V. L. Christensen

Factors associated with early embryonic mortality

This paper describes the patterns of embryonic mortality in poultry species and summarises the various causes of mortality. Descriptions of the morphological stages of development at each of these time periods are given. Data are presented describing factors affecting development at oviposition, egg storage and incubation.

Broiler embryo bone development is influenced by incubator temperature, oxygen concentration and eggshell conductance at the plateau stage in oxygen consumption1

1. Four experiments were conducted to evaluate the effects of temperature (TEM) and oxygen (O2) concentrations during the last 4 d of incubation on bone development. Fertile eggs from two strains were obtained that either exhibited Low or High eggshell conductance (G). 1The mention of trade names in this publication does not imply endorsement of the products mentioned nor criticism of similar products not mentioned. 2. Four experimental cabinets provided either four TEM (36, 37, 38 or 39°C) or four O2 concentrations (17, 19, 21 or 23% O2). Data were analysed as a 2 × 2 factorial design. In the fourth experiment, two temperatures (36 and 39°C), two O2 concentrations (17 and 23%) and the same Low and High G strains were evaluated in a 2 × 2 × 2 factorial design. 3. Body weights (BW) and residual yolks were obtained, both legs were dissected. Femur, tibia and shank weights, length and thickness were recorded. Relative asymmetry (RA) of each leg section was calculated. 4. The results indicated that elevated TEM during incubation increased RA between the two legs, mainly in the Low G strain. Chickens at the lowest O2 concentrations had lighter and shorter tibias, lighter shanks, and increased RA of femur length compared to chickens in the 23% O2. In the fourth experiment no interactions were observed between O2 and TEM. High TEM depressed BW of Low G broilers, but no significant effect of treatments was observed on BW of High G broilers. Nevertheless, the high TEM or low O2 independently caused reduced femur and tibia weights and length, shank length and thickness, and both low O2 and high TEM together increased RA in shank weight. 5. These results suggest that late incubation conditions affect long bone development in broilers.

Length of the plateau and pipping stages of incubation affects the physiology and survival of turkeys.

1. The hypothesis tested was that accelerating the rate at which a turkey embryo passes through the Plateau and Pipping stages (incubation days 25 to 28) affects growth and embryonic mortality. 2. Eggs from 4 genetic lines were incubated similarly until 24 d of incubation. The eggs were divided at random, half incubated at 36.8 degrees C (CON) and the remaining half incubated at 37.3 degrees C (FAST). 3. The passage time for each developmental stage was recorded at 4 h intervals. Tissues were collected and measured for growth, and glycogen concentration at each developmental stage in response to the accelerated development. 4. The FAST treatment accelerated passage through the Plateau in some lines but not others, but time to pip was shortened in all genetic lines. Embryonic survival rate was affected differently by FAST. Slower growth in response to FAST enhanced survival. 5. In conclusion the rate at which genetically different embryos grow, mature and survive may involve choices among the 3 processes.

Effects of Incubator Temperature and Oxygen Concentration During the Plateau Stage of Oxygen Consumption on Turkey Embryo Long Bone Development

Temperature (TEM) and O(2) concentrations during the plateau stage of oxygen consumption are known to affect yolk utilization, tissue development, and thyroid metabolism in turkey embryos. Three experiments were conducted to evaluate these incubation effects on long bone development. Fertile eggs of Nicholas turkeys were used. In each trial, standard incubation conditions were used to 24 d, when the eggs containing viable embryos were randomly divided into 4 groups. Four experimental cabinets provided 4 TEM (36, 37, 38, or 39 degrees C) or 4 O(2) concentrations (17, 19, 21, or 23% O(2)). In the third experiment, 2 temperatures (36 and 39 degrees C) and 2 O(2) concentrations (17 and 23%) were evaluated in a 2 x 2 factorial design. Body and residual yolk weights were obtained. Both legs were dissected, and shanks, femur, and tibia weights, length, and thickness were recorded. Relative asymmetry of each leg section was calculated. Chondrocyte density was evaluated in slides stained with hematoxylin and eosin. Immunofluorescence was used to evaluate the presence of collagen type X and transforming growth factor beta. Hot TEM caused reduction of tibia weights and increase of shank weight when compared with cool TEM. The lengths of femur, tibia, and shanks were reduced by 39 degrees C. The relative asymmetry of leg weights were increased at 38 and 39 degrees C. Poult body and part weights were not affected by O(2) concentrations, but poults on 23% O(2) had bigger shanks and heavier tibias than the ones on 17% O(2). High TEM depressed the fluorescence of collagen type X and transforming growth factor beta. The O(2) concentrations did not consistently affect the immunofluorescence of these proteins. The chondrocyte density was affected by TEM and O(2) in resting and hypertrophic zones. In the third experiment, high TEM depressed BW, leg muscle weights, and shank length. Low O(2) reduced tibia and shanks as a proportion of the whole body. We concluded that incubation conditions affect long bone development in turkeys.

Development during the first seven days post-hatching.

The neonate has basic needs that must be satisfied at the time of hatching if survivability and maximum potential are achieved. Some of these basic needs are fresh air, clean water, proper feed, and heat. The developmental state at hatching of the neonate differs among all avian species (Nice, 1962). The post-hatching period for altricial neonates is more critical than for precocial birds because they hatch in a less mature state. Differing amounts of maternal care are therefore essential for each species (Nice, 1962). Although embryonic growth among species is very similar, no two physiological systems seem to mature at the same rate (Ricklefs and Starck, 1998). In addition, maturity may be a function of the egg conductance constant (Ar and Rahn, 1978), which is determined by egg mass, eggshell conductance (or functional properties) and the length of the incubation period (see Box 1 later), all of which may constrain neonatal maturity. When viewed energetically, the difference between maturity types resides in the different water concentrations in eggs and hatchlings, in the density of chemical potential energy in the dry matter of true hatchlings, and in the different amounts of energy transferred from the egg to the spare yolk (Ar et al., 1987). On the basis of species comparisons of posthatching growth of all birds (Lilja, 1983; Ricklefs, 1987), it has been suggested that the rate of growth after hatching is at least partially determined by the pattern of organ growth. It appears that a high rate of growth is correlated to early growth of ‘‘supply organs’’ (oesophagus, proventriculus, gizzard, intestines, heart and liver) at the expense of ‘‘demand organs’’ (breast, wings, legs and feathers). These changes begin very early in development (Lilja and Olsson, 1987), and Schmalhausen (1930) hypothesised that growth and organ function come into conflict when growth occurs too slowly or too rapidly. Even under the most optimum conditions, a newly hatched bird is not free from stress. This is impossible because the absence of stress is death (Selye, 1951). However, hatchlings possess abilities to cope with stress via adrenal cortical hormones (Davis and Siopes, 1989). Hatchlings have different blueprints for growth and maturation that must occur within a predetermined time frame. Many times this blueprint does not include adjustments that need to occur in maturational and growth processes in an imperfect environment. The objective of this paper is to define a ‘‘physiologically normal’’ hatchling and to describe the principles involved in the maturation and growth of several organ systems during the initial stages of life. At least six physiological systems exist that require maturation during the last week of incubation or, in the case of altricial species, the initial days of life outside the shell. These systems are: (1) the circulatory system (heart and blood); (2) the kidney and body fluids system; (3) the digestive system; (4) body temperature regulation; (5) the respiratory system; and (6) the immune system. These systems are discussed here using published data to help clarify important points.

The mRNA for zona pellucida proteins B1, C and D in two genetic lines of turkey hens that differ in fertility

The avian inner perivitelline layer (IPVL) contains zona pellucida protein-B1 (ZPB1), zona pellucida protein-C (ZPC) and zona pellucida protein-D (ZPD). These three proteins may be involved in sperm binding to the IPVL. ZPB1 is produced by the liver and transported to the developing preovulatory follicle, while ZPC and ZPD are synthesized and secreted by the granulosa cells of the preovulatory follicle. The mRNA of ZPB1, ZPC, and ZPD was investigated in two lines of turkey hens selected for over 40 generations for either increased egg production (E line) or increased body weight (F line). Total RNA was extracted from the liver and from 1cm(2) sections of the granulosa layer around the germinal disc and a nongerminal disc area of the F(1) and F(2) follicles of hens from each genetic line. Northern analysis was performed using chicken cDNA probes for all three ZP proteins. Hepatic mRNA for ZPB1 was greater (P<0.05) in turkey hens from the E line than the F line. Although, there was no difference in ZPC mRNA between the germinal disc and nongerminal disc region of the two largest follicles in E line hens, ZPC mRNA was greater in the nongerminal disc region compared to the germinal disc region in the two largest follicles obtained from the F line hens. There were no differences in ZPD mRNA between the germinal disc and nongerminal disc regions of the F(1) and F(2) follicles for either genetic line. The results suggest that the greater rates of fertility previously observed in eggs from the E line hens compared with the F line of hens may be related to differential amounts of the potential sperm binding proteins ZPB1 and ZPC.

Genetic Control of Embryonic Cardiac Growth and Functional Maturation in Turkeys

Turkey experimental lines E (selected 44 yr for increased total egg production) and F (selected 38 yr for increased 16-wk BW) were mated reciprocally with the randombred control lines from which they were derived (RBC1 and RBC2, respectively), and the pureline and reciprocal cross poults were compared for their BW, heart weight, heart rates, myocardial glycogen and lactate concentrations, and plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activities. The CK and LDH were used as indicators of cardiac insufficiency. Orthogonal contrasts of the data from the pureline and reciprocal cross data were used to estimate additive genetic effects, reciprocal effects (confounded maternal and sex-linked effects), and heterosis for each of the traits measured. Long-term selection for increased egg production in the E line has reduced embryo heart weight and has altered the energy metabolism of the myocardium. The differences in energy metabolism may be due to the more rapid heart rates. Conversely, long-term selection for increased 16-wk BW has significantly decreased the heart rate of F line embryos and has not changed the weight of the heart relative to the BW until the embryo has passed through the plateau stage. The F line embryos show a different energy metabolism that relies much more on gluconeogenesis. Embryo deaths occur more frequently in turkey embryos when the energy metabolism of the myocardium shows elevated glycogen to lactate ratios as it did in the pure E and F lines.

Effects of long term storage on the egg, embryo and chick.

When most people think of a chicken egg, they think of something nutritious to eat. From an avian embryo’s point of view the egg provides the same function. At the time the egg is laid, if the oöcyte has been successfully fertilised, almost all of the nutrients required for the embryo to grow and develop are enclosed within the egg. There are two main elements missing; one is oxygen and the other is heat. The importance of both of these factors in embryonic growth and development are addressed elsewhere in this book (see French, 2002; Ar and Deeming, 2002). In most commercial poultry breeding operations, hatching eggs are produced by flocks of breeders housed in barns. Male and female broiler breeders are housed together so that natural mating can occur, while turkey breeder hens are housed separately from the toms and therefore must be artificially inseminated. Most broiler breeder eggs are laid in nests and collected by mechanical belts while turkey breeder eggs are still hand-collected. Irrespective of the method of insemination or egg collection, once the eggs are collected they are stored in on-farm cool stores, usually for a few days, until they can be transported to the hatchery. This is done as daily egg transport from the breeder farms to the hatchery would be inefficient. Once transported to the hatchery, the eggs are again stored in large egg stores. Egg storage at the hatchery usually occurs for two reasons. First, hatching eggs are stored until enough eggs are available to fill large incubator racks. Second, stock-piling of eggs occurs in anticipation of fluctuations in egg production or demand for broiler chicks during the production year. Storage of fertile eggs both on farm and at the hatchery occurs at temperatures below 21 C. This can prevent the growth of bacteria, but the main purpose is to stop development of the very young embryo. After oviposition, temperature is the primary catalyst which influences embryonic development (see French, 2002). As assessed by microscopic embryonic staging methods (Eyal-Giladi and Kochav, 1976), storage of fertile Single Comb White Leghorn eggs for various lengths of time at 14 C stopped all observable embryonic development (Table 1; Fasenko et al., 1992). Using a similar microscopic staging technique developed for turkey embryos (Gupta and Bakst, 1993), it was shown that turkey embryonic development during storage for 3, 7 or 14 days at 15 C continued from Stage 7 (the stage most common at the time of lay) to a Stage of 8, and then ceased (Bakst and Gupta, 1997). The minimum temperature above which embryonic development occurs (physiological zero) has been reported at two different levels. Edwards (1902) reported the minimum temperature for embryonic development to be 21 C, while Funk and Biellier (1944) found this minimum temperature to be 28 C. Previous researchers have hypothesised that the minimum temperature for embryonic development is not the same for all developing tissues of the early growing embryo (Kaufman, 1948). Therefore the objective of storing eggs at temperatures well below physiological zero is to prevent abnormal growth of the embryo which could occur if eggs were held at temperatures between physiological zero and normal incubation temperatures of 37.5 C. The generally accepted optimal range for hatching egg storage in the industry is between 14 and 18 C. Different storage temperatures and conditions are recommended depending on the length of storage (See reviews by Proudfoot and Hamilton, 1990; and Meiijerhof, 1992). AVIAN BIOLOGY RESEARCH 2 (1/2), 2009 73–79

Zona pellucida protein B2 messenger ribonucleic acid expression varies with follicular development and granulosa cell location1

ABSTRACT The freshly ovulated ovum in avian species is surrounded by a protein layer called the inner perivitelline layer (IPVL). The IPVL contains zona pellucida proteins and 6 distinct zona pellucida genes have been identified (ZPA, ZPB1, ZPB2, ZPC, ZPD and ZPX1) in the chicken. In the present research, the expression of the mRNA for ZPA, ZPB2, and ZPX1 was investigated in 2 lines of turkey hens selected for either increased egg production (E line) or increased body weight (F line). Theca and granulosa cell expression of the mRNA for ZPA and ZPB2 was also investigated in hierarchical and prehierarchical follicles from broiler breeder hens. Granulosa tissue was collected from F1 through F4 and F1 through F10 follicles in E line and F line hens, respectively. A one cm2 section of the granulosa layer around the germinal disc (GD) and an equivalent sized nongerminal disc (NGD) area was also collected from the F1 and F2 follicles from other hens from each genetic line. Granulosa and theca tissue was collected from hierarchical and prehierarchical follicles of broiler breeder hens. Total RNA was extracted from the samples. Minor groove‐binding probes and primers for detecting ZPA, ZPB2, and ZPX1, were made for real‐time PCR analyses. Expression of ZPA, ZPB2, and ZPX1 was detected in all follicle sizes from both genetic lines of hens. No significant differences in ZPA and ZPX1 mRNA expression were detected between the GD and NGD granulosa cells. However, the expression of the mRNA for ZPB2 was significantly greater in the GD granulosa cells when compared to the NGD granulosa cells in F1 and F2 follicles from E line and F line hens. In broiler breeder hens, the mRNA expression of ZPA and ZPB2 was greatest in the smallest prehierarchical follicles. The results suggest that higher expression of ZPB2 in the germinal disc area may be important for the preferential binding of sperm to this region of the IPVL.