Predictors of success of semen cryopreservation in chickens.

Semen cryopreservation is very important for the ex situ management of genetic diversity in birds but it is rarely used. This is partly because of the highly variable success rates, and this emphasizes the need for predictors of semen freezability. This study evaluated the ability of semen quality tests to predict the success rates of semen cryopreservation in chickens and the relationships between each test. Individual variations of in vitro quality tests of semen were compared to the fertility obtained with fresh and cryopreserved semen. The in vitro semen quality tests represented viability, integrity, motility (percentage of viable and morphologically normal cells (PVN); mass motility (MMOT) and different motion parameters including percentage of motile spermatozoa (PMOT)) and biophysical tests (OSM, resistance to osmotic stress; membrane fluidity (FLUID)). Different in vitro tests were significantly correlated between each other for fresh (MMOT, PVN and FLUID, many criteria of objective motility) and cryopreserved semen (MMOT, different objective motility parameters, PVN). Fertility was significantly correlated with PVN for fresh semen and PVN and different objective motility criteria for cryopreserved semen. Membrane fluidity, followed by PVN, PMOT and MMOT, measured on fresh semen samples was positively correlated with fertility obtained with cryopreserved semen. The combination of the first three tests explained 85% of the variability of fertility observed with cryopreserved semen. In conclusion, we showed that different in vitro tests of semen quality are of predictive value for the success rate of semen cryopreservation in the chicken, the most accurate being membrane fluidity.

The evolution of sperm morphometry in pheasants.

Post-copulatory sexual selection is thought to be a potent evolutionary force driving the diversification of sperm shape and function across species. In birds, insemination and fertilization are separated in time and sperm storage increases the duration of sperm-female interaction and hence the opportunity for sperm competition and cryptic female choice. We performed a comparative study of 24 pheasant species (Phasianidae, Galliformes) to establish the relative importance of sperm competition and the duration of sperm storage for the evolution of sperm morphometry (i.e. size of different sperm traits). We found that sperm size traits were negatively associated with the duration of sperm storage but were independent of the risk of sperm competition estimated from relative testis mass. Our study emphasizes the importance of female reproductive biology for the evolution of sperm morphometry particularly in sperm-storing taxa.

Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species.

In order to improve the genetic management of bird species within the European Endangered Programs (EEP), a research project on artificial insemination and cryopreservation of Galliformes semen has been developed. The aim of the program is to create a sperm cryobank for threatened bird species. During this study, semen was collected from 17 pheasant species and specific characteristics of ejaculates were analyzed (volume, sperm concentration, motility, pH). Artificial insemination with fresh semen was performed in nine species and with frozen semen in eight species. Inseminations with frozen and thawed semen were made in 17 species. Viability of fresh and frozen semen was assessed in vitro using double stains, eosin and nigrosin. The effect of pH (7-8.5) on viability of fresh and frozen/thawed spermatozoa was also studied. Chicks hatched in eight and three species after insemination with fresh and frozen/thawed semen, respectively. Species varied widely in semen viability: 1-30% of spermatozoa survived freezing and thawing. There was a negative correlation between the viability of frozen spermatozoa and semen pH. In our experimental conditions, the pH of diluents had no effect on semen viability. However, semen with the highest pH had the lowest quality after freezing and thawing. These experiments demonstrated the feasibility of using a very simple and inexpensive method to achieve artificial insemination and cryopreservation of semen in endangered pheasant species.

Antibody response to Newcastle disease vaccination in a flock of young houbara bustards (Chlamydotis undulata).

Twelve young houbara bustards (Chlamydotis undulata) were vaccinated with a lentogenic strain of Newcastle disease virus. Another seven birds were kept in close contact with the treated flock but were not vaccinated. Antibody levels were measured in all birds with hemagglutination inhibition test over the course of 1 yr. Antibody formation with no side effects was observed in 18 birds. The duration and amplitude of the antibody response differed between the groups.

Cross-protection test of an avian poxvirus isolated from houbara bustards.

An avian poxvirus was isolated previously from the houbara bustard (Chlamydotis undulata). We carried out a cross-protection test on 66 captive-bred canaries. Thirty-five canaries were vaccinated with a commercial canary poxvirus (CP) vaccine. Three weeks later all 66 birds were assigned randomly to six different groups: group Ia (n = 14) was vaccinated and challenged with houbara bustard poxvirus (HP) strain; group Ib (n = 13) was vaccinated and challenged with a CP strain; group Ic (n = 7) was vaccinated and not inoculated; group IIa (n = 14) was nonvaccinated and challenged with HP strain; group IIb (n = 11) was nonvaccinated and challenged with a CP strain; and group IIc (n = 7) was not vaccinated and not challenged. Vaccinated groups (Ia, Ib, Ic) had no losses and remained healthy. All of the birds (100%) in group IIb died within 10 days, and 10 birds (71.4%) of group IIa died within 20 days. The nonvaccinated control group (IIc) remained healthy. Poxvirus was isolated from the liver, digestive tract, lungs, and inoculation lesions of nonvaccinated dead CP- and HP-challenged birds. Secondary bacterial infections were higher among nonvaccinated HP-challenged birds (85.7%) than in nonvaccinated CP-challenged birds (25%). The results of this experiment reveal a degree of immunogenic relatedness between CP and HP strain and support the recommendation that houbara bustards be vaccinated with a CP vaccine.

Seasonal variation of LH, sex steroids, body mass, molt, display, and laying in two subspecies of Houbara bustard, Chlamydotis undulata macqueenii and Chlamydotis undulata undulata, housed in outdoor cages under natural conditions.

Subspecies of the Houbara bustard are resident in North Africa (Chlamydotis undulata undulata) or are partial latitudinal migrants in Asia (Chlamydotis undulata macqueenii). Houbara breed in arid or semiarid zones, and the reproductive biology of wild Houbara is largely unknown. Body mass, molt, courtship display, laying, and plasma concentrations of LH, testosterone, and progesterone were measured in captive-bred birds of both subspecies, housed in outdoor cages under natural conditions, over a period of 1 year and 6 months, at Taif, Saudi Arabia (21 degrees 15' N). Captive Houbara bustards show a seasonal breeding pattern with a laying period from January to July. Males began displaying well in advance of any eggs laid and display long after the termination of laying. In females, gonadal activity and molt were mutually exclusive; however, in males, slight overlap between sexual display and molt was observed. In females, seasonal variation of LH and progesterone was synchronous with laying activity. In males both testosterone and LH plasma concentrations were highest in January (testosterone approximately 8 ng/ml; LH approximately 3 ng/ml) at the beginning display period. This could correspond, in the wild, to the establishment of territories. Levels dropped significantly before the females started laying but stayed at an intermediate level throughout the breeding season (testosterone approximately 3 ng/ml; LH approximately 1.5 ng/ml). In the middle of August, during molt, and following heavy rainfall, LH and testosterone peaked (testosterone approximately 2.3 ng/ml; LH approximately 1.8 ng/ml), suggesting a possible impact of rainfall on Houbara gonadal activity. Houbara stopped laying before the maximum ambient temperature reached 32 degrees, indicating that high temperature could be a factor terminating laying activity. No major differences were found between the two subspecies in the timing or duration of the reproductive period; only the annual variation of the body mass varied slightly between the subspecies.

Debilitating cutaneous poxvirus lesions on two captive houbara bustards (Chlamydotis undulata).

Poxvirus was isolated from cutaneous nodules on two young Houbara bustards (Chlamydotis undulata) bred in captivity in Saudi Arabia. Birds were emaciated and presented nodules on tibiotarso-tarsometatarsal joints, toes, and the carpal joint. Diagnosis was confirmed by histopathology, virus isolation on inoculated chorioallantoic membranes of embryonated chicken's eggs, and electron microscopy. Progressive leg lesions were extensive and interfered with walking, significantly debilitating the birds. Successful excisions of these lesions were performed.

Concurrent avian pox and Newcastle disease infection in a Houbara bustard (Chlamydotts undulatd).

A case of acute respiratory disease was observed on a 3-month-old Houbara bustard (Chlamydotis undulatd) at the National Wildlife Research Center (NWRC) breeding unit. Newcastle disease (ND) and avian pox viruses were isolated from the lungs, spleen and trachea of the affected bird. A heavy secondary bacterial infection was also noticed. The clinical and pathological findings observed in this case are described. It is the first description of the isolation of ND virus from a Houbara bustard. The epidemiology of the disease in the breeding flock is discussed.

Artificial insemination in Houbara bustards (Chlamydotis undulata): influence of the number of spermatozoa and insemination frequency on fertility and ability to hatch.

Between 1989 and 1992, artificial insemination was used in the reproduction of two subspecies of Houbara bustard (Chlamydotis undulata macqueenii and Chlamydotis undulata undulata) at the National Wildlife Research Center, Taif. The laying period was between January and July and the mean annual egg production in C. undulata macqueenii was 10.2 and in C. undulata undulata was 7.6. No differences were found in sperm production between the two subspecies: the mean volume of ejaculate was 0.08 ml; the mean sperm concentration was 350 x 10(6) spermatozoa ml-1; and the mean number of spermatozoa per ejaculate was about 20 x 10(6). Large intra- and inter-individual variation was found in sperm parameters. Intra-individual variation in number of spermatozoa per ejaculate was due mainly to seasonal variation. The mean quantity of spermatozoa produced per week by fully sexually mature Houbara bustards was 165 x 10(6). There was no significant difference in fertility or in ability to hatch between the two subspecies. Overall, in 1992, mean fertility was 69.3% and 49.2% of eggs hatched. Females showed a median sperm storage duration of 10 days and a maximum sperm storage duration of 22 days. A positive correlation was found between fertility and quantity of spermatozoa inseminated (R = 0.99, P < 0.001). Sperm storage duration was related to the number of spermatozoa inseminated. The best results (85% fertile eggs) were obtained when more than 10(6) spermatozoa were inseminated between 3 and 6 days before laying. Analysis of hatching showed that embryo mortality increased when inseminations were performed more than 10 days before laying.