLE', T.A. ARMBRUST, M. A. BRYAN, and A. M. SAXTON D e p m e n t ofAnimal Science, The Universityof Tennessee, Knoxville, TN 37760 Phone: (423) 9747226 F M : (423) 974-7448
Research Report 57
GRIZZLE et al.
tions, as is the case in the mammal [3], and that it could also affect egg production and fertility. This paper describes the third in a series of studies designed to look at the compounding effects of water contaminants on the production characteristics of domestic poultry. This experiment addressed the effect of low-level chronic exposure to water nitrates plus E. coli on the reproductive performance of broiler breeder hens.
MATERIALS AND METHODS ANIMALS W Ohundred forty Arbor Acres broiler breeder hens were raised from day-old chicks on restricted daily intake diets per breeder recommendation. At 20 wk of age, all birds were randomly assigned to 1.5 x 3.0 m floor pens, 10 hens plus 1rooster per pen. Each pen was equipped with two hanging tube feeders and a 10-compartment nest box. Birds received a standard breeder layer diet, which was formulated per breeder recommendations (Table 1). Supplemental light was proTABLE 1. Composition of breeder layer diet INGREDIENT
%O
Yellow corn
67.48
Soybean oil meal (48%)
11.53
Dehydrated alfalfa meal (17%)
6.99
Fish meal - Menhaden
5.00
Ground limestone
7.31
Vitamin-Tracemineral mixA
1.00
Dicalcium phosphate (22% Ca;18.5% P)
0.27
Salt
0.33
DL-Methionine
0.09
Crude protein (%) Metabolizableenergy (Cal/Kg)
15.00 2800.00
Methionine + Cystine (%)
0.62
L-Lysine (%)
0.80
Calcium (%)
3.30
Total phosphorus (%)
0.50
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
50 mgL nitrite ( N 0 2 ) over a 28-day period. However, the length of the production cycle in breeder or layer buds offers greater opportunity for long-term chronic effects of water contaminates. Unfortunately there is confusion in the literature when describing nitrogen contamination of feed or water supplies. Nitrate refers to the amount of NO3 in the water, while nitrate-nitrogen (NO3-N) refers to the nitrogen (22.58%) contribution to nitrate. These terms have been used interchangeably, and it is not always clear whether the author(s) meant nitrate or nitrate-nitrogen. The National Academy of Sciences has set the safe upper limit of nitrate-nitrogen in human drinking water at 10 mg/L [6]. Bacterial contamhation of poultry water supplies is a significant problem, particularly if open trough or bell-type drinkers are used. The presence of coliform bacteria in water supplies is usually an indication of fecal contamination, which may be the result of poor well construction or maintenance [A. Under certain conditions, some strains of E.coli are capable of respiratory invasion, with subsequent blood and pericardial sac involvement [SI. This infection can be established if respiratory disease agents such as pathogenic pleuropneumonia-like organisms (Mycoplasma), Infectious Bronchitis Virus, or Newcastle disease virus are present, as these agents enable the bacteria to enter the respiratory tract via an aerosol route [8]. Airsacculitis, pericarditis, and perihepatitis appear more extensive in the presence of a mixed infection, Mycoplasma and E. coli, rather than a single agent [9,10]. This condition is more prevalent in young birds than old, during periods of high stress, e.g., during the establishment of dominance in young birds (crowding), and during extremes in weather, particularly cold [11, 121. Chlorinationof water supplies has been shown to improve gumboro and airsacculitis conditions [13]. The effect of water nitrate and E. coli interaction on the breeder animal is relatively unknown. The size and maturity of the animal should make it less susceptible to nitrate toxicity than the younger, more immunologically incompetent bird. It is certainlylogical to think that chronic nitrate-nitrogen exposure would lower the resistance of breeder hens to respiratory diseases and secondary E. coli infec-
WATER QUALITY AND BR. BREEDERS
vided for a total of 16 hr/day. The experiment began in late December when hens reached 26 wk of age and 15% hen day egg production (HDP). The experiment continued until October, when hens reached 66 wk of age and 40 wk egg production. All roosters were removed after 20 wk of egg production, and were replaced with younger 26-wk-old birds of the same strain. Replacement birds had been raised on the same restricted intake diet as the original flock and had received 16 hr of tight/day from 20 wk of age. W o hens and no roosters died during the experiment.
A minimum of 50 eggs/pen were set, and eggs were kept separate by pen. At 15,25,and 40wk of egg production (41,51, and 66 wk of age), 10 hens per treatment were killed by cervical dislocation. The ovary was removed, and the size of the largest (Fl), third largest (F3), and fifth largest (F5) preovulatory follicle was measured. Follicles greater than 10 mm, 6-8 mm, 4-6 mm, 1-3 mm, and those atretic were counted. The designation F1, F2,M,etc., describes the size and hierarchal order of ovulation as described by Warren and Scott [17]. Atretic follicles were identified as those in a collapsed and hemorrhagic condition [181. Data were analyzed by the mixed model procedure of SAS [19,20].
WATER TREATMENTS Hens were assigned to one of eight water treatments consisting of 0, 10,20, or 40 mg/L added sodium nitrate and 0 or 100 CFU/mL E. coli (ATTC #11775 from urine). Municipal water contained 1.90 mg/L nitrate-nitrogen by analysis [14]. Final calculated nitrate-nitrogen levels were 1.90, 3.55, 5.19, or 10.38 mg/L. E.coli treatment level was chosen per a Tennessee poultry farm survey that found coliform contamination of well water to 100 CFU/mL [15]. Each treatment was replicated3 times for a total of 30 hens per treatment. Water was supplied to each pen via a 20-L plastic bucket positioned outside of the pen and connected by plastic tubing to hard cup drinkers, three drinkers per pen. Drinkers were shielded from pen contamination by a wooden shelf positioned 6 in. above the drinkers. Water buckets were emptied and filled with fresh water treatments daily. Bacteria were grown as overnight cultures and were counted by spectrophotometric methods before inoculation. Water samples were collected once per month and cultured on Violet Red Bile Agar with Mug (Difco, Detroit, MI), an agar selective for coliform bacteria. The API 20E system (BioMerieux Vitek, Hazelwood, MO) further verified bacterial identification. DATA COLLECTION Data were collected on HDP and monthly fertility and fertile hatchability [16] of eggs until hens reached 40 wk of egg production. Eggs were set on the morning of the 4th Monday of every month from January through September after a preceding 10-daycollection period (14-day period during the last 60 days of the experiment, August and September).
RESULTS AND DISCUSSION Nitrate-nitrogen or bacteria alone did not affect (P2.05) overall HDP from weeks 5 to 40. However, a significant interaction occurred between nitrate-nitrogen and bacteria (Table 2). Least square means comparison showed that nitrate-nitrogen at 10.38 mg/L in the presence of added bacteria depressed HDP during the 40-wk production period. However, when not combined with bacteria, nitrate-nitrogen to 10.38 mg/L did not affect HDP. More careful examination of the data shows that the highest HDP occurred in the 1.90 mg/L nitrate-nitrogen hens consuming E. coli, and the lowest rate of egg production in those consuming 10.38 mg/L nitratenitrogen and E. coli, which explains the difference observed. There is room to question whether this data truly indicates a casual relationship between egg production and the nitrate-bacteria interaction, especially since the negative control (1.90 mg/L nitratenitrogedno E. coli) rate of egg production was the second-lowest of all treatments and very close to the lowest value observed (10.38 mg/L nitrate-nitrogen and E. coli). These findings are consistent with those of A d a m et al. [5], who were not able to show a reduction in egg production during a 28-day period due to 300 mg/L water nitrate. However, Bentley et al. [21] reported that 300 mg/L water nitrate reduced early egg production in laying hens. As would be expected, a significant ( P s .OS) main effect due to reproductive age @-day period) was found. Average HDP for the 2nd, 3rd, 4th, 5th, and 6th 28-day periods of egg production (February, March, April,
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
58
Research Report 59
GRIZZLE et al.
BACIERIA
HEN-DAY EGG
NITRAm-NITROGEN
PRODUCTION^
(%)
60.16f2.48ab
1.90 mg/L
None
3.55 m g L
61.63f2.54ab
5.19 mg/L
62.56f 2.03ab
10.38 mdL
64.28 f 2.03ab
1.90 mg/L
65.262 2.03a
3.5.5 mg/L
61.4.5?2.03ab 64.11+2.03ab 59.43+2.03b
*Mean f standard error a’bLeastsquare means within the same column with different superscripts are significantly different (P1.05).
May, or June, respectively) were 66.62, 66.14, 66.44, 68.85, and 63.25%. HDPs for these periods were all significantly higher ( P I .OS) than those for the 7th, 8th, and 9th 28-day periods (July, August, and September; 58.26, 58.81, and 50.52%, respectively). Since these data were averaged across all treatments, the lowered HDP during the last three time periods of the experiment was considered an effect of reproductive age and high summer temperatures. Least square means compari-
sons showed differences among treatments during the summer months (Pr.05). However, it was not clear whether the differences were attributable to the additive effect of temperature stress on the three-way interaction among nitrate-nitrogen, bacteria, and age. Neither water-borne nitrate-nitrogen nor bacteria affected (Pr.05) follicle size or number (Table 3). Follicle number decreased ( P I .OS) with age as would be expected. There was no change in the size of the F1 and F3
None
35.45
27.78
16.47
0.76
4.36
10.19
24.66
12.15
E. d i
34.57
27.52
16.61
0.67
3.30
10.80
22.50
13.29
0.38
0.66
0.70
0.16
0.49
0.73
1.51
1.01
Pooled SEMA
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
5.19 mg/L 10.38 m d L
WATER QUALITY AND BR. BREEDERS
follicles as the age of the hen increased. The fifth largest preovulatory follicle (F5) decreased in size, and was smaller (P5.OS) after 25 wk of egg production and numerically smaller (P 1.OS) after 40 wk of egg production. Likewise, there were fewer follicles greater than 10 mm after 25 and 40 wk of egg production (PS.05). Among the small follicles (Le., those less than 9 mm), the number of 4-6 mm follicles decreased, and those atretic increased over the experimental period (Ps.05). Gilbert et al. [18] suggested that the number of follicles in each size class regulates rate of lay. As the hen ages, fewer follicles advance into the larger size classes due to increased atresia. Our data indicates that atresia targets 4-6 mm follicles. This is in contrast to Gilbert et al. [18], who suggested that 7-8 mm follicleswere most susceptible to atresia. The decrease in the F5 follicle during the reproductive cycle of these hens probably indicates that fewer follicles are advancing toward ovulation, and corresponds to, or is the cause of, the declining rate of egg production. The size of the F1 and the F3 follicles changed very little during 40 wk of egg production, indicating that an optimal size must be reached before ovulation can occur. Every 28 days, a minimum 150 eggs per treatment (50 eggs/pen) were set for evaluation of fertility and fertile hatchability. Overall fertility or fertile hatchability was not affected (Pr.05) by E. coli or nitrate-nitrogen alone. Fertility and fertile hatchability decreased as the reproductive age of the hen increased, and
was lowest after 30 wk egg production ( P S .05; Table 4). This was also the hottest month of the experiment (July) for mean house high and low temperatures. The decrease observed during this month seemed attributable more to high temperatures than to the age of the hen, particularly since fertility increased as temperatures cooled in late summer and early fall. Fertile hatchability also declined as the age of hen increased, to 69.86% after 16 wk of egg production (Table 4). During May, after roosters were replaced, fertile hatchability increased slightly but then declined to 62.01% in July and was lower (PS.05) than in any other time period. During the fall, fertile hatchability once again increased as temperatures cooled. Least square means comparisons of treatments during the summer months showed differences in fertility and fertile hatchability ( P I .OS), but as with egg production, the results may have been compounded by the temperature effect rather than the treatment effect. Analysis of least square means did not show an interactive effect of nitrate-nitrogen and bacteria on fertility (Table 5). Although this result points to the younger males used to spike the flock, it also demonstrates that prolonged exposure to low-level nitrate-nitrogen and E. coli as used in this study did not affect the hens' ovum viability. In contrast, fertile hatchability was affected by water nitrate treatment (Table 5). Among hens drinking only nitrate-nitrogen-contaminated water, fertile hatchability was significantly
I
MONTHSET
,
WEEKEGG PRODUCIION~
AVERAGE HOUSE TEMPERATURE LOW "C
HIGH "C
FERTILITY
FERTILE HATCHABILITY
%
76
Janualy
4
7.2
16.8
95.45rt 1Ma
76.9 21.98b
Februaty
8
6.9
16.7
95.91f l . M a
76.47k1.98'
12
5.2
17.3
95.36k l.04ab
73.78f1.98"
1 March April
16
10.3
20.0
94.90%1.04ab
69.865 1.98'
Mav
20
16.9
28.8
96.13k 1.07a
74.83L2.03b
July
30
24.9
34.9
92.822 1.09b
62.01k2.md
34
22.5
32.2
94.56&1.0?'
85.8352.03a
38
19.4
29.9
95.74kl.OP
83.04f2.03a
IAugust
September
*June data lost to hatchery fire
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
60
Research Report GRIZZLE et al.
61
TABLE 5. Interactive effect of water nitrate and bacteria on percent fertility and fertile hatchability of eggs
a*bL.eastsquare means in the same column with different superscripts are significantly diffcrent (PS.05).
less ( P r .05) for those consuming 10.38 mg/L nitrate-nitrogen compared to control hens consuming only municipal water with 1.90 mg/L nitrate-nitrogen. Interestingly, there was no change in fertile hatchability among hens drinking E. coli and any level of nitrate-nitrogen. The reduction in fertile hatchability among hens consuming only nitrate-nitrogen-contaminated water may have resulted from impaired or limited vitamin A availability to the developing embryo despite dietary vitamin A levels (15,400 IU/kg diet) that were more than adequate. Adams ef al. [5] reported a significant ( P S .05) %-day decline in liver vitamin A and /3-carotene among poults drinking 200 mgL nitrite, but not among laying hens. The short duration of their experiment, the difference in species, or the tolerance of nitrite and increased liver stores of vitamin A characteristic of adults may explain their results. Early studies by Tomov [22] reported leg weakness and plaques on the esophageal mucosa indicative of vitamin A deficiency in hens fed high levels of nitrate and nitrites (100 to 200 mgL sodium nitrite or 1000or 2000 mgL potassium nitrate). Eggs were reported to contain blood spots. The question of why hens consuming only nitrate-nitrogen and not those consuming both nitrate and bacteria experienced reduced hatchability is intriguing. The bacteria in the water supply may have removed the nitrate by metabolizing it. However, these bacteria are capable of nitrate reduction, and the resulting nitrite is more detrimental to animal health than the original nitrate [23]. Whether detri-
mental amounts of nitrite formed at the low levels of nitrate-nitrogen used in this study is questionable. A more plausible explanation may come from Adams et al. [5],who reported that nearly half of the water nitrite was lost from trough waterers when the environmental temperature was 21-24°C. Therefore it is possible that even if bacterial reduction of nitrate to nitrite occurred, much of the resulting nitrite may have been lost during the last 4 months of the experiment when house temperatures were over 21°C (Table 6). Coliform bacterial counts from water samples taken from the buckets were much higher during the summer months, but did not exceed 250 CFU/mL at any sampling period (data not shown). Whether 250 coliform colonies/mL. TABLE 6. Average daily water consumption per hen for a 3@-day period from June 12 to July 17
NITRATE NITROGEN
None
619.18k17.01
Erali
631.54 k 16.94
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
I
JAPR WATER QUALITY AND BR. BREEDERS
represents sufficient numbers to remove significant amounts of nitrate-nitrogen from the water supply, or whether this is high enough to compromise the birds' performance, is not known. As reported by Good [4], only 50 coliform colonies/ml of water is permitted in potable water. Neither bacteria nor nitratenitrogen affected water consumption during a 30-day sampling period from June 17 to July 12 (P2.05;Table 6). Average maximum daily temperature was 28.7"Cduring this time. We had anticipated that birds consuming 10.38 mg/L nitrate-nitrogen would consume more water because of the sodium contribu-
tion. However, there was in fact no difference (Pr.05) in water consumption, perhaps because of the increased temperature. This experiment confirms that broiler breeder performance is affected by water nitrates at 10.38 mglL. The compound effect of water nitrate-nitrogen and E. coli compromised egg production, an effect perhaps attributable to immune suppression and opportunistic disease organisms. Fertile hatchability was reduced by nitrate-nitrogen, which may cause reduced vitamin A availability to the embryo. Further research is planned in this area.
CONCLUSIONS AND APPLICATIONS 1. Broiler breeder egg production was not changed by water nitrate or bacteria alone. However, the combination of E. coli and 10.38 mg/L nitrate-nitrogen reduced HDP measured over an entire production cycle. 2. Neither water nitrate nor bacteria affected fertility of hatching eggs. 3. The treatments of 10.38 mglL nitrate-nitrogen reduced fertile hatchability of eggs; E. coli either alone or in combination with nitrate-nitrogen did not affect fertile hatchability. 4. Reduction in fertile hatchability associated with high levels of water nitrate-nitrogen may be mediated by augmenting vitamin A available for embryonic development.
REFERENCES AND NOTES 1.Barton, T.L, LH. Hileman, and T.S.Nelson, 1986. A survey of water quality on Arkansas broiler farms and its effect on performance. Mimeo, 36 pp. Proc. 21st Natl. Meeting Poultry Health and Condemnations, Univ. Arkansas, Fayetteville, AFL 2. Marrelt, LE and M.L. Sunde, 1968. The use of turkey poults and chickens as test animals for nitrate and nitrite toxicity. Poultry Sci. 68(2):511-519. 3. Wood, P A , 1980. The molecular pathology of chronic nitrate intoxication in domestic animals: A hypothesis. Vet. Human Toxic. 2226-27. 4. Good, B., 1985.Water ualityaffectspoultryproduction. Poultry Dig. 44(517):1~&109. 5. Adam, AW.,RJ. Emerick, and C.W. Carlson, 1966. Effects of nitrate and nitrite in the drinkin water on chicks, poults, and laying hens. Poultry Sci. 4!:12151222. 6. National Academy of Sciences, National Research Council, Assembly of Life Sciences, 1981. The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds. Natl. Acad. Sci., Washington, DC. 7. Carter, T.A.and RE Sneed, 1986. Drinking water for poultry. Poultry Science and Technology Guide No. 42, North Carolina State Univ., Raleigh, NC. 8. Gross,W.B., 1962.Blood cultures, blood counts, and temperature records in an experimentally produced "air ' ' rpliinfection sac disease" and uncomplicated Eschenchla of chickens. Poultry Sci. 41:691-700. 9. Hamdy, AH. and CJ. Blanchard, 1969. Effect of lincomycin and spectinomycin water medication on
chickens experimenta1l.y infected with and Eschenchla d. Poultry Sci. 48:17031708. 10. Gross, W.B., 1958. The role of E. & in the cause of chronic res irato diseases and certain respiratory diseases. Am. Vet.%es. 19:448452.
f
11.Gross, W.B. and G. Colmano, 1969. The effect of social isolation on resistance to some infectious diseases. Poultry Sci. 48514-520. 12.Yoder, H.W., Jr., 1991. Mycoplasmosis. Pages 196235 in: Diseases of Poultry. B.W. Calnek, H.J. Barnes, C.W.Beard,W.M. Reid,andH.W. Yoder, eds. IowaState Univ. Press, Ames, IA. 13. Cam, L E , D.W. Murphy, and C.J. Wabeck, 1988. Chlorination of drinkin water for broilers. Livestock environment 111. Pages $79-285 in: Proc. 3rd Internatl. Livestock Environment Symp., Toronto, Canada. ASAE Publ. 1-88.St. Joseph, MI. 14. American Public Health Assn., 1975. Standard Methods for the Examination of Water and Wastewater. 14th Edition., Amer. Public Health Assn., Washington, DC. 15. Burcham, T.N.,H.C. Goan, P.H. Denlon, andC.L Ahrens, 1991. Quality of ground water on Tennessee oultry farms. Mimeo, 4 p Proc. 2nd Tenn. Water kesources Conf., Knoxville,
h.
16. Fertility was determined by candling all eggs at 7 days incubation; those showing embryonic development and the establishment of vascular development were determined to be fertile. Eggs showing development were returned to the incubator, transferred on the evening of
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
62
Research Report GRIZZLE et al. the 18th day of incubation, and removed on the morning of the 21st day of incubation. All eggs that did not show development and those deemed dead were broken out and examined for the presence of embryonic development. These were examined by visual inspection, and not with a stereo microscope. Any egg showing obvious s i p s of development, Le., those with enlarged germinal disk, visible area pellucida and primitive streak, blood islets, or obvious differentiation were declared fertile but dead, and records were changed according1 Fertility was calculated as the number of fertile eggs Gertile and alive + fertile but dead) at 7 days/number of eggs set. Fertile hatchabilitywas calculated as the percent chicks hatched from fertile eggs. 17. Warren, D.C. and H.M. Scott, 1935. The time factor in egg formation. Poultry Sci. 14:195-207.
(m
19. SAS Inslitute, Ine, 1996. The mixed procedure. Pages 531-656 in: SAWSTAT Software: Changes and Enhancements through release 6.11. SAS Institute, Inc., Cary, NC. 20. The design of the experiment was a three-factor split plot, with bacteria assigned the main plot, nitrate level the sub-plot, and reproductive age the sub-sub plot. Data from each individual pen were entered as replicate treatments. values of the eight nitrate-nitrogen&. The mixed model procedure correctly estimates error terms used to test effects in the model. For the model used in this experiment, incorrect standard errors and painvise tests among means would occur if a standard futed effects
analysis were used (i.e., SAS general linear models procedure). Least square means were separated by Fisher’s Least Significant difference test (a = 0.05). Power calculations showed that with an anticipated mean square error of 20, approximately30 birds per treatment would be required to detect a 4 percentage point difference in e production at a = 0.05 with 95.0% confidence. Theregre the number of hensltreatment used in the experiment was considered adequate. Weeks 1-4 eggproduction data were omitted from the data deck to remove any potential bias due to the inconsistent manner in which the flock came into production. This was considered to be partly due to subzero weather during this time; even though the house was heated, 4 of 24 pens had not reached 40% hen-day egg production (from a startingpoint of 15%) by the secondweek of data collection. 21. Bentley, AB., Q.B. Kinder, G.B. Garner, and J.E. Savage, 1965. Effect of nitrate in water on performance of laying hens. Poultry Sci. 44.1351. 22. Tomov, A, 1965. Effect of prolonged intake of nitrates and nitrites on hens and the quality of their eggs. Vet. Med. Nauki (Sofia) 2:31>321. 23. Bruning-Fann, C.S. and J.B. Kaneene, 1993. The effects of nitrate, nitrite, and N-nitroso compounds on animal health. Vet. Hum. Toxicol. 35(3):237-250.
ACKNOWLEDGEMENT The authors thank Arbor Acres Farm, Inc., Blairsville, GA for providing all birds used in this study.
Downloaded from http://japr.oxfordjournals.org/ at University of Birmingham on March 23, 2015
18. Gilbert, AB., M.M. Perry, D. Wadington, and M A Hardie, 1983. Role of atresia in establishing the follicular hierarchy in the ovary of the domestic hen domesticus). J. Reprod. Fert. 69221-227.
63