K.L. KNIGHT1
T.A. SCOTT2
1Faculty of Agriculture, Food and Natural Resources, University of Sydney, NSW
2Department of Veterinary Science, University of Sydney, Camden NSW, Australia
Infertility and embryonic mortality are major losses to the poultry industry and classification of true fertility by identifying very early embryonic mortality is difficult, particularly when eggs have been incubated and removed after candling. Preliminary studies were carried out with the purpose of providing a definitive diagnostic tool for classifying the fertility status of incubated chicken eggs.
After determining that propidium iodide staining and fluorescence microscopy was successful in determining fertility, a second trial was carried out to quantify the number of fertile eggs that were classified as "infertile" following six days of incubation. Eggs from Baiada Poultry Pty Limited (n=9600) were stored for two different periods of time (3 and 7d) prior to incubation and placed in two different positions in a standard egg trolley in an incubator (front and back). The germinal discs of 347 eggs were stained with propidium iodide and viewed under a fluorescence microscope to identify light points indicative of fertile (DNA) eggs. Overall, 11.85% of eggs initially observed to be infertile by visual inspection were found to be fertile.
The effect of pre-incubation storage length, incubation position and analysis day on the ability to determine fertility was not significant. These results suggest that propidium iodide staining of the germinal disc and fluorescence microscopy is an effective tool in determining true fertility of chicken eggs.
Introduction
The infertile or fertile status of an unincubated egg cannot always be determined with the naked eye and Kosin (1944) reported that there would be a three percent error rate when candling eggs for 'clears'. Some eggs that are detected as infertile at candling often contain embryos that died before or soon after oviposition; yet do not display the typical characteristics of fertile eggs. Breakout analysis post candling is beneficial in that many obviously fertile eggs would be screened out whereby removing the tedium and losses associated with analysis on fresh, unincubated eggs. There are many factors that contribute to poor hatchability and embryonic mortality and much research has been carried out into determining and minimising its causes.
Some research has been undertaken in defining true fertility to aid in the understanding of where along the reproductive pathway losses due to embryonic mortality occur. Liptoi et al. (2004) determined true fertility in duck embryos by simultaneously using outer perivitelline (OPVL) sperm counting and propidium iodide (PI) staining of the germinal disc to differentiate infertile eggs from very early dead embryos. Nonetheless, true fertility remains an ambiguous area due to the inaccurate and difficult methods currently available for detection of very early embryonic mortalities.
Materials and methods
A total of 9600 eggs from Cobb 500 birds were divided evenly into two groups and placed under cold storage (18°C) at Marsden Park Hatchery. Group one was held for 3 days and group two was held for 7 days before setting in Petersime incubators. Eggs from the two storage times were further divided according to position in the incubator, either front or back. After incubation for six days, all eggs were candled and 1206 infertile and early dead embryos (12.56% of total eggs) were set aside for classification. Of these, 574 (47.6 %) were from the 3-day storage group and 632 (52.4 % of total) were from the 7-day storage group. Live embryos were placed back into the incubator for continued growth and hatch. Eggs were transported to the lab and stored at 4°C until classification. The four groups were those stored for 3 days and placed at the front of the incubator (3F), or the back of the incubator (3B), and those stored for 7 days and stored at the front (7F) or the back (7B) of the incubator. One tray (approximately 30 eggs) from each group was stacked in the order 3F, 7F, 3B, and then 7B and placed back in 4°C storage. Analysis was carried out on each stack at random to minimise the effects of time on the stain and on the eggs.
a) Experimental procedure
The 798 eggs identified as infertile by candling were refrigerated and stored large end up to allow movement of the germinal disc (GD) to the uppermost point on the yolk. A total of 347 of the eggs were examined from the above 798 eggs classified as infertile (and confirmed by breakout) were randomly selected. A portion of the shell at the large end was removed and the outer perivitelline membrane pulled back to expose the germinal disc. A visual assessment was then made to determine fertility according to Mauldin (1998). As described by Liptoi et al. (2004), the germinal discs seemingly infertile or uncertainly fertile were transferred to a microscope slide for further analysis. A scalpel was used to make a small incision through the inner perivitelline membrane on the edge of the GD and it was then removed with a small, flat-ended surgical tool. Propidium iodide (Sigma P4170, Sigma-Aldrich Co., PO Box 14508 St. Louis Missouri 63103 USA) a DNA-specific red fluorescent dye was used to stain the GD at a concentration of 5μg PI / ml 0.9% NaCl solution. For each slide, 5μl of the solution was used and a coverslip placed on top.
b) Microscope analysis
True fertility of an embryo is determined by the presence of cell nuclei (Liptoi et al., 2004). If the egg is fertile, PI will stain and illuminate the nuclei but if the egg is infertile, there is no nucleus and the slide will display a dark red background without lighting points (Liptoi et al., 2004).
Examples of fertile eggs are illustrated in Figure 1. Propidium iodide acts by intercalating between base pairs of DNA. The cells of fertile eggs are diploid and it is estimated that at the time of oviposition, embryos are at the 30,000 to 70,000 cell stage (Emanuelsson, 1965). On the contrary, infertile egg cells remain haploid and there is no nucleus to be stained. Preliminary studies involved preparing and viewing numerous slides (n=225) under a fluorescence microscope (Olympus BX61; 400x magnification) to gain an understanding of the differences between infertile and fertile eggs. Analysis of infertile eggs was then made to identify true fertility. The remaining eggs were classified as membrane, blood ring, black eye, errors, cracked shell, shell problem or contamination according to and for the purposes of Marsden Park Hatchery.
Results
It was hypothesised that a number of eggs that are classified as infertile by breakout analysis would be classified as fertile upon microscopic analysis. Observations of fertility and embryo viability are summarised in Figure 2. All of the eggs were candled and 1206 were set aside as infertile or embryonic mortalities. After visual assessment, 798 (8.31%) were found to be infertile, 408 (4.09%) were embryonic mortalities and there were 15 errors (fertile live embryos).
Of the 347 eggs that were infertile upon visual inspection, 41 (11.85%) were found to be fertile after propidium iodide staining and fluorescence microscopy. Statistical analysis using regression of binomial proportions found that none of the factors or factor interactions had any significant effect on the number of fertile eggs (P = 0.127). The distribution of eggs identified as infertile macroscopically that were later classified as fertile but very early mortality for the two storage and egg positions is presented in Figure 3. The differences in position were not significant, but would suggest further follow up is required.
Conclusions
The success of the poultry industry depends on the reproductive capacity of its birds and the health and survivability of its chicks. In particular, breeders and hatcheries aim to optimise the conditions that produce the greatest number of viable chicks. However, on average, 15% of eggs are non viable with 10% of these being infertile with the remaining 5% lost as embryonic mortality (Mauldin, 1998). The availability of a method to determine true fertility would be beneficial for isolating reproductive or management problems, which lead to very early embryonic mortality.
In the past, determining the fertility status of an egg has been difficult. Many claim that true fertility can be determined with candling and microscopic examination whereas others believe that the only way to be accurate is to carry out breakout analysis on an entire sample. In any case, there are likely a number of eggs that are incorrectly assessed as "infertile" when the true status is fertile.
A fluorescent dye, propidium iodide (PI) and fluorescence microscopy was used to stain germinal discs in an attempt to determine true fertility of chicken eggs. The presence of cell nuclei and hence fluorescence, is representative of a fertile egg whereas infertile eggs do not possess a nucleus and the slide will simply stain dark red.
On the basis of these preliminary trials, propidium iodide staining with the use of fluorescence microscopy technology was found to be an effective tool for determining true fertility in chicken eggs. Further analysis was then carried out to try and quantify the number of false assessments made. Almost 12% of eggs that were classified as infertile upon visual or macroscopic inspection were found to be fertile. In this study neither length of preincubation storage of eggs, incubation position nor the day that the analysis took place were significant in scoring the number of eggs found to be fertile. This trial has provided a definitive tool allowing further studies to be carried out into the causes of embryonic mortality and in particular, very early embryonic mortality.
Acknowledgements
We gratefully acknowledge Baiada Poultry Pty Limited for the provision of eggs for the project and thank the staff for their help with candling. We also acknowledge the support of Drs P. Groves and J. Ruiz for their helpful input in developing the project. This manuscript was extracted from K.L. Knights 4th year honours thesis.
References
Emanuelsson, H. (1965). Experimental Cell Research, 39: 386-399.
Kosin, I.L. (1944). Poultry Science, 23: 266-269.
Liptoi, K., Varga, A. and Barna, J. (2004). Acta Veterinaria Hungarica, 52: 227-233.
Mauldin, J.M. (1998). Breakout analysis guide for hatcheries. The University of Georgia, College of Agricultural and Environmental Sciences Cooperative Extension Service.
From Proceedings of the "18th Australian Poultry Science Symposium", New South Wales, Australia.
