- Email: [email protected]
Stage of goose embryo development at oviposition depending on genotype, flock age, and period of laying
E. L ukaszewicz,∗,1 M. Laso´ n,∗ A. Kowalczyk,∗ J. Rosenberger,∗ K. Andres,† and M. Bakst‡ ∗
Division of Poultry Breeding, Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, 51-630 Wroclaw, Poland; † University of Agriculture in Krakow, 33-332 Krakow, Poland; and ‡ USA Poultry consultant, Retired USDA-ARS Beltsville, MD 20705, USA periment 1: Individual breed differences were evident with Stage X EGK embryos comprising 42.4, 33.3, and 38.7% in the eggs examined from the WK, Bi, and Za, respectively. For all breeds combined, 38.8% of the embryos were in Stage X, but in the next order in WK there was stage XI (18.2%), while in geese from the genetic reserve it was stage XIII (Bi – 33.3; Za – 29.0%). Experiment 2: In eggs of 1-, 2-, and 3-year-old WK geese, the majority of embryos (38.7, 32.4 and 42.2%, respectively) were in Stage X. In contrast, in 4-yearold geese the embryos were in Stage XI (36.1%). Experiment 3: In eggs collected in March and in June most of embryos were in Stage X (33.7% and 43.6%, respectively). In addition, more developmentally advanced stages (XI-XIII) were similar in both periods. However, embryos in Stage 2 HH were only observed in eggs collected at the end of laying season. Interestingly, earlier stages (VI-IX) were observed exclusively in the eggs collected in March.
ABSTRACT Embryo development and chick quality are influenced by parental genotype, age, nutrition, environment, and flock management. The aim of study was to determine if genotype, age of goose or eggs laid near the onset of egg production vs. eggs laid near the end of reproduction influence the stage of embryo at oviposition. Three experiments were undertaken. To compare genotypes (Experiment 1) 150 eggs were collected from 3-year-old commercial line White Koluda (WK) geese and from two breeds involved in a genetic resources conservation program, Zatorska (Za) and Bilgoraj (Bi). Age comparison (Experiment 2) was conducted with 200 eggs collected from 1-, 2-, 3-, and 4-year-old WK geese. To compare laying periods (Experiment 3), 150 WK eggs were collected at the first week of March and 100 at the second half of June. Eggs were stored for 72 h at 16◦ C, staged using Eyal-Giladi and Kochav (EGK, Roman numerals) and Hamburger and Hamilton (HH, Arabic numerals) procedures. Ex-
Key words: goose, age, genotype, laying period, embryo staging 2019 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pez225
INTRODUCTION
(Fujihara et al., 2001). The most popular, mainly in the Asian countries, commercial breeds were derived from wild swan goose (Anser cygnoides), while these bred in Europe originate from greylag goose (Anser anser) (Crawford, 1990). Both, wild species and derived breeds differ widely in body weight and conformation, feather color and, what is the most important from economic point of view, in the reproductive characteristics. Low reproductive indices limit geese development as an intensively managed poultry species. Comparing to other poultry species geese are characterized by short and seasonal reproduction period (in the European countries it lasts about 6 mo, usually from late January till July), low number of hatching eggs (from 15 to 60, depending on breed), hatchability below 80%, high embryo mortality, and as the consequence—a low number of goslings per female (Nitsan et al., 1988; Bednarczyk and Rosi´ nski, 1999; L ukaszewicz 2002; Rosi´ nski et al., 2006a,b). However, contrary to chickens, turkeys or even ducks, goose reproductive flock can be used for 3 to 6 seasons (Bieli´ nski and Rosi´ nski, 1988).
Geese were probably the first domesticated avian species (Crawford, 1990), and despite long lasting genetic selection, their behavior, reproductive performance, body size and composition are still comparable with wild goose ancestors. On one hand this limits a wider use of goose as economically unprofitable poultry species, but on the other one, due to an increasing interest in production and consumption of original and safe poultry origin products obtained from flock kept in accordance with animal welfare, goose production can become quite profitable. Also increasing awareness and public interest in environmental protection and animal welfare gradually returning to sustainable agricultural production system may also contribute to the increased importance of geese production (especially in Europe) C 2019 Poultry Science Association Inc. Received January 2, 2019. Accepted March 26, 2019. 1 Corresponding author: [email protected]
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Stage of goose embryo development at oviposition depending on genotype, flock age, and period of laying
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L UKASZEWICZ ET AL.
was to determine if the stage of goose embryo development in freshly laid eggs depends on genotype, flock age and period of laying season.
MATERIALS AND METHODS Three independent experiments were undertaken. Experiment 1: Genotype effect—50 eggs of comparable weight were collected from 3-year-old commercial line White Koluda (WK) geese and from two breeds involved in a genetic resources conservation program, Zatorska (Za) and Bilgoraj (Bi) (both flocks were also 3-year-old). The WK, Za and Bi geese do not have common ancestors, the genetic distance estimated from microsatellite markers was as follows: WK–Za - 0.185; WK–Bi - 0.229; Za–Bi - 0.374 (Wrzaszcz, 2011). Experiment 2: Effect of goose age—it was conducted with 200 eggs of comparable weight collected from 1-, 2-, 3-, and 4-year-old WK geese, 50 eggs per age group. In Experiment 1 and 2, the eggs were collected at the first decade of June. Experiment 3: Effect of the laying period—to compare the laying periods, 150 WK eggs were collected at the first week of March and 100 WK eggs were collected at the second half of June. In June at 8 am, at the day of eggs collection, at the farm location, the external temperature was 19◦ C, i.e., close to the physiological zero (Edwards, 1902). In every experiment the weight of eggs collected varied between 140 and 150 g. Despite proper management and ensuring the right number of nests per one female, it is often observed that some nests are empty, while in others there are several eggs. Therefore, for our purposes, the eggs were collected only from nests with single eggs and, thus, not exposed to possible heating by subsequent females laying the egg. All flocks were mated naturally at a ratio of one gander to 4 females, kept in semi-intensive system with free access to limited green paddocks. In each experiment all geese were fed with the commercial mixtures of the same nutritive value— 15% of crude proteins and 2,650 kcal metabolic energy. Good quality water and food were available ad libitum. In every experiment, the eggs were collected at 8 am of the same day and transported to the laboratory within 2 to 4 h after collection, and in air-conditioned vehicle. All eggs were stored prior to staging for 72 h at 16◦ C. After determining the egg status—fertile or unfertile and yolk separation, the blastoderm was isolated (under stereo microscope Nikon SMZ800 equipped with NIS Elements software) according the method elaborated by Gupta and Bakst (1993), and briefly described in our previous publication (Lukaszewicz et al., 2017). The stage of embryo was determined according to EyalGiladi and Kochav (EGK, Roman numerals) and Hamburger and Hamilton (HH, Arabic numerals) procedures. A total of 600 eggs were examined in this study. The results obtained in each experiment were expressed as the proportion of the particular stages of embryo
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An improvement of any of afore-mentioned critical stages of goose breeding can contribute to a significant increase in efficiency and profitability of goose production. One of such elements relates with egg storage and incubation process. Aiming to achieve the highest rates of chick hatchability is particularly important for species with low egg production and long incubation period during which the embryos are exposed to mortality for prolonged length of time. The relatively small number of birds in one parent flock (usually one flock consists of 600 to 1500 females) limits the supply of hatching eggs. The period of the year when geese lay their eggs typically starts when external temperatures are below 0◦ C and finishes when temperatures are above 25◦ C, which can affect the conditions during egg storage, which significantly affect later embryonic development and the quality of hatched goslings (Fasenko, 2007). It is well known that prolonged egg storage (beyond 7 D) affects negatively the efficiency of chick hatchability (Bakst and Akuffo, 2002; Bakst et al., 2012; Goliomytis et al., 2015; Pokhrel et al., 2018). Badowski (2009) indicated that the hatchability of goslings from set eggs stored for 21 D at temperature 10◦ C and relative air humidity 70% was lower by 31.6 percentage points, on average (85.3% vs. 53.7%) when compared to eggs stored for 7 D. It has been already proved that higher stage of chicken embryo development prior incubation, obtained by SPIDES (Short Periods of Incubation During Egg Storage) reduced embryo mortality during egg storage in broiler breeders (Daymond et al., 2013). The stage 3 HH, (Hamburger and Hamilton, 1951), i.e., when early primitive streak is formatted, had the most beneficial effect on hatchability of stored eggs. This stage could be obtained by 4, short (4 h each) preincubations of 21 D stored eggs. Single 6 h preincubation advanced embryo development to stage XIV EGK (Eyal-Giladi and Kochav, 1976), but the hatchability was worse, even than in the untreated controls. Our earlier experiment on stages of embryo development at oviposition in the freshly laid eggs collected from 3-year-old geese (Lukaszewicz et al., 2017) indicated great varieties in distribution of the developmental stages—from stage VI EGK to stage 2 HH. Sellier et al. (2006) suggested that the stage of blastoderm development at oviposition might be controlled genetically and/or the degree of female domestication. Mather and Laughlin (1979) and Fasenko et al. (1992) found that older broiler breeder hens tended to produce more developmentally advanced embryos at oviposition that were also more resistant to storage than less developed embryos (citation by Daymont at al., 2013). Knowledge about the significance of embryo stage at oviposition on further success of hatching and chicks’ quality, the huge differences in goose breeds, period of their utilization as the reproductive flock and changing environmental conditions during laying period inspired us to perform presented studies. The aim of this study
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STAGE OF GOOSE EMBRYO AT OVIPOSITION
Stages of goose embryo development EGK stages Breed White Koluda Bilgoraj Zatorska In total
HH stage
Blastoderms n/%
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
2
33 100 21 100 31 100 85 100
1 3.03 – – – – 1 1.18
– – – – – – – –
– – – – – – – –
3 9.09 – – – – 3 3.53
14 42.42 7 33.33 12 38.71 33 38.82
6 18.18 3 14.29 2 6.45 11 12.94
4 12.12 2 9.52 5 16.13 11 12.94
3 9.09 7 33.33 9 29.03 19 22.35
– – 1 4.76 1 3.23 2 2.35
2 6.06 1 4.76 2 6.45 5 5.88
Table 2. Stages of goose embryo development depending on flock age (number and % distribution). Stages of goose embryo development Flock age (years) 1 2 3 4 In total
EGK stages Blastoderms n/%
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
HH stage 2
31 100 37 100 33 100 36 100 137 100
– –
1 3.22 2 5.41 – – 1 2.78 4 2.92
– –
2 6.45 – – 3 9.09 – – 5 3.65
12 38.71 12 32.43 14 42.42 10 27.78 48 35.04
4 12.90 10 27.03 6 18.18 13 36.11 33 24.09
4 12.90 7 18.92 4 12.12 7 19.44 22 16.06
5 16.13 4 10.81 3 9.09 4 11.11 16 11.68
– – – – – – – – – –
3 9.68 – – 2 6.06 – – 5 3.65
1 2.70 1 3.03 – – 2 1.46
1 2.70 – – 1 2.78 2 1.46
development in relation to total number of collected blastoderms.
Statistical Analysis To verify if the frequency of particular stages of embryo development is related to the tested experimental factor (genotype, age and period of the laying season), the chi-squared test was used. All statistical comparisons were made considering a P < 0.05 significance level.
RESULTS AND DISCUSSION Out of 600 goose eggs used in this experiment 360 embryos were isolated successfully, i.e., in 60%, however we were not interested in blastoderm collection efficiency, and we did not focused on egg fertility level. The effectiveness of blastoderm isolation resulted from both, the level of egg fertilization (especially of Bilgoraj goose that was within 70%), as well as the ability to collect them. Although goose eggs are 2 to 3 times larger than chicken eggs, the germinal disc is similar in size, but the goose egg yolk is much denser and while separating and cleaning the blastoderms from adhering yolk, several of them were lost. Regardless of the analyzed factor, blastoderms between stage VI EGK to stage 2 HH appeared in all evaluated groups however, the dominant stage, as in the unincubated eggs of other poultry species (Dupuy et al., 2002;
Sellier et al., 2006; Bakst and Wade, 2014) was stage X EGK. It has been stated in 36.9% of blastoderms of all groups, on average, and in the next order there was stage XI (21.4%) and XII (15.3%). Nevertheless, the effect of goose genotype (Table 1), flock age (Table 2) and period of the laying season (Table 3) in distribution of particular developmental stages could be observed, however, the existed differences were statistically not significant (P < 0.05). In the Experiment 1 (genotype effect), similar as in other two presented experiments, and in our previous studies (Lukaszewicz et al., 2017), the majority of embryos (38.8%, on average) were at stage X EGK, but in the next order in the commercial line WK there was stage XI (18.2%), while in goose involved in Polish genetic resources conservation program the embryos were at more advanced stage—stage XIII (Bilgoraj—33.3; Zatorska—29.0%), which is characterized by complete hypoblast formation (Eyal-Giladi and Kochav, 1976). In WK eggs, the embryos were the least developed, majority of them did not exceed Stage X EGK (Table 1). Out of 3 evaluated genotypes only WK is the commercial line selected since 1962—the male strain (W-33) toward improving the meat traits and the female strain (W-11) toward the laying performance (Klopotek, 2018). Coleman and Siegel (1966) found that in the divergent selection of chickens toward low and high body weight at the 8th week of life, in the line of high body weight the embryos at the time of oviposition were less developed and more sensitive during longer egg storage than low body weight line.
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Table 1. Stages of goose embryo development depending on genotype (number and % distribution).
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L UKASZEWICZ ET AL.
Stages of goose embryo development EGK stages Period of season March June In total
HH stage
Blastoderms n/%
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
2
83 100 55 100 138 100
2 2.41 – – 2 1.45
4 4.82 – – 4 2.90
2 2.41 – – 2 1.45
2 2.41 – – 2 1.45
28 33.73 24 43.64 52 37.68
20 24.10 13 23.63 33 23.91
16 19.28 6 10.91 22 15.94
9 10.84 7 12.73 16 11.59
– – – – – –
– –
Moreover, intensive selection toward growth rate and meat traits causes decrease in the reproductive characteristics (Sadeghi et al., 2013). Sawicka et al. (2015) studying the effect of different selection pressure on the number of live and apoptotic BCs in the stage X EGK of quail embryos, concluded that impact of selection may be carried out even during the early stage of embryonic development. Bakst et al. (2012) studying blastoderm of freshly laid and incubated eggs of 2 chicken broiler breeder lines, stated slight, but significant differences in blastoderms diameter when line averages were compared. If we consider that higher stage of embryo development at oviposition reduces its mortality during egg storage (Daymond et al., 2013), we can speculate that the embryos of Bi and Za goose that are kept in more primitive environmental condition than commercial WK flocks, and which were not genetically selected, are more advanced at oviposition in order to better survive the unfavorable conditions before the onset of eggs incubation? The same can relates to age of goose flock. Unlike chicken or turkey breeders, goose can be kept as the reproductive flock for 4, and even more years, but from the third breeding season, both the quality of semen, egg fertilization and goslings hatchability decrease (Lukaszewicz, 2002). In the second experiment (age effect), the embryo isolated from eggs of 1-, 2- and 3-year-old WK goose were at stage X EGK (38.7; 32.43 and 42.2%, respectively), while these collected from eggs of 4-year-old goose were slightly more advanced— stage XI EGK (36.11%) (Table 2). As above, taking into consideration that higher stage of development at the start of diapause period is more beneficial, it may be assumed that in older goose flocks the lower fertility rates are compensated by higher stage of embryo development at oviposition. Finally, event can decrease unfavorable low number of goslings obtained from older female (Nowaczewski et al., 2018). It has been also confirmed by Pokhrel et al. (2018) who stated that freshly laid eggs from old broiler breeder flocks are in a more advanced developmental stage than blastoderms from young flocks. Analyzing the effect of laying period (first week of March vs. second half of June) we stated the highest percentage of embryos at stage X EGK. The proportion of more advanced stages (XI-XIII) was similar in both
5 9.09 5 3.62
periods, while the lower stages (VI-IX) were observed exclusively in the eggs collected in March, and stage 2 HH was found only in the eggs laid at the end of laying season. This indicates the impact of the laying period (more probably related to the daily temperatures outside and inside the building) on the stage of embryo development in the unincubated eggs. As mentioned previously, in June the external, environmental temperature was close to physiological zero (20◦ C), while in March it was around 5◦ C. The question is how far the stage of goose embryo development in the freshly laid egg affects the ability of eggs to storage conditions, and later on, their hatchability, hatched gosling quality and performance. We can assume that obtained results encourage us for further research on factors affecting the stage of goose embryo development at oviposition, the possibilities of applying SPIDES (Short Period of Incubation During Egg Storage) (Dymond et al., 2013) during short (up to 7 D) and prolonged (up to 21 D) storage and its impact on gosling hatching efficiency and further performance.
ACKNOWLEDGMENTS The experiments and article processing charge were supported partly by the Ministry of Science and Higher Education for WUELS statutory activity and by Wroclaw Centre of Biotechnology, program The Leading National Research Centre (KNOW) for years 2014 to 2018. Supporting institutions had no role in study design, data collection, and analysis.
REFERENCES Badowski, J. 2009. Hatchability of goslings from eggs stored for up to three weeks before incubation. Proc. XXI Int. Poult. Symp. PB WPSA “Science for Poultry Practice—Poultry Practice For Poland, September7–9:220. Science”, Wroclaw-Szklarska Poreba, Bakst, M. R., and V. Akuffo. 2002. Impact of egg storage on embryo development. Avian Poult. Biol. Rev. 13:219–228. Bakst, M. R., V. Akuffo, D. Nicholson, and N. French. 2012. Comparison of blastoderm traits from 2 lines of broilers before and after egg storage and incubation. Poult. Sci. 91:2645–2648. Bakst, M. R., and A. J. Wade. 2014. New observations regarding staging turkey embryos from oviposition through primitive streak formation. Avian Biol. Res. 7:99–105.
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Table 3. Stages of goose embryo development depending on the period of the laying season (number and % distribution).
STAGE OF GOOSE EMBRYO AT OVIPOSITION
Klopotek, E. 2018. Influence of the physical form of oat grain on the fattening results and carcass quality of White Koluda geese. Sci. Ann. Polish Soc. Anim. Prod. 14:23–36. L ukaszewicz, E. 2002. Cryoconservation of gander semen. Zesz. Nauk. AR Wroclaw 440:1–111 (in Polish, abstract in English). L ukaszewicz, E., M. Laso´ n, J. Rosenberger, A. Kowalczyk, and M. Bakst. 2017. Goose embryonic development from oviposition through 16 hours of incubation. Poult. Sci. 96:1934–1938. Mather, C. M., and K. F. Laughlin. 1979. Storage of hatching eggs: the interaction between parental age and early embryonic development. Br. Poult. Sci. 20:595–604. Nitsan, Z., I. Nir, and A. Dvorin. 1988. Reproductive performance of laying geese kept in flocks or in individual pens. Proc. Intern. Symp. Waterfowl Product, The Satellite Confer. XVIII WPC, Beijing, China, September11–18: 278–280. Pokhrel, N., E. Ben-Tal Cohen, O. Genin, M. Ruzal, D. SelaDonenfeld, and Y. Cinnamon. 2018. Effects of storage conditions on hatchability, embryonic survival and cytoarchitectural properties in broiler from young and old flocks. Poult. Sci. 97: 1429–1440. Nowaczewski, S., B. Grzegrz´olka, K. Kukulski, and B. Kolano´s. 2018. Effect of eggshell colour on early embryo development in ringnecked pheasant (Phasianus colchicus). Anim. Sci. Pap. Rep. 36:75–86. Rosi´ nski, A., S. Nowaczewski, H. Kontecka, M. Bednarczyk, G. Elminowska-Wenda, H. Bieli´ nska, and A. Maczy´ nska. 2006a. Analysis of the laying rhythm and reproductive traits of geese. Folia Biol. (Krak´ ow) 54:145–152. Rosi´ nski, A., S. Nowaczewski, H. Kontecka, J. Samolak, H. Bieli´ nska, and J. Badowski 2006b. Analysis of the laying rhythm in White R geese. Arch. Gefl¨ ugelk. 70:187–192. Koluda Sadeghi, R., A. Pakdel, M. Moradi Shahrbabak, and H. Moradi Shahrbabak. 2013. Divergent selection effect on reproductive trait in Japanese quails. Res. J. Poult. Sci. 6:18–22. Sawicka, D., K. Samek, L. Chojnacka-Puchta, A. Witkowski, S. and M. Bednarczyk. 2015. Changes in Knaga, M. Debowska, quail blastodermal cell status as a result of selection. Folia Biol. (Krak´ ow) 63:63–67. Sellier, N., J. P. Brillard, V. Dupuy, and M. R. Bakst. 2006. Comparative staging of embryo development in chicken, turkey, duck, goose, guinea fowl, and Japanese quail assessed from five hours after fertilization through seventy-two hours of incubation. J. Appl. Poult. Res. 15:219–228. Wrzaszcz, L . 2011. Evaluation of the genetic diversity of geese population based on DNA polymorphism. PhD thesis, Siedlce, Poland.
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Bednarczyk, M., and A. Rosi´ nski. 1999. Comparison of egg hatchability and in vitro survival of goose embryos of various origins. Poult. Sci. 78:579–585. Bieli´ nski, K., and A. Rosi´ nski. 1988. Influence of the age of White Italian geese on their reproductive performance. Proc. Int. Symp. Waterfowl Product., The Satellite Conf. XVIII WPC, Beijing, China, September11–18:223–227. Coleman, J. W., and P. B. Siegel. 1966. Selection of body weight at eight weeks of age. 5. Embryonic state at oviposition and its relationship to hatchability. Poult. Sci. 45:1003–1011. Crawford, R. D. 1990. Origin and history of poultry species, Chapter 1. pages 1–41 in Poultry Breeding and Genetics. R. D. Crawford ed. 1st edition. Elsevier. Dupuy, V., B. Nersessian, and M. R. Bakst. 2002. Embryonic development from first cleavage through seventy-two hours incubation in two strains of Pekin duck (Anas platyrhynchos). Poult. Sci. 81:860–868. Dymond, J., B. Vinyard, A. D. Nicholson, N. A. French, and M. R. Bakst. 2013. Short periods of incubation during egg storage increase hatchability and chick quality in long-stored broiler eggs. Poult. Sci.92:2977–2987. Edwards, C. L. 1902. The physiological zero and the index of development for the egg of the domestic fowl (Gallus domesticus). Am. J. Physiol. 6:351–397. Eyal-Giladi, H., and S. Kochav. 1976. From cleavage to primitive streak formation - complementary normal table and a new look at 1st stages of development of chick. 1. General morphology. Dev. Biol. 49:321–337. Fasenko, G. M. 2007. Egg storage and the embryo. Poult. Sci. 86:1020–1024. Fasenko, G. M., R. T. Hardin, F. E. Robinson, and J. L. Wilson. 1992. Relationship of hen age and egg sequence position with fertility hatchability, viability, and preincubation embryonic development in broiler breeders. Poult. Sci. 71:1374–1383. Fujihara, N., Y. M. Xi, and M. J. Zhang. 2001. Genetic resource conservation in poultry reproduction. J. Appl. Anim. Res. 19:33– 47. Goliomytis, M., T. Tsipouzian, and A. L. Hager-Theodorides. 2015. Effects of egg storage on hatchability, chick quality, performance and immunocompetence parameters of broiler chickens. Poult. Sci. 94:2257–2265. Gupta, S. K., and M. R. Bakst. 1993. Turkey embryo staging from cleavage through hypoblast formation. J. Morphol. 217:313–325. Hamburger, V., and H. L. Hamilton. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49–92.
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Division of Poultry Breeding, Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, 51-630 Wroclaw, Poland; † University of Agriculture in Krakow, 33-332 Krakow, Poland; and ‡ USA Poultry consultant, Retired USDA-ARS Beltsville, MD 20705, USA periment 1: Individual breed differences were evident with Stage X EGK embryos comprising 42.4, 33.3, and 38.7% in the eggs examined from the WK, Bi, and Za, respectively. For all breeds combined, 38.8% of the embryos were in Stage X, but in the next order in WK there was stage XI (18.2%), while in geese from the genetic reserve it was stage XIII (Bi – 33.3; Za – 29.0%). Experiment 2: In eggs of 1-, 2-, and 3-year-old WK geese, the majority of embryos (38.7, 32.4 and 42.2%, respectively) were in Stage X. In contrast, in 4-yearold geese the embryos were in Stage XI (36.1%). Experiment 3: In eggs collected in March and in June most of embryos were in Stage X (33.7% and 43.6%, respectively). In addition, more developmentally advanced stages (XI-XIII) were similar in both periods. However, embryos in Stage 2 HH were only observed in eggs collected at the end of laying season. Interestingly, earlier stages (VI-IX) were observed exclusively in the eggs collected in March.
ABSTRACT Embryo development and chick quality are influenced by parental genotype, age, nutrition, environment, and flock management. The aim of study was to determine if genotype, age of goose or eggs laid near the onset of egg production vs. eggs laid near the end of reproduction influence the stage of embryo at oviposition. Three experiments were undertaken. To compare genotypes (Experiment 1) 150 eggs were collected from 3-year-old commercial line White Koluda (WK) geese and from two breeds involved in a genetic resources conservation program, Zatorska (Za) and Bilgoraj (Bi). Age comparison (Experiment 2) was conducted with 200 eggs collected from 1-, 2-, 3-, and 4-year-old WK geese. To compare laying periods (Experiment 3), 150 WK eggs were collected at the first week of March and 100 at the second half of June. Eggs were stored for 72 h at 16◦ C, staged using Eyal-Giladi and Kochav (EGK, Roman numerals) and Hamburger and Hamilton (HH, Arabic numerals) procedures. Ex-
Key words: goose, age, genotype, laying period, embryo staging 2019 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pez225
INTRODUCTION
(Fujihara et al., 2001). The most popular, mainly in the Asian countries, commercial breeds were derived from wild swan goose (Anser cygnoides), while these bred in Europe originate from greylag goose (Anser anser) (Crawford, 1990). Both, wild species and derived breeds differ widely in body weight and conformation, feather color and, what is the most important from economic point of view, in the reproductive characteristics. Low reproductive indices limit geese development as an intensively managed poultry species. Comparing to other poultry species geese are characterized by short and seasonal reproduction period (in the European countries it lasts about 6 mo, usually from late January till July), low number of hatching eggs (from 15 to 60, depending on breed), hatchability below 80%, high embryo mortality, and as the consequence—a low number of goslings per female (Nitsan et al., 1988; Bednarczyk and Rosi´ nski, 1999; L ukaszewicz 2002; Rosi´ nski et al., 2006a,b). However, contrary to chickens, turkeys or even ducks, goose reproductive flock can be used for 3 to 6 seasons (Bieli´ nski and Rosi´ nski, 1988).
Geese were probably the first domesticated avian species (Crawford, 1990), and despite long lasting genetic selection, their behavior, reproductive performance, body size and composition are still comparable with wild goose ancestors. On one hand this limits a wider use of goose as economically unprofitable poultry species, but on the other one, due to an increasing interest in production and consumption of original and safe poultry origin products obtained from flock kept in accordance with animal welfare, goose production can become quite profitable. Also increasing awareness and public interest in environmental protection and animal welfare gradually returning to sustainable agricultural production system may also contribute to the increased importance of geese production (especially in Europe) C 2019 Poultry Science Association Inc. Received January 2, 2019. Accepted March 26, 2019. 1 Corresponding author: [email protected]
1
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez225/5478568 by Georgetown University Law Center E.B. Williams Library user on 26 August 2019
Stage of goose embryo development at oviposition depending on genotype, flock age, and period of laying
2
L UKASZEWICZ ET AL.
was to determine if the stage of goose embryo development in freshly laid eggs depends on genotype, flock age and period of laying season.
MATERIALS AND METHODS Three independent experiments were undertaken. Experiment 1: Genotype effect—50 eggs of comparable weight were collected from 3-year-old commercial line White Koluda (WK) geese and from two breeds involved in a genetic resources conservation program, Zatorska (Za) and Bilgoraj (Bi) (both flocks were also 3-year-old). The WK, Za and Bi geese do not have common ancestors, the genetic distance estimated from microsatellite markers was as follows: WK–Za - 0.185; WK–Bi - 0.229; Za–Bi - 0.374 (Wrzaszcz, 2011). Experiment 2: Effect of goose age—it was conducted with 200 eggs of comparable weight collected from 1-, 2-, 3-, and 4-year-old WK geese, 50 eggs per age group. In Experiment 1 and 2, the eggs were collected at the first decade of June. Experiment 3: Effect of the laying period—to compare the laying periods, 150 WK eggs were collected at the first week of March and 100 WK eggs were collected at the second half of June. In June at 8 am, at the day of eggs collection, at the farm location, the external temperature was 19◦ C, i.e., close to the physiological zero (Edwards, 1902). In every experiment the weight of eggs collected varied between 140 and 150 g. Despite proper management and ensuring the right number of nests per one female, it is often observed that some nests are empty, while in others there are several eggs. Therefore, for our purposes, the eggs were collected only from nests with single eggs and, thus, not exposed to possible heating by subsequent females laying the egg. All flocks were mated naturally at a ratio of one gander to 4 females, kept in semi-intensive system with free access to limited green paddocks. In each experiment all geese were fed with the commercial mixtures of the same nutritive value— 15% of crude proteins and 2,650 kcal metabolic energy. Good quality water and food were available ad libitum. In every experiment, the eggs were collected at 8 am of the same day and transported to the laboratory within 2 to 4 h after collection, and in air-conditioned vehicle. All eggs were stored prior to staging for 72 h at 16◦ C. After determining the egg status—fertile or unfertile and yolk separation, the blastoderm was isolated (under stereo microscope Nikon SMZ800 equipped with NIS Elements software) according the method elaborated by Gupta and Bakst (1993), and briefly described in our previous publication (Lukaszewicz et al., 2017). The stage of embryo was determined according to EyalGiladi and Kochav (EGK, Roman numerals) and Hamburger and Hamilton (HH, Arabic numerals) procedures. A total of 600 eggs were examined in this study. The results obtained in each experiment were expressed as the proportion of the particular stages of embryo
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An improvement of any of afore-mentioned critical stages of goose breeding can contribute to a significant increase in efficiency and profitability of goose production. One of such elements relates with egg storage and incubation process. Aiming to achieve the highest rates of chick hatchability is particularly important for species with low egg production and long incubation period during which the embryos are exposed to mortality for prolonged length of time. The relatively small number of birds in one parent flock (usually one flock consists of 600 to 1500 females) limits the supply of hatching eggs. The period of the year when geese lay their eggs typically starts when external temperatures are below 0◦ C and finishes when temperatures are above 25◦ C, which can affect the conditions during egg storage, which significantly affect later embryonic development and the quality of hatched goslings (Fasenko, 2007). It is well known that prolonged egg storage (beyond 7 D) affects negatively the efficiency of chick hatchability (Bakst and Akuffo, 2002; Bakst et al., 2012; Goliomytis et al., 2015; Pokhrel et al., 2018). Badowski (2009) indicated that the hatchability of goslings from set eggs stored for 21 D at temperature 10◦ C and relative air humidity 70% was lower by 31.6 percentage points, on average (85.3% vs. 53.7%) when compared to eggs stored for 7 D. It has been already proved that higher stage of chicken embryo development prior incubation, obtained by SPIDES (Short Periods of Incubation During Egg Storage) reduced embryo mortality during egg storage in broiler breeders (Daymond et al., 2013). The stage 3 HH, (Hamburger and Hamilton, 1951), i.e., when early primitive streak is formatted, had the most beneficial effect on hatchability of stored eggs. This stage could be obtained by 4, short (4 h each) preincubations of 21 D stored eggs. Single 6 h preincubation advanced embryo development to stage XIV EGK (Eyal-Giladi and Kochav, 1976), but the hatchability was worse, even than in the untreated controls. Our earlier experiment on stages of embryo development at oviposition in the freshly laid eggs collected from 3-year-old geese (Lukaszewicz et al., 2017) indicated great varieties in distribution of the developmental stages—from stage VI EGK to stage 2 HH. Sellier et al. (2006) suggested that the stage of blastoderm development at oviposition might be controlled genetically and/or the degree of female domestication. Mather and Laughlin (1979) and Fasenko et al. (1992) found that older broiler breeder hens tended to produce more developmentally advanced embryos at oviposition that were also more resistant to storage than less developed embryos (citation by Daymont at al., 2013). Knowledge about the significance of embryo stage at oviposition on further success of hatching and chicks’ quality, the huge differences in goose breeds, period of their utilization as the reproductive flock and changing environmental conditions during laying period inspired us to perform presented studies. The aim of this study
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STAGE OF GOOSE EMBRYO AT OVIPOSITION
Stages of goose embryo development EGK stages Breed White Koluda Bilgoraj Zatorska In total
HH stage
Blastoderms n/%
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
2
33 100 21 100 31 100 85 100
1 3.03 – – – – 1 1.18
– – – – – – – –
– – – – – – – –
3 9.09 – – – – 3 3.53
14 42.42 7 33.33 12 38.71 33 38.82
6 18.18 3 14.29 2 6.45 11 12.94
4 12.12 2 9.52 5 16.13 11 12.94
3 9.09 7 33.33 9 29.03 19 22.35
– – 1 4.76 1 3.23 2 2.35
2 6.06 1 4.76 2 6.45 5 5.88
Table 2. Stages of goose embryo development depending on flock age (number and % distribution). Stages of goose embryo development Flock age (years) 1 2 3 4 In total
EGK stages Blastoderms n/%
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
HH stage 2
31 100 37 100 33 100 36 100 137 100
– –
1 3.22 2 5.41 – – 1 2.78 4 2.92
– –
2 6.45 – – 3 9.09 – – 5 3.65
12 38.71 12 32.43 14 42.42 10 27.78 48 35.04
4 12.90 10 27.03 6 18.18 13 36.11 33 24.09
4 12.90 7 18.92 4 12.12 7 19.44 22 16.06
5 16.13 4 10.81 3 9.09 4 11.11 16 11.68
– – – – – – – – – –
3 9.68 – – 2 6.06 – – 5 3.65
1 2.70 1 3.03 – – 2 1.46
1 2.70 – – 1 2.78 2 1.46
development in relation to total number of collected blastoderms.
Statistical Analysis To verify if the frequency of particular stages of embryo development is related to the tested experimental factor (genotype, age and period of the laying season), the chi-squared test was used. All statistical comparisons were made considering a P < 0.05 significance level.
RESULTS AND DISCUSSION Out of 600 goose eggs used in this experiment 360 embryos were isolated successfully, i.e., in 60%, however we were not interested in blastoderm collection efficiency, and we did not focused on egg fertility level. The effectiveness of blastoderm isolation resulted from both, the level of egg fertilization (especially of Bilgoraj goose that was within 70%), as well as the ability to collect them. Although goose eggs are 2 to 3 times larger than chicken eggs, the germinal disc is similar in size, but the goose egg yolk is much denser and while separating and cleaning the blastoderms from adhering yolk, several of them were lost. Regardless of the analyzed factor, blastoderms between stage VI EGK to stage 2 HH appeared in all evaluated groups however, the dominant stage, as in the unincubated eggs of other poultry species (Dupuy et al., 2002;
Sellier et al., 2006; Bakst and Wade, 2014) was stage X EGK. It has been stated in 36.9% of blastoderms of all groups, on average, and in the next order there was stage XI (21.4%) and XII (15.3%). Nevertheless, the effect of goose genotype (Table 1), flock age (Table 2) and period of the laying season (Table 3) in distribution of particular developmental stages could be observed, however, the existed differences were statistically not significant (P < 0.05). In the Experiment 1 (genotype effect), similar as in other two presented experiments, and in our previous studies (Lukaszewicz et al., 2017), the majority of embryos (38.8%, on average) were at stage X EGK, but in the next order in the commercial line WK there was stage XI (18.2%), while in goose involved in Polish genetic resources conservation program the embryos were at more advanced stage—stage XIII (Bilgoraj—33.3; Zatorska—29.0%), which is characterized by complete hypoblast formation (Eyal-Giladi and Kochav, 1976). In WK eggs, the embryos were the least developed, majority of them did not exceed Stage X EGK (Table 1). Out of 3 evaluated genotypes only WK is the commercial line selected since 1962—the male strain (W-33) toward improving the meat traits and the female strain (W-11) toward the laying performance (Klopotek, 2018). Coleman and Siegel (1966) found that in the divergent selection of chickens toward low and high body weight at the 8th week of life, in the line of high body weight the embryos at the time of oviposition were less developed and more sensitive during longer egg storage than low body weight line.
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Table 1. Stages of goose embryo development depending on genotype (number and % distribution).
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L UKASZEWICZ ET AL.
Stages of goose embryo development EGK stages Period of season March June In total
HH stage
Blastoderms n/%
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
2
83 100 55 100 138 100
2 2.41 – – 2 1.45
4 4.82 – – 4 2.90
2 2.41 – – 2 1.45
2 2.41 – – 2 1.45
28 33.73 24 43.64 52 37.68
20 24.10 13 23.63 33 23.91
16 19.28 6 10.91 22 15.94
9 10.84 7 12.73 16 11.59
– – – – – –
– –
Moreover, intensive selection toward growth rate and meat traits causes decrease in the reproductive characteristics (Sadeghi et al., 2013). Sawicka et al. (2015) studying the effect of different selection pressure on the number of live and apoptotic BCs in the stage X EGK of quail embryos, concluded that impact of selection may be carried out even during the early stage of embryonic development. Bakst et al. (2012) studying blastoderm of freshly laid and incubated eggs of 2 chicken broiler breeder lines, stated slight, but significant differences in blastoderms diameter when line averages were compared. If we consider that higher stage of embryo development at oviposition reduces its mortality during egg storage (Daymond et al., 2013), we can speculate that the embryos of Bi and Za goose that are kept in more primitive environmental condition than commercial WK flocks, and which were not genetically selected, are more advanced at oviposition in order to better survive the unfavorable conditions before the onset of eggs incubation? The same can relates to age of goose flock. Unlike chicken or turkey breeders, goose can be kept as the reproductive flock for 4, and even more years, but from the third breeding season, both the quality of semen, egg fertilization and goslings hatchability decrease (Lukaszewicz, 2002). In the second experiment (age effect), the embryo isolated from eggs of 1-, 2- and 3-year-old WK goose were at stage X EGK (38.7; 32.43 and 42.2%, respectively), while these collected from eggs of 4-year-old goose were slightly more advanced— stage XI EGK (36.11%) (Table 2). As above, taking into consideration that higher stage of development at the start of diapause period is more beneficial, it may be assumed that in older goose flocks the lower fertility rates are compensated by higher stage of embryo development at oviposition. Finally, event can decrease unfavorable low number of goslings obtained from older female (Nowaczewski et al., 2018). It has been also confirmed by Pokhrel et al. (2018) who stated that freshly laid eggs from old broiler breeder flocks are in a more advanced developmental stage than blastoderms from young flocks. Analyzing the effect of laying period (first week of March vs. second half of June) we stated the highest percentage of embryos at stage X EGK. The proportion of more advanced stages (XI-XIII) was similar in both
5 9.09 5 3.62
periods, while the lower stages (VI-IX) were observed exclusively in the eggs collected in March, and stage 2 HH was found only in the eggs laid at the end of laying season. This indicates the impact of the laying period (more probably related to the daily temperatures outside and inside the building) on the stage of embryo development in the unincubated eggs. As mentioned previously, in June the external, environmental temperature was close to physiological zero (20◦ C), while in March it was around 5◦ C. The question is how far the stage of goose embryo development in the freshly laid egg affects the ability of eggs to storage conditions, and later on, their hatchability, hatched gosling quality and performance. We can assume that obtained results encourage us for further research on factors affecting the stage of goose embryo development at oviposition, the possibilities of applying SPIDES (Short Period of Incubation During Egg Storage) (Dymond et al., 2013) during short (up to 7 D) and prolonged (up to 21 D) storage and its impact on gosling hatching efficiency and further performance.
ACKNOWLEDGMENTS The experiments and article processing charge were supported partly by the Ministry of Science and Higher Education for WUELS statutory activity and by Wroclaw Centre of Biotechnology, program The Leading National Research Centre (KNOW) for years 2014 to 2018. Supporting institutions had no role in study design, data collection, and analysis.
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Table 3. Stages of goose embryo development depending on the period of the laying season (number and % distribution).
STAGE OF GOOSE EMBRYO AT OVIPOSITION
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