The normal stages of development for the California valley quail

One challenge in avian embryology is establishing a standard developmental timetable, primarily because eggs incubated for identical durations can vary in developmental progress, even within the same species. For remedy, avian development is classified into distinct stages based on the formation of key morphological structures. Developmental stages exist for a few galliform species, but the literature is lacking a description of normal stages for California valley quail (Callipepla californica). Thus, the objective of this study was to stage and document the morphological and structural development of California valley quail. Over two laying seasons, 390 eggs were incubated at 37.8֯ C in 60% RH for ≤23 days. Eggs were opened every ≤6 hours to document embryonic development, including, blastoderm diameter, anterior angle of nostril to beak tip, and lengths of wing, tarsus, third toe, total beak, total foot, and embryo. California valley quail embryos were staged and compared to domestic chicken (Gallus gallus domesticus), the staging standard for galliformes, as well as Japanese quail (Coturnix japonica), blue-breasted quail (Synoicus chinensis) and northern bobwhite quail (Colinus virginianus). This study produced the first description of the 43 normal stages of development for California valley quail. Compared with other galliformes, the California valley quail has a different number of stages and displays developmental heterochrony in stages 1–24, and morphological and developmental differences in stages 25–hatch. The observed differences emphasize the importance of staging individual avian species instead of relying on poultry animal models or close relatives for developmental reference. This is extremely important in species-specific embryological studies that evaluate critical windows of development or evaluate the impacts of environmental change on avian development. This study also suggests that staging frequencies of ≤6 hours and egg transport protocols should be standardized for future staging studies.


Introduction
Avian developmental embryology has been a topic of study for >5,000 years, when ancient Egyptians began to artificially incubate bird eggs and evaluate their daily progress by observing embryonic development [1]. Aristotle firmly cemented the discipline by systematically describing daily changes in the morphology and physiology of developing avian embryos [1]. Subsequent studies assessed the morphology of avian embryos to better understand embryonic development in varying environments, to compare development between species [1,2], and to contribute to the poultry and medical industry [3,4]. Daily observations of avian embryological development are fascinating but have severe limitations; mainly that eggs incubated for identical durations can vary in their degree of development, even within the same species [5,6]. Thus, when making comparisons, inter-or intra-specific, incubation time is an inaccurate measure of developmental progress. One remedy is to classify avian development into distinct, definable stages based on development, or visibility, of key morphological structures [6]. Domestic chickens (Gallus gallus domesticus) have historically served as the primary animal model for avian embryology. Developmental stages described for other galliform species were based on the stages of chicken embryos defined by Hamburger and Hamilton [7]. For example, early stages 1-6 were characterized by formation of the primitive streak and notochord [8]. Stages 7-14 were identified by somite formation and limb development [9][10][11].  were associated with external structures, including visceral arches, digit formation, and pigment development [6,12,13]. Final stages 40-46 were characterized by growth of the beak and third toe [14,15]. However, stages vary among galliforms both in rate and timing [6,7,[16][17][18]. For example, Japanese quail (Coturnix japonica) embryos have 46 stages of development across 16.5 days of incubation [6], blue-breasted quail (Synoicus chinensis) embryos have 39 stages across 17 days [18], and northern bobwhite quail (Colinus virginianus) have 41 stages of development across 23 days of incubation (Fig 1) [17]. Interestingly, rates of development for bobwhite quail and chickens have no observed differences in development through stage 6 (*25 hours). However, bobwhites lag chicken embryos by 12-16 hours at stage 14, and *24 hours by stage 24 [17]. Similarly, the gastrulation process and somite formation occur in the same order but, in the latter parts of incubation, normal stages of the chicken embryo are not applicable to other quail species.
This differential development demonstrates the need for a description of normal stages of development for each species in avian embryological studies. To date, no description of developmental stages exists for California valley quail (Callipepla californica). Thus, the objective of this study was to stage and document the morphological and structural development of California valley quail.

Results
The first description of normal stages of development for the California valley quail was documented. Daily development of the California quail embryo is shown through images of opened eggs.

Stages of development
The California valley quail has 43 normal stages of development, from pre-incubation through hatch (Table 1). Stages 1-6 were identified by primitive streak formation and gastrulation. Stages 7-14 were charted by the number of somite pairs, where every third somite pair defined a stage. Beyond stage 14, somites became increasingly more difficult to identify due to mesoderm growth on the anterior of the embryo. Other morphological structures including visceral arches, limb-buds, and external structures were identified and used to stage embryos. As development progressed, total body length, when extended, initially increased then gradually decreased at~72 h of incubation. This was caused by cranial and cervical rotation toward the embryo's trunk and tail region. Once rotation reached its maximum, total body length, when extended, increased for the remainder of incubation. Final stages of incubation were charted on the length of beak and third toe.  Stage 10 (48-54 h): 10 somite pairs. Ten somite pairs visible. The first somite pair is beginning to disappear and will not be counted in subsequent stages [7,18]. Blood islands have increased. The first signs of heart and cranial rotation. Three primary brain vesicles and optic vesicles present. Embryo length: 4.90 ±0.17 mm (N = 8; Fig 3D).
Stage 13 (54 h): 19 somite pairs. Nineteen somite pairs visible. Telencephalon is enlarged and has turned farther to the right. Heart muscle is more prominent than the previous stage ( Fig 4C).
Stage 15 (66 h): 24 somite pairs. Twenty-four somite pairs present. Optic cup and double contour has formed. Vitelline veins are much more prominent than previous stages. Third branchial arch is present. The forebrain and hindbrain create an acute angle. Rotation is more accentuated than previous stages. Amnion extends to the somite pairs 7-10. Embryo length: 7.22 ±0.47 mm (N = 2; Fig 5A).

Stage 18 (78-84 h).
First appearance of wing-bud. Darkening ridge of cells demarcate the beginning of wing formation. Tail bud has rotated to the right. Amnion enclosed except for small oval on the trunk. Embryo length: 6.85 ±0.91 mm (N = 6; Fig 5D).
Stage 29 (8-9 d). Elbow joint more pronounced; knee bent at knee-joint. Second digit and third toe have lengthened. Digital plate is more angular in appearance than previous stage. Neck between collar and mandible has lengthened. Beak is distinct and much more prominent than previous stage. No egg tooth present. Beak length: 1.56 ± 0.05 mm (N = 4; Fig 8C).

Discussion
The California valley quail embryo has 43 normal stages of development, which differs from the number of developmental stages from avian models and close galliform relatives. Domestic chickens and Japanese quail embryos have 46 stages of development [6,7], blue-breasted quail have 39 stages [18], and northern bobwhite quail have 41 stages [17]. The chronology of development for the California valley quail differs from other quail in stages 1-24 (1-5 d; Fig 12A), and the morphological and structural development differs from the domestic chicken in stages 25-hatch (6-23 d; Fig 12B). When incubation time is standardized, California valley quail development is similar to other staged galliforms (Fig 12C). When development is standardized, California quail lag behind other staged galliforms (Fig 12D). The observed differences in the number and timing of stages of development emphasize the importance of staging avian species of interest instead of relying on animal models and close relatives for reference. Caution should be exercised when extrapolating data from poultry species like chickens and Japanese quail to other birds due to the multiple generations of selection and domestication >10,000 years ago [20]. This is extremely important in species-specific embryological studies that access critical windows of development [21] and evaluate the impacts of environmental change on avian development [22][23][24][25].
The observed differences in the number of stages of development also emphasize the need for establishing standard observation frequencies when staging avian embryos. We documented embryos in increments of �6 hours, similar to studies that staged the domestic chicken [7], Japanese quail [6], and blue-breasted quail [18]. However, northern bobwhite quail were only observed every 24 hours when staged [17], resulting in the possibility of missed stages. Future embryological studies, including a more thorough investigation of northern bobwhite development, would benefit from examining embryos in increments of �6 hours to document all morphological stages more accurately.
Although we compared developmental timing and the number of stages between galliforms, avian development is influenced by temperature and humidity during incubation, which vary in embryological staging studies. Incubation temperatures for domestic chickens where 39.4˚C, 37.5˚C, and 38˚C, with no humidity reported [7], Japanese quail were incubated at 38˚C with 70% RH [6], and blue-breasted quail at 37.7˚C with 70% RH [18]. Northern bobwhites were incubated at 37.5˚C with 60% RH [24], and a 31.7˚C wet bulb, or~66% RH [17]. We incubated California valley quail at 37.5˚C with 60% RH; the same as northern bobwhites [24]. It is unknown how these slight differences influence the comparison of stages since incubation temperature and humidity are species dependent.
Normal stages of development are required to determine the impacts of stressors and teratogens on embryo development [21]. Factors such as heat stress from drought [21,22] and hazardous pesticides have been shown to drastically affect the development of other quail species [23][24][25], and have major consequences for population sustainability [26,27]. California valley quail are considered highly vulnerable to climate change due to increased fire risk, and spring heat waves disrupting reproduction [28]. The stages produced in this study could help determine the impact of high temperatures on developing California valley quail embryos.
One often overlooked factor in staging studies is the condition of the eggs from hatchery to laboratory. During this study, several eggs failed to develop and hatch. Although this is normal during studies with eggs, varying shipping conditions could have influenced hatchability [7,31]. Shipping, and receiving temperatures can exceed physiological zero [29], and reach threatening or lethal levels [22][23][24][25][26]. This may result in altered development [21,23,24], affecting the recorded embryological stages. Our eggs were packaged in foam within cardboard boxes, shipped during the spring within 24 hours of collection, and quickly processed on the receiving end. Since spring temperatures were mild and the packaging offered some thermal buffering, we don't suspect that shipping conditions greatly influenced our results. However, future research directed at documenting normal stages of development would benefit from controlling and recording hatchery, shipping, and receiving conditions. We hope that this series of normal staging and recommendations will be useful as a laboratory aid for future embryological investigations.

Materials and methods
All research was approved by the Texas A&M University-Commerce's Institutional Animal Care and Use Committee; Animal Use Protocol P21-026. Quail Research Laboratory at Texas A&M University-Commerce. In 2020, 3 shipments (N = 290 eggs) were delivered over 12 weeks, April-June. In 2021, 1 shipment (N = 100 eggs) was delivered in May. Staggered shipments in 2020 provided sufficient time to incubate eggs in each shipment. Upon arrival, eggs were inspected for fine-line cracks and broken shells by candling. Eggs with cracks or broken shells were discarded. After inspection, eggs were weighed to the nearest 0.01 g using a portable balance (Scout SPX222, Ohaus, Parsippany, New Jersey), labeled with a permanent marker, and incubated at 37.5˚C with~60% RH in a cabinet incubator (1502 Digital Sportsman, GQF, Savannah, GA, USA). Eggs were automatically turned every hour to mimic natural incubation [18] and opened daily, during specific time periods, to examine embryos and record key morphological characteristics and structures.

Staging embryonic development
To stage California valley quail embryo during incubation, key morphological characteristics and structures were documented for 50 time segments (Fig 13). For each time segment, 5 eggs were randomly selected, placed on an 11-mm rubber stage under a dissecting microscope (M80/IC90E, Leica Camera, AG, Wetzlar, Germany), and opened by carefully removing 2-4 mm of the egg's blunt end with a 25-gauge needle and fine-pointed forceps. Once opened, blue methylene (0.1-0.25 ml) was injected directly under the embryo using a 25-gauge syringe to enhance visibility and increase contrast.
Measurements recorded from opened eggs included blastoderm diameter, anterior angle of nostril to beak tip, and lengths of wing, tarsus, third toe, total foot, total beak, and embryo, when extended [7,14,30]. Limb-bud length was measured proximal to distal and width was measured anterior to posterior [7]. The first pair of somites of the series was not clearly defined; therefore, not counted [7,18]. Photographs were taken of all opened eggs and developing embryos. Visibility of photographed embryo was improved by adjusting contrast, exposure, highlight, shadows, whites, blacks, and clarity using image editing software (Photoshop measured, and compared to developmental studies of other galliforms [6,7,17,18]. This study is different from other descriptions of normal embryonic staging because embryos were photographed in ovo instead of analyzing preserved or fixed embryos. Once the first year of research was concluded, all data were analyzed, and stages were determined according to structural developments and chronological findings. Any missing or poorly defined stages were prioritized for year 2. Embryos incubated �10 days were euthanized in a closed chamber with 90% CO 2 for �20 minutes [31][32][33]. Embryos incubated >20 days were handled in accordance with Texas A&M University-Commerce IACUC protocol P21-026. All statistical analyses were performed using Microsoft Excel (Version 2011, Redmond, WA). All measurements are reported as mean ±SD. Statistical decisions were made at an alpha <0.05.