Transcriptome analysis of blastoderms exposed to prolonged egg storage and short periods of incubation during egg storage.

BACKGROUND: Cool temperature egg storage prior to incubation is a common practice in the broiler industry; however, prolonged egg storage causes increased embryonic mortality and decreased hatchability and growth in surviving chicks. Exposing eggs to short periods of incubation during egg storage (SPIDES) reduces the adverse consequences of prolonged storage. SPIDES increases blastodermal cell viability by reducing apoptosis, though the counteracting mechanisms are unclear. To define the impact of prolonged storage and SPIDES, transcriptome analysis compared gene expression from blastoderms isolated from eggs exposed to the following treatments: control (CR, stored at 17 °C for 4 days), prolonged storage (NSR, stored at 17 °C for 21 days), SPIDES (SR, stored at 17 °C for 21 days with SPIDES), and incubated control (C2, stored at 17 °C for 4 days followed by incubation to HH (Hamburger-Hamilton) stage 2, used as the ideal standard development) (n = 3/group). Data analysis was performed using the CLC Genomics Workbench platform. Functional annotation was performed using DAVID and QIAGEN Ingenuity Pathway Analysis. RESULTS: In total, 4726 DEGs (differentially expressed genes) were identified across all experimental group comparisons (q < 0.05, FPKM> 20, |fold change| > 1.5). DEGs common across experimental comparisons were involved in cellular homeostasis and cytoskeletal protein binding. The NSR group exhibited activation of ubiquitination, apoptotic, and cell senescence processes. The SR group showed activation of cell viability, division, and metabolic processes. Through comparison analysis, cellular respiration, tRNA charging, cell cycle control, and HMBG1 signaling pathways were significantly impacted by treatment and potential regulatory roles for ribosomal protein L23a (RPL23A) and MYC proto-oncogene, BHLH transcription factor (MYC) were identified. CONCLUSIONS: Prolonged egg storage (NSR) resulted in enriched cell stress and death pathways; while SPIDES (SR) resulted in enriched basic cell and anti-apoptotic pathways. New insights into DNA repair mechanisms, RNA processing, shifts in metabolism, and chromatin dynamics in relation to egg storage treatment were obtained through this study. Although egg storage protocols have been examined through targeted gene expression approaches, this study provided a global view of the extensive molecular networks affected by prolonged storage and SPIDES and helped to identify potential upstream regulators for future experiments to optimize egg storage parameters.

Observations of turkey eggs stored up to 27 days and incubated for 8 days: embryo developmental stage and weight differences and the differentiation of fertilized from unfertilized germinal discs.

For logistical reasons, egg storage prior to incubation is a growing practice in the commercial turkey industry. Yet the consequence of increasing egg storage over 7 d is a progressive increase in embryo mortality. The objective of this study was to provide the information necessary to differentiate an early dead embryo from an unfertilized egg after 8 days of incubation (DOI). Five groups of eggs each from inseminated and virgin hens were stored for progressively increasing periods of time (5-d or less, 6 to 10 d, 11 to 15 d, 16 to 20 d, and 21 to 27 d) and incubated. At 8 DOI, eggs were examined and the stage of development (Hamburger and Hamilton, 1951) and embryo weights in normally developed eggs were determined. There was a significant negative correlation between the stage of development and embryo weight with increasing storage periods. All remaining eggs from the inseminated and virgin hens were broken-out and the appearance of the yolk and the fertilized and unfertilized germinal discs examined. The yolks of both hen groups with unfertilized ova maintained a homogeneous uniform yellow-orange color. In contrast, yolks of ova that had been fertilized, with or without early-dead embryos, and yolks from virgin hens that showed evidence of parthenogenetic development (3%) had a heterogeneous appearance. Using fluorescence microscopy, the heterogeneous appearance was due to sheets of aberrant cells and less frequently dispersed cells and folds of the perivitelline layer. It was concluded that clear egg breakouts need to be performed to more accurately assess the impact of egg storage on embryonic mortality. Furthermore, such breakouts should be performed with a high intensity light directed across the surface of the germinal disc to clearly differentiate the subtle differences between an early-dead embryo and an unfertilized germinal disc.

Impact of broiler egg storage on the relative expression of selected blastoderm genes associated with apoptosis, oxidative stress, and fatty acid metabolism.

Cool temperature storage of eggs prior to incubation is a frequent practice by commercial broiler hatcheries. However, continued storage beyond 7 d leads to a progressive increase in the rate of early embryonic mortality. In this study, we examined the relative expression of 31 genes associated with fatty acid metabolism (8), apoptosis (7), and oxidative stress (16) pathways to better understand the basis of embryo mortality during egg storage. A total of 642 broiler eggs in 2 separate trials were subjected to the following egg treatments: stored 4 d (Control 1, C1); stored 21 d but subjected to short periods of incubation during egg storage (SPIDES); stored un-manipulated 21 d (NonSPIDES, NS); and stored 4 d then incubated for 10 h to advance the embryos to the same developmental stages as the SPIDES embryos (Control 2, C2). Hatchability trials (277 eggs) confirmed the efficacy of SPIDES compared to NS treatments in both trials. To determine relative expression of 31 selected genes, 365 blastoderms were isolated, staged, and flash frozen in batches of 5 to 10 blastoderms per vial (7 vials per egg treatment) prior to RNA extractions. Analysis of gene expression was performed using qRT-PCR and the results presented as relative expression normalized to C1. The relative expression of genes in which the SPIDES and C2 treatments were significantly up- or down-regulated in tandem indicated that the stage-specific expression of those genes was maintained by the SPIDES treatment. This study provides the relative gene expressions of blastodermal cells before and after prolonged egg storage as well as insight as to how SPIDES impacts blastodermal cell gene expression.

Lectin staining of the uterovaginal junction and sperm-storage tubule epithelia in broiler hens.

Mammalian sperm bind to terminal carbohydrates associated with glycoconjugates on the apical surface of oviduct epithelial cells in the caudal region of the oviduct and undergo cellular and molecular modifications associated with capacitation prior to ovulation. In contrast, chicken sperm are stored for up to 23 d in sperm-storage tubules (SST) localized in the uterovaginal junction (UVJ). Little is known of the cellular and molecular mechanisms that regulate sperm storage in and release from the SST. The purpose of this study was to identify glycoconjugates associated with the SST epithelial cell surface using lectins. Virgin hens and hens of higher and lower fertility in egg production for 6 to 16 wk were used in this study. Sections of UVJ mucosa containing SST were stained with fluorescent conjugated lectins and examined by confocal microscopy. Carbohydrate moieties associated with the UVJ and SST epithelia differed in their lectin binding patterns. No differences in the lectin binding patterns within the 2 epithelia were discernible between the virgin and younger and older hens. Minor differences were observed between the higher and lower fertility hens. Only lectins specific for galactose and N-acetylgalactosamine moieties were localized to the luminal surface of the SST. While resident sperm may be closely apposed to the SST epithelial cell apical microvilli, it is unlikely that sperm binding to the microvilli via terminal carbohydrates associated with glycoconjugates is a requisite for prolonged storage. However, the possibility of SST epithelial cell communication with resident sperm via shedding microvillous vesicles characterized by surface glycoconjugates with terminal galactose and N-acetylgalactosamine moieties is currently being investigated.

Short periods of incubation during egg storage increase hatchability and chick quality in long-stored broiler eggs.

It is recognized that cool egg storage for 8 d or longer, commonly employed in broiler parent and commercial layer production, reduces hatchability. In this study, we investigated the efficacy of short periods of incubation during egg storage (SPIDES) in the restoration of hatchability of broiler hatching eggs stored for 21 d. Prolonged cool storage reduced hatchability of untreated eggs from 92 to 71%. The SPIDES treatment, which consisted of four 4-h preincubations at 4- to 5-d intervals during storage, reduced the incubation time and restored hatchability to 84% by lowering both early and late embryo mortality (P = 0.0002). The SPIDES-treated embryos exhibited higher proportions of viable cells after each preincubation (P = 0.02), potentially alleviating the negative effects of storage-induced cell death on embryo development. After completion of 4 preincubations, SPIDES embryos were advanced to intermediate primitive streak formation, a developmental stage previously associated with embryo mortality during storage. In contrast to reported preincubation methods imposed on-farm immediately before the eggs are first cooled, the SPIDES technique permits 4 d of cool storage before the initial preincubation treatment, introducing flexibility in the incubation protocol and enabling cool storage up to 3 wk with much improved hatch rates than would usually be expected. Although SPIDES chicks exhibited a BW equivalent to that of embryos derived from unstored eggs at hatch, the initial relative growth was increased as a result of SPIDES, generating a higher BW over the first 4 wk posthatch (P < 0.05). Single preincubations of 6 and 12 h at 4 d of storage caused similar advances in embryo stage to the SPIDES treatment, but the hatchability was worse than in the untreated controls, suggesting small multiple preincubations during storage have a greater benefit than a single incubation performed on d 4 of storage. Future research regarding the cellular and molecular basis of physiological stress reduction in SPIDES embryos will yield new insights into the alleviation of early embryo mortality associated with egg storage.

Comparison of blastoderm traits from 2 lines of broilers before and after egg storage and incubation.

We examined the impact of egg storage on the hatchability of eggs from 2 lines (A and B) of commercial broiler breeders with known differences in fertility (line B slightly higher) and hatchability following egg storage (line A slightly lower). Eggs from both lines were stored in a 16°C room for 3 to 4, 10 to 12, and 17 d, the blastoderms were isolated, evaluated, and statistically compared. No significant interactions (line × duration of storage) were observed in the following traits: blastoderm diameter, total and percentage of viable blastodermal cells, stage of blastoderm development, and embryo weight and stage of development after 7 d of incubation. However, significant differences were observed when comparing line averages across storage treatments (diameter and total number of blastodermal cells) and storage treatment averages across lines (percentage of viable cells, and embryo weight and stages following incubation). To ascertain the basis for the observed differences in fertility, perivitelline sperm-hole numbers were determined in eggs within 72 h of lay (3 to 4 d storage group). Eggs from line B had a significantly higher number of eggs having greater numbers of sperm holes than eggs from line A. It is concluded that no single blastoderm trait or combination of traits can be definitively associated with the inter-line differences in hatchability following storage of eggs. Alternatively, differences in PVL sperm-hole counts between lines may be associated with the reported differences in fertility between the 2 lines.

Oviductal expression of avidin, avidin-related protein-2, and progesterone receptor in turkey hens in relation to sperm storage: effects of oviduct tissue type, sperm presence, and turkey line.

The sperm storage tubules (SST) of the turkey hen, which are located in the uterovaginal junction (UVJ) of the oviduct, maintain viable sperm for up to 10 wk after a single insemination. The mechanisms of this in vivo sperm storage are poorly understood. Our objective was to evaluate mRNA and protein expression of avidin and 2 avidin-associated factors, avidin-related protein-2 (AVR2) and progesterone receptor, in the oviducts of 2 different lines to determine the extent to which they were sperm responsive and tissue specific. At 38 wk of age, Hybrid Grade Maker and Converter turkey hens were artificially inseminated with diluted semen (AI) or were sham-inseminated with extender alone (SI). Forty-eight hours after insemination, total RNA was extracted from the UVJ epithelium (containing SST) and vaginal epithelium (VGE) of SI and AI hens. Real time-polymerase chain reaction data showed a clear tissue region-specific effect on gene expression in the turkey hen oviduct, with much greater (P < 0.0001) expression in the UVJ compared with VGE region for avidin and AVR2 mRNA in both lines and for progesterone receptor mRNA in the Converter line. In contrast to real-time PCR data, in situ hybridization of SI and AI tissues showed that the presence of sperm increased avidin mRNA in the SST and UVJ surface epithelium in the Converter hens. Immunohistochemistry confirmed the presence of avidin protein in the epithelium of the UVJ in both lines; however, whereas avidin protein was localized in the SST of SI-Grade Maker hens, this protein was not detected in the SST of Converter hens. The upregulation of avidin and AVR2 mRNA within the sperm storage region indicates the involvement of avidin, and perhaps avidin analogs, in the sustained storage of sperm in the SST, possibly through the binding of biotin to avidin. The absence of avidin protein in the SST and VGE of Converter hens in the presence of increased mRNA may indicate a rapid turnover of protein.

Physiology and endocrinology symposium: role of the oviduct in maintaining sustained fertility in hens.

In poultry, sperm transferred by natural mating or AI into the distal end of the vagina immediately begin their ascent to the uterovaginal junction (UVJ) at the anterior end of the vagina. However, due to an intense selection process in the vagina, less than 1% of the sperm transferred actually reach the UVJ. Those sperm that do reach the UVJ enter numerous tubular invaginations of the surface epithelium of the vagina located in the UVJ mucosa, collectively referred to as the sperm-storage tubules (SST). Sperm residing in the SST lumen are capable of surviving up to several weeks while retaining their fertilizing capacity. Resident sperm are released gradually from the SST while the hen is in egg production, ascend to the site of fertilization, and interact with the next ovulated ovum. In this manner, given the absence of an estrus to synchronize ovulation with copulation, poultry are ensured a population of sperm at the site of fertilization around ovulation. Over the past decade, several new and diverse observations have been published addressing the microanatomy of the UVJ and SST, and the cellular and molecular mechanisms orchestrating oviductal sperm selection and storage. These include the role of sperm mobility in selection and transport, SST numbers in different poultry species and lines of high and low fertility, roles of the immune system and possibly neuroendocrine-like cells in the vagina in sperm selection and storage, and the roles of aquaporins and a fluid exchange mechanisms contributing to sperm release from the SST. The objective of this paper is to review and integrate these observations into a comprehensive understanding of the cellular and molecular events influencing the fate of sperm in the oviduct of the hen, particularly with regard to oviductal sperm selection and storage.

Comparisons of sperm storage tubule distribution and number in 4 strains of mature broiler breeders and in turkey hens before and after the onset of photostimulation.

The biological basis of sustained fertility in broiler and turkey hens is their capacity to store sperm in the oviductal sperm storage tubules (SST) located in the uterovaginal junction. The objectives of this study were to determine if the numbers of SST varied between 4 strains of broiler breeders and determine the number of SST in the turkey before (less than 9 d of photostimulation) and after (up to 22 d of photostimulation and laying) photostimulation. No statistical differences were observed in SST numbers in the 4 strains of broilers examined or in turkey hens before and after the onset of egg production. The mean numbers of SST for broilers and turkeys were 4,893 and 30,566, respectively. We conclude that any differences between the fertility of the 4 broiler breeder strains examined cannot be explained by differences in SST numbers. However, differences in the duration of fertility between broilers and turkeys are, in part, related to their respective numbers of number of SST. Furthermore, we conclude that turkey SST are morphologically differentiated and functional before the onset of photostimulation and while the oviduct is morphologically undeveloped.

Morphology of the turkey vagina with and without an egg mass in the uterus.

In this study we examined the gross anatomy of the uterus and vagina in turkeys in egg production. With no uterine egg mass, removal of the tunica serosa that enclosed the uterus revealed deep periodic in-folding of the muscularis transversely circumscribing the sac-like segment. When the connective tissue embracing the neutral buffered formalin fixed vagina was completely teased free, the exposed tubular segment was shaped as a counter-clockwise spiral or as a series of angular, random bends. The uterovaginal junction was flush with the uterine mucosa or projected slightly into the uterine lumen. With a uterine egg mass, the deep in-foldings of the uterus were abolished. The only alteration to the morphology of the vagina was that the uterovaginal junction appeared dilated and pressed into its juncture with the uterus. Whether an egg mass was present or not, uterovaginal junction folds that projected into the uterus possessed sperm storage tubules. An egg mass in the uterus compressed the uterovaginal junction folds, and its mucosa became contiguous with the uterine mucosa. Finally, from an evolutionary perspective, in the turkey and possibly other species possessing a nonintromittent phallus, vaginal pleomorphism may have been driven primarily by the need to accommodate the overall length of the vagina in a limited abdominal space and to a lesser extent on sexual selection.

Turkey sperm reside in the tubular glands in the urodeum following artificial insemination.

A turkey hen in egg production requires 48 h after the last insemination to maximize the number of sperm in the uterovaginal junction sperm-storage tubules. Where the sperm that continue to fill the oviductal sperm-storage sites during this 48-h period reside remains unknown. Histological sections of the juncture of the vagina with the urodeum, the central compartment of the cloaca, revealed deep tubular glands containing periodic acid-Schiff-positive secretory material. When examined 36 h after the last artificial insemination, sperm were observed in the lumen of the tubular glands associated with the urodeum. We suggest that sperm reside in the tubular glands within the urodeum and are released in association with the secretory activity of the tubular glands. These sperm then may ascend the vagina to continue to populate the sperm-storage tubules. Alternatively, the sperm in the tubular glands of the urodeum may be evidence of spermatorrhea and have no functional role in the fertilization process.

Serotonin localization in the turkey vaginal but not sperm storage tubule epithelia.

Elucidating the cellular and molecular mechanisms regulating sperm selection and transport in the vagina of the hen had been the focus of a limited amount of research over the past decade. New observations indicate the presence of nonneuron endocrine cells in the epithelia lining the lumina of the turkey hen vagina and uterovaginal junction. Although no cells in the vagina or uterovaginal junction surface epithelia exhibited argentaffin staining, typical of cells containing neurosecretory granules, cells restricted to the vaginal and uterovaginal junction but not the sperm storage tubule epithelia were immunoreactive positive to serotonin. We speculate that if released into the vaginal lumen and submucosa, serotonin could augment cilia and sperm tail beat frequencies and facilitate smooth muscle contraction, respectively. If this is the response to sperm at insemination, it would represent the first evidence of a local control mechanism responding to sperm in the turkey vagina.

Alkaline phosphatase reactivity in the vagina and uterovaginal junction sperm-storage tubules of turkeys in egg production: implications for sperm storage.

1. Currently there remains contradictory information on the localisation and possible role of alkaline phosphatase (AP) in the chicken and Japanese quail oviducts. 2. Using turkeys with a hard-shelled egg in their uteri, vaginal and uterovaginal junction mucosae were stretched and fixed as whole mounts prior to the histochemical localisation of AP activity. 3. Scattered AP reactive cells were observed in the vaginal and uterovaginal junction surface epithelia and intense AP reactivity of the sperm-storage tubule (SST) epithelium, localised to its apical border. 4. We suggest that such AP reactivity in hens in egg production may reflect cell differentiation and proliferation in the vagina and SST and possibly a mechanism for the transfer of lipid from the SST epithelia to resident sperm.

Morphological and histochemical characterization of the seminiferous epithelial and Leydig cells of the turkey.

Unlike mammals, there is little fundamental information about spermatogenesis in birds. This study was undertaken to clarify the morphology, histochemistry, and lectin affinity of the seminiferous epithelial cells and Leydig cells in pre-pubertal (8- to 15-week old) and adult (40- to 44-week old) domestic turkeys. In adult turkeys, three types of spermatogonia were defined based on their chromatin distribution and nuclear morphology: the dark type A (A(d)); the pale type A (A(p)); and the type B. The A(d) is the least numerous and least conspicuous and consequently difficult to locate. Based on its spatial distribution and overall morphology, type A(d) spermatogonia were postulated to be the spermatogonia stem cells in the turkey. Antibodies to c-kit were localized to spermatogonia in the pre-pubertal and to a lesser extent in adult males. Peanut agglutinin (PNA) was specific for spermatocytes in the pre-pubertal males and spermatogonia and early spermatocytes in adult males. Wheat-germ agglutinin (WGA) highlighted Sertoli cells in both age groups. Bandeiraea simplicifolia I, soybean agglutinin, and winged-pea agglutinin staining were limited to the wall of the seminiferous tubule and some extra-tubular cell types. Concanavalin A staining was diffuse and not cell-specific and, therefore, could not be used to selectively identify a particular cell type. It was concluded that WGA and PNA could aid in identifying specific cell types in the seminiferous epithelium of testis from pre-pubertal and mature turkeys. Only Leydig cells were alkaline phosphatase reactive in the mature turkey testes. The information from this study is being used to adapt techniques for the isolation and partial purification developed for mammalian spermatogonia to avian spermatogonia and other specific cell types in the testes.

Aquaporins are observed in the duct epithelia of the epididymal region of the large white turkey.

The cellular and molecular mechanisms regulating the reuptake of the testicular fluid supporting sperm exiting the testes in the bird are not known. The presence of aquaporins, proteins involved in transmembrane water transport, was investigated. Observations were limited to the ductuli efferentes, collecting ducts, and ductus epididymis. Interestingly all of these ducts were positive for aquaporins-2, -3, and -9 but not aquaporin-7. When positive, aquaporin was observed localized over the whole cell or the apical plasma membrane of the nonciliated cells and the apical plasma membrane and cilia of the ciliated cells. This study is the first to clearly demonstrate the presence of aquaporins-2, -3, and -9 in the epididymal region of any bird. We assume the aquaporins play a role in concentrating the sperm and in the promotion of sperm maturation in the epididymal region.

Localization of aquaporins in the sperm storage tubules in the turkey oviduct.

Oviductal sperm storage tubules (SST), located at the uterovaginal junction, are the primary site of sperm storage in turkeys. Sperm reside within these storage sites and may be released via a dynamic interaction between sperm mobility and a fluid current generated by the SST epithelial cells. In this study, aquaporins 2, 3, and 9 (proteins that form water channels in the plasmalemma of a variety of cells) were immunocytochemically localized within the apical aspect of the epithelial cells that form the SST. These observations support the contention that the SST epithelial cells are capable of water exchange that may interact with sperm residing within the SST.

Impact of egg storage on carbonic anhydrase activity during early embryogenesis in the turkey.

Carbonic anhydrase is an enzyme that plays important roles in the conversion of carbon dioxide to bicarbonate, acid-base balance, and in subembryonic fluid formation in the early Japanese quail embryo. While turkey egg storage longer than 10 d is known to increase the rate of embryo mortality, little is known of the biological mechanisms that contribute to this phenomenon. In this study, we examined the impact of turkey egg storage on carbonic anhydrase activity in the freshly laid egg through 72 h of incubation. Carbonic anhydrase activity, which was not affected by egg storage for 21 d at 18 degrees C, was first observed in the germ wall, that area of yolk subjacent to the area opaca, after 24 h incubation. By 48 and 72 h, the yolk sac had formed with the yolk sac endoderm and was strongly positive for carbonic anhydrase. In contrast, mesodermal and ectodermal layers were negative. Our observations support recent studies showing carbonic anhydrase activity associated with the endodermal cell of the yolk sac in Japanese quail embryos and that such activity appears to be involved with subembryonic fluid formation in the turkey. This work also demonstrated that if an embryo survives cold egg storage, carbonic anhydrase activity does not appear to be affected.

Potential relationships between physical traits and male broiler breeder fertility.

Genetic selection in primary broiler breeders may modify skeletal structure, possibly impeding semen transfer, and could alter the size and degree of fluctuating asymmetry (FA) of bilateral traits associated with fertility. Hence, we hypothesized specific morphometric traits could predict differential fertility. Sixty primary broiler breeder males from Strains A and B (n = 30/strain) were individually housed with an average of 10 females per male. Male fertility and sperm penetration (SP) through the perivitelline layer were estimated on fresh eggs. At 50 wk, BW, keel length (KL), posterior pelvic width and length (PPW, PPL), dorsal pelvic width and length (DPW, DPL), tarsometatarsal length and width (TL, TW), comb length and width (CL, CW), and wattle length, width, and area (WL, WW, WA) were measured. Results indicated that Strain A had smaller BW, KL, WL, WW, WA, CL, CW, PPL, DPL, and DPW. A higher degree of FA was found in Strain A TL and WL (P < 0.05), yet DPW FA was greater for Strain B (P < 0.001). In addition, DPW FA negatively correlated with Strain B fertility (r = -0.369; P < 0.01); however, other FA measurements did not correlate with estimated fertility or SP. Strain A WL correlated with SP (r = 0.383; P < 0.01) and fertility (r = 0.346; P < 0.01). Results indicate DPW alteration may impact semen transfer upon copulation, as Strain A fertility negatively correlated with DPW (r = -0.298; P < 0.05). This research provides evidence that morphometric traits might be useful to predict fertility in broiler breeders.

Embryonic development from first cleavage through seventy-two hours incubation in two strains of pekin duck (Anas platyrhynchos).

Embryonic mortality is a significant problem plaguing the commercial duck industry worldwide. Yet, an objective means to stage development of the duck embryo is lacking. Such a staging procedure, which is described in this study, is essential for the critical and reproducible assessment of embryo development. The morphological features associated with duck embryo development are very similar to those of the chicken, although the duck embryo develops more slowly. The staging scheme presented here provides objective morphological criteria describing the embryonic development of the duck.

Phenotypic traits as reliable indicators of fertility in male broiler breeders.

Genetic selection procedures applied to improve broiler performance may negatively impact the subsequent reproductive efficiency of breeders, particularly in males. Identification of traits that reliably indicate individual male fertility would facilitate selection for reproduction. We hypothesized that physical traits, such as comb area, relative testicular weight, and testicular weight asymmetry, may correlate with fertility in two male-selected primary broiler breeder strains (A and B). Thirty males per strain, individually housed with an average of 10 females, were evaluated at five age periods within the 30-to-50-wk breeding cycle. Flock fertility by candling eggs at Day 19 of incubation and sample fertility by visual assessment of the germinal disc were determined. Sperm penetration (SP) through the perivitelline layer was assessed. Comb area was evaluated by image analysis at 40 and 50 wk, and relative testicular weight was measured at 50 wk. Strain A sample and flock fertility (P < 0.001) and SP values (P < 0.0001) were significantly lower than Strain B. Both strains had a significant decline of fertility and SP with age (P < 0.0001). Strain A comb area correlated with sample fertility (P < 0.05), flock fertility (P < 0.05), and relative testicular weight (P < 0.01). Conversely, Strain B relative testicular weight correlated with sample fertility (P < 0.0001) and flock fertility (P < 0.001). Significant correlations were not found between testicular weight asymmetry and other reproductive traits. Results suggest comb area may be a reliable indicator of male fertility in Strain A.