Cryoconservation of avian gonads in Canada.

Avian genetic resources have declined dramatically over the past half century as the cost of maintaining populations has exceeded the perceived benefit of keeping them. Despite the early importance of poultry in the development of cryopreservation techniques, very little avian germplasm has been conserved. Cryopreservation and recovery of avian gonads preserve the W chromosome and overcome problems of freezing and recovering semen or conserving and manipulating embryonic cells, and the use of vitrification procedures for preserving gonads minimizes cellular damage. On the basis of research demonstrating the biological possibility of cryopreserving and transplanting avian gonads, 5,125 testicles and 2,667 ovaries from 10 populations of Japanese quail, 9 populations of chickens, and 1 population of Chilean tinamou were cryopreserved and sent to the Canadian Animal Genetic Resources program for long-term storage. These gonads represent 20 of the 33 distinct avian populations currently maintained at Canadian public institutions of agricultural research.

Growth of subcutaneous chicken testicular transplants.

Avian genetic resources have declined dramatically over the past half century, partly because the poultry community has been slow to adopt cryoconservation of avian germplasm. Techniques for gonadal cryopreservation and functional recovery have recently been developed but only some have been optimized. Chicks were castrated at 2 or 6 d and testicles were autotransplanted subcutaneously in one piece after disruption of the tunica membranes to optimize transplantation procedures without the complication of tissue rejection or immunosuppression. At 22 wk of age, the roosters were euthanized and growth of the testicular tissue was evaluated. Mortality with castration at 2 d was high but was much reduced with castration at 6 d. Transplantation of whole testicles subcutaneously on the back of chicks, without complete removal of the tunica membranes, yielded good growth of tissue with transplantation at 2 or 6 d of age. These results will contribute to the use of testicular cryopreservation and transplantation as an effective conservation strategy for avian germplasm. Further definition of the age of treatment will improve the overall efficiency.

Chimeric plumage coloration produced by ovarian transplantation in chickens.

Ovaries from Rhode Island Red donors were transplanted orthotopically into White Leghorn recipients. At maturation, recipients were mated with Rhode Island Red roosters to test the origin of their ovaries, using plumage coloration as a marker. A chick with chimeric plumage coloration was produced, indicating mechanisms that produce follicles with both donor and recipient ovarian contents. This study suggests that ovarian transplantation could be useful for in vivo studies of cytological and molecular mechanisms involved in avian folliculogenesis.

A simple vitrification method for cryobanking avian testicular tissue.

Cryopreservation of testicular tissue is a promising method of preserving male reproductive potential for avian species. This study was conducted to assess whether a vitrification method can be used to preserve avian testicular tissue, using the Japanese quail (Coturnix japonica) as a model. A simple vitrification method that included dimethyl sulphoxide, ethylene glycol, and sucrose as cryoprotective agents, and allowed the storage of tissue in a sealed macrotube was applied to the testicular tissue from 1-wk-old Japanese quail. The vitrified tissue was warmed at room temperature or at 40°C. After warming, tissue was implanted onto the chorioallantoic membrane of 8- to 9-d-old chicken embryos and the vascularization of the grafts was evaluated. When compared with fresh tissue, the tissue that had been warmed at 40°C showed no difference in vascularization. The tissue that had been warmed at room temperature was significantly less vascularized than the fresh tissue. Vitrification of testicular tissue and storage in macrotubes provide a promising model for preservation and recovery of male germplasm of avian species.

Comparison of bones of 4 strains of laying hens kept in conventional cages and floor pens.

The maintenance of bone strength has been an important issue in the debate over cage use for laying hens. Bone strength depends on adequate mechanical load and cages restrict movement. Four laying crosses (Lohmann White, Lohmann Brown, H&N White, and Rhode Island Red × Barred Plymouth Rock cross hens) were housed in conventional cages or in floor pens equipped with perches and nest boxes to measure the effect of the housing system on bone strength. Approximately 15 hens of each genotype from each housing system were killed at 50 wk of age and the radius and tibia of each were removed for analysis. There were no differences between the Lohmann White and H&N White (White Leghorn) hens, likely because of their similar genetic background. The Lohmann Brown and the cross hens (brown-egg layers) were larger and they had heavier bones, but the bone density was not different from that of the other lines. The radius was heavier for hens kept in floor pens than for those kept in cages, but the tibia was not. When hens were kept in floor pens, both bones had greater cortical bone density and cross-sectional area, but the difference between housing systems in cortical bone cross-sectional area was much greater for the radius than it was for the tibia. Although the movement of hens in cages is limited, they spend a great deal of time standing, which puts a mechanical load on the tibia. Hens in floor pens are able to stretch their wings or fly, in contrast to hens kept in cages, which likely explains why the difference between housing systems in cortical bone was greater for the radius than for the tibia.

Production of donor-derived offspring after ovarian transplantation between Muscovy (Cairina moschata) and Pekin (Anas platyrhynchos) ducks.

Transplantation of ovarian tissue between different breeds of chickens or Japanese quail can result in normal egg production and give rise to donor-derived offspring with a high frequency. The feasibility of interspecific ovarian transplantation was evaluated by transplanting ovarian tissue from Muscovy ducks to Pekin ducks just after hatch, with subsequent treatment of the recipients with an immunosuppressant. Among the 8 mature Pekin ducks that had received ovarian tissue from Muscovy ducklings, 2 produced fertile eggs when mated with Muscovy drakes, and one of these produced only Muscovy ducks, which were derived from the transplanted Muscovy ovary. The other produced mule ducks that were hybrids of the host Pekin duck and the Muscovy drake. Further development of interspecific transplantation of ovaries may provide a simple method for the conservation and propagation of endangered birds.

Comparative costs of programmes to conserve chicken genetic variation based on maintaining living populations or storing cryopreserved material.

1. There have been substantial losses of chicken lines kept for research in recent years and the objective of this research was to critically review alternative methods of preserving genetic resources. 2. The costs of programmes using living populations, semen cryopreservation and reconstitution, and ovary and semen cryopreservation and reconstitution were evaluated over 20 years using biological parameters of cryopreservation and population reconstitution that were derived from the literature. 3. Keeping live populations was most cost effective for periods of up to three years, but keeping live populations is increasingly difficult to justify with longer periods and any research population that will not be used within five years should be cryoconserved and in situ maintenance discontinued. 4. The rapid reconstitution possible using ovaries and semen would allow the inclusion of cryopreserved material in a short-term research project with the cost of recovery included in the budget. The low cost of cryoconservation suggests that all avian material should be conserved and reconstituted when needed for research.

Evaluation of wet-feeding wheat-based diets containing Saccharomyces cerevisiae to broiler chickens.

1. This experiment investigated the effects of water and Saccharomyces cerevisiae added to wheat-based diets on gastrointestinal, blood and performance parameters of broiler chickens. 2. A total of 160 one-d-old male broiler chicks were given air-dry or wet diets, with or without S. cerevisiae supplementation (0 and 20 g/kg air-dry feed) ad libitum to 42 d. 3. Feeding broilers with a diet mixed with water in a ratio of 1·2 : 1·0 increased body weight, feed intake, abdominal fat, carcase weight, feed transit time and blood HDL (high density lipoprotein) (without yeast). Supplementation with S. cerevisiae increased DM digestibility but reduced ileal pH, ileal coliform population and abdominal fat content. 4. There was a significant interaction between S. cerevisiae and wet feeding, with S. cerevisiae supplementation inducing a significant increase in body weight and feed intake but a reduction of relative abdominal fat and ileal pH of broilers fed on wet diets. 5. It is concluded that wet feeding improved growth performance by increasing feed intake and that the addition of a culture of S. cerevisiae had a growth stimulating effect, as the inclusion of yeast in wet wheat-based broiler diets generated greater responses than yeast in dry-based diets.

Formation of testicular structure under skin after auto-transplantation of dispersed testicular cells in the chicken.

Transplantation of immature testis tissue under the skin provides a simple approach for the establishment of donor-derived-only spermatogenesis in birds. There are reports on the reconstruction of testicular structures from dissociated testicular cells in mammalian species. The present study aimed to evaluate whether, in birds, the transplantation of testicular cells under the skin could form a new testis and lead to fertility. Testicular cells from newly hatched chicks were auto-transplanted under the neck skin as a cell suspension or cell pellet. Testicular tissue was auto-transplanted under the neck skin as a control study. At the age of 1 year, both suspension and pellets of testicular cells formed testicular tissue under the skin. Only 11.3% of seminiferous tubules contained primary or normal lumens with normal spermatogenesis in testicular cell transplants while 80% of seminiferous tubules contained normal lumens in the testis tissue transplant. This technique, if further developed, would provide a novel system to manipulate and study spermatogenesis in birds.

Evaluation of glycerol removal techniques, cryoprotectants, and insemination methods for cryopreserving rooster sperm with implications of regeneration of breed or line or both.

A series of experiments was designed to evaluate the quality of cryopreserved rooster sperm and its fertility so that programs needing to bank germplasm and recreate animals can do so utilizing a minimal amount of cryopreserved semen. In experiment 1, rooster semen from the National Animal Germplasm Program genebank was thawed and glycerol was removed using a discontinuous Accudenz column or by stepwise dilution. The postthaw sperm motilities, plasma membrane integrity, and concentration were determined before and after deglycerolization. Line differences in postthaw sperm concentration and progressive motility were observed before deglycerolization (P<0.05). After glycerol removal, the sperm that was centrifuged through Accudenz had greater total motility (37 vs. 33% sperm; P<0.05), but use of the stepwise dilution method recovered more sperm per milliliter (320.4x10(6)) compared with the Accudenz method (239.2x10(6) sperm; P<0.05; range across 6 lines of 165.7 to 581.0x10(6) sperm/mL). In experiment 2, rooster semen was cryopreserved using Lake's diluent containing either dimethyl acetamide (DMA) or glycerol as the cryoprotectants. Postthaw analysis revealed that the samples cryopreserved with glycerol survived freezing better, determined by total motility (47.8 and 15.1% glycerol and DMA samples, respectively; P<0.05) and annexin V analyses (1.6 and 11.3% membrane-damaged sperm for glycerol and DMA samples, respectively; P<0.05). Differences in sperm motilities (total and progressive motility) and velocities (path velocity, straight-line velocity, curvilinear velocity) were observed between the 2 cryoprotectant treatments once the glycerol had been removed from those samples cryopreserved with glycerol, of which the glycerol samples had significantly more motile sperm and higher velocities (P<0.05). The fertility of the samples frozen using the 2 cryoprotectants was tested using a single insemination (intravaginal or intramagnal) of 200x10(6) sperm and the fertility (number of live embryos) was evaluated over 18 d. Overall, the intravaginal inseminations had lower fertility than the intramagnal inseminations (P<0.05). In the intravaginal inseminations, the sperm cryopreserved using DMA resulted in lower fertility, but there were no differences in fertility in the intramagnal inseminations due to cryoprotectant (P>0.05). These results indicate that reasonable postthaw sperm quality and fertility can be derived using cryopreserved rooster semen. By utilizing this information, estimations can be made for storing sufficient material for line or breed, or both, recreation programs.

Invasive and noninvasive measurement of stress in laying hens kept in conventional cages and in floor pens.

Measurements of the heterophil:lymphocyte (H/L) ratio (invasive technique) and corticosterone in yolk and albumen (noninvasive techniques) were used to measure stress in 3 commercial laying strains, Lohmann White (LW), H&N White (HN), Lohmann Brown (LB), and a noncommercial cross (CR) between Rhode Island Red (male) and Barred Plymouth Rock (female), kept in conventional cages or floor pens. All chicks were reared in their respective environments, and 450 and 432 pullets were placed at 18 and 7 wk of age in cages and floor pens, respectively. Blood from 12 hens per strain was taken at 19, 35, and 45 wk of age in each housing system. A total of 100 heterophils and lymphocytes were counted and their ratio (H/L ratio) was calculated. Corticosterone was measured in yolk and albumen from 12 hens per strain in each housing system at 22 and 45 wk of age. The H/L ratio was within the normal range. The interaction between environment and strain for the H/L ratio showed that in both environments, LB and CR hens had a higher H/L ratio than LW and HN layers. In cages, there were significant differences in H/L ratios between LW and HN hens that were likely due to genetic differences. The LW hens had significantly lower corticosterone concentrations in yolk than LB hens. In cages but not floor pens, yolk corticosterone concentrations at wk 22 were significantly higher than at wk 45. In floor pens but not cages, albumen corticosterone at wk 22 was higher than at wk 45. The H/L ratios suggest that none of the hens were unduly stressed, and corticosterone levels in yolk and albumen support the suggestion that hens adapted to their environments with age. Although measurement of yolk corticosterone and the H/L ratio may be comparable, the measurement of corticosterone level in the albumen may differ because it is secreted over a short time.

Production performance and egg quality of four strains of laying hens kept in conventional cages and floor pens.

Production performance and egg quality were compared between 4 strains of beak-trimmed layers: 3 commercial strains-Lohmann White (LW), H&N White (HN), Lohmann Brown (LB)-and a noncommercial cross between Rhode Island Red (male) and Barred Plymouth Rock (female) in conventional cages and in floor pens. All chicks were reared and 857 pullets were housed at 18 wk of age in their respective environments. Body weight, hen-day egg production, feed consumption and efficiency, and egg quality were measured at wk 20, 30, 40, and 50. In floor pens, the location of eggs was recorded for 4 consecutive days at 4-wk intervals between 20 and 50 wk of age. Eggs from cages, nest-boxes, and the floor were tested for Escherichia coli and coliform contamination at 38 and 42 wk of age. Mortality was recorded during the rearing and laying periods. Housing systems significantly influenced BW and mortality but not feed consumption or feed efficiency. The interaction between environment and strain was significant for hen-day egg production at wk 20 to 30 and for BW at wk 30, 40, and 50. Hens in floor pens had greater BW, egg and yolk weights, and yolk color than those in cages. Commercial hens produced more eggs than the cross hens. Overall, HN hens had the best production performance, whereas cross hens had better egg quality. In floor pens, LW and HN hens laid most of their eggs in nest boxes, whereas LB and cross hens laid half of their eggs on the floor. Eggs from cages had lower E. coli and coliform contamination than those from nest-boxes and the floor, and E. coli contamination was greater for LB eggs than for LW eggs. Significant strain differences were found for the use of nest-boxes, with a high percentage of floor eggs for brown egg strains. This study suggests that genotype x environment interactions should be considered when alternative housing systems are proposed.

Bone fracture incidence in end-of-lay high-producing, noncommercial laying hens identified using radiographs.

Bone fractures in laying hens are both a welfare and an economic concern for the poultry industry. The aim of this study was to use radiographs to quantify fracture incidence in 6 lines of noncommercial high-producing laying hens. A total of 451 hens (n = 71 to 78) were killed at 47 wk (White Leghorn-Black, White Leghorn-Blue) or 65 wk of age [Barred Plymouth Rock (BR), White Leghorn-Burgundy (WL-BUR), Columbian Plymouth Rock, Rhode Island Red (RIR)]. Radiographs were obtained with hens in 2 positions (lateral and ventrodorsal) and were used to identify fractures in the skeleton. Data on scallop-shaped indentations (possibly fractures) of the keel bone were also collected. After radiography, the left wings were removed for analysis of humeri, radii, and ulnae. Data for the 2 age groups were analyzed separately. The overall incidence of hens with at least 1 fracture was 6.6 and 15.7% in the 47- and 65-wk-old hens, respectively. Fracture incidence in 47-wk-old hens was not different between White Leghorn-Black and White Leghorn-Blue lines. Significant line differences were observed in the 65-wk-old hens, with at least 1 fracture found in 29.5% of RIR hens versus 9.5 and 4.2% observed in Columbian Plymouth Rock and WL-BUR lines, respectively. Fracture incidence in BR hens (18.2%) was greater than in WL-BUR hens. Fractures in RIR hens occurred predominantly in the furculum and wing bones, whereas pubic bones were most affected in BR hens. The proportion of hens with scallop-shaped indentations of the keel ranged from 36.1 to 88.2% and differed between lines in both age groups. High egg production did not seem to be associated with bone fragility in these lines. Two of the older lines (RIR and WL-BUR) had similar egg production, number of eggs to 60 wk, and egg shell weights at 4 ages but had a significantly different fracture incidence. The line differences in fracture incidence may have been affected by calcium metabolism, bone structure, and body weight.

Long-term production of donor-derived offspring from chicken ovarian transplants.

A surgical technique for orthotopic transplantation of ovarian tissue was recently developed for chickens, and short-term production of donor-derived offspring from the transplants was demonstrated. The present study evaluated the long-term reproductive function of the transplanted ovaries. In addition, ovarian tissue was transplanted into chicks from eggs that had been treated with busulfan (BU) after 24 h of incubation to reduce the growth of host germ cells. The success rate of ovarian transplantation was not different between BU- and non-BU-treated birds, but the ratio of donor-derived to host-derived offspring was significantly higher in the BU-treated birds. Over the 18-mo period each successfully transplanted hen produced up to 396 eggs with between 50 to 98% of donor-derived progeny. These results demonstrate that when transplanted ovaries attach and develop they can maintain their reproductive function for an extended period of time. These transplantation techniques provide a unique opportunity for development of a practical, efficient protocol for the cryopreservation of germplasm of chickens and may provide an opportunity for regenerating populations of wild birds.

Radiodensity in the central cavity of humeri in high-producing non-commercial laying hens.

1. Line differences in the incidence of the presence of radiodense material in the central cavities of humeri of 6 different lines of laying hens were observed using radiographs. 2. Internal humeral radiodense material was observed in over 60% of hens in two lines and 18% of hens in two other lines measured at 65 weeks of age. There was no difference in the incidence (15, 16%) between the last two lines observed at 47 weeks of age. 3. In the 4 lines studied at 65 weeks of age the presence of radiodense material was associated with increased humerus dry weight and ash weight in two lines, while percentage-ash was reduced in all 4 lines. 4. Results from this and previous studies suggest that humeral medullary bone could be an appreciable source of variability in experimental data collected to assess skeletal health.

Production of offspring from cryopreserved chicken testicular tissue.

Cryopreservation of avian germplasm provides a means of genetic banking for future needs in biological research and animal production. The sperm of birds can be cryopreserved and used to fertilize eggs. However, the fertility of frozen-thawed avian semen is generally much lower than that of mammalian semen and varies among species or among lines, reducing the value of semen for the preservation of genetic resources. In the present study, a simple freezing protocol was used to cryopreserve testicular tissue of day-old chicks, and after subsequent transplantation, the frozen-thawed testicular tissue developed functional seminiferous tubules that produced sufficient sperm to fertilize eggs, resulting in donor-derived offspring. This study provides an alternative to semen cryopreservation for storage of the male germline in birds.

Offspring produced from orthotopic transplantation of chicken ovaries.

The loss of avian genetic variation and the threat of disease lend urgency to the cryopreservation of remaining poultry stocks. However, techniques for freezing ova and embryos are not available for birds, and the recovery of genetic material has been a major obstacle to cryopreservation. To overcome this problem, we transplanted chicken ovarian tissue just after hatch with or without subsequent treatment of the recipient with an immunosuppressant. Nine of 12 hens in the nonimmunosuppressed group and 6 of 9 birds in the immunosuppressed group produced eggs, whereas 3 hens in each group produced donor-derived offspring. These results suggest that transplantation of ovarian tissue of chickens is possible if performed just after hatch. This finding should allow efficient cryopreservation of female germ cells in chickens with regeneration in live birds. In addition, ovarian transplantation could be useful for studies in genetics or developmental biology or could provide convenient access to the female germline for genetic manipulation.

Effect of strain of layer and age at photostimulation on egg production, egg quality, and bone strength.

Bone strength in layers is a concern for economic reasons and animal welfare concerns. Bone characteristics were investigated in 3 strains of hens: Babcock B-300, a small-bodied commercial white-egg layer; ISA-Brown, a commercial brown-egg layer; and an unselected Brown Leghorn line (BL). After being reared together in a single pen with 8 h of light per day, hens were caged with 14 h of light per day. Half of the hens were caged at 18 wk of age and the other half at 20 wk of age, resulting in a 2-wk difference in the age at photostimulation. Body weights, egg production, feed efficiency, and egg quality were measured throughout production. At 15, 25, 50, and 74 wk of age, hens were euthanized for sampling of the radius and the humerus. Breaking strength of the radius and humerus was measured, and the area and density of trabecular (largely medullary bone) and cortical bone were measured using quantitative computed tomography. Egg production and feed conversion of ISA-Brown hens was as good as or better than that of Babcock B-300 hens, and both commercial strains had higher production than the BL. Photostimulation late delayed sexual maturity and improved albumen and shell characteristics but had only minor effects on egg production and did not affect the yolk weight. The delayed photostimulation resulting from caging 2 wk later affected the radius by increasing the area of the trabecular space at 50 wk of age and the density of the bone in the trabecular space at 74 wk of age. Breaking strength of the humerus at 25 wk of age was greater for the birds that were photostimulated late but was not different later in the trial. The humerus, but not the radius, of the BL had a greater breaking strength than that of the commercial strains, suggesting that selection has decreased humeral breaking strength.

The technique of orthotopic ovarian transplantation in the chicken.

A surgical technique has been established for orthotopic transplantation of ovarian tissue in newly hatched chickens. In these trials survivability of the chicks after surgical manipulation was 100%. The size and orientation of the ovary at 2 wk of age suggested that the graft was attached and had undergone development. This technique should allow the development of a cryopreservation protocol of chicken ovarian tissue for the conservation of poultry genetic material.

A study on the interaction of xylanase and phytase enzymes in wheat-based diets fed to commercial white and brown egg laying hens.

A trial was conducted to investigate potential interactions between phytase and xylanase enzymes in wheat-based laying diets. Hens (480 ISA-White and 480 ISA-Brown) were distributed into 160 experimental units and fed one of 10 diets containing 75 to 77% wheat from 33 to 64 wk of age with a diet change at 49 wk. Two diets were adequate in P content (0.3 and 0.25% available P in the 2 phases) with or without xylanase (0 or 2,000 U/kg; Avizyme 2300, Danisco Animal Nutrition, Marlborough, Wiltshire, United Kingdom), and 8 diets had reduced P (0.2 and 0.15% available P) with or without xylanase and phytase (0, 300, 500, and 700 ppu/kg; Phyzyme 5000G, Danisco Animal Nutrition). Egg production was higher for ISA-Brown than for ISA-White hens, and ISA-Brown hens were larger. Eggs from ISA-Brown hens had lower albumen height; higher egg, shell, and albumen weights; and lower yolk weight than those from ISA-White hens. Egg production was not affected by the diet. In P-reduced diets without xylanase, phytase significantly increased BW gain in the first period with no change in feed intake or feed efficiency. In P-adequate diets, xylanase increased egg and albumen weight and albumen height. In P-reduced diets with xylanase, increasing levels of phytase increased egg and albumen weight. This trial demonstrated no negative interactions between these enzymes for production traits and no interactions between the diet and strain of hen. These data suggest that poultry producers can use these enzymes individually or together in feed for the Brown and White egg layers used in this study without concern for the strain of hen.