An efficient method of guinea fowl sperm cryopreservation.

France is the only country that practices pedigree selection of guinea fowl for meat production. The increasing risk of line extinction for sanitary or breeding failure reasons makes clear the need for an efficient method of reproductive cell cryopreservation in this species. However, an efficient method of guinea fowl sperm freezing in secured packaging is still lacking. The aim of the present study was to develop such a method. Based on results previously obtained in chickens, different cryoprotectants and freezing/thawing processes were tested and then adapted to guinea fowl. Semen quality was measured by semen viability evaluation and then by fertility measured after intravaginal artificial insemination. The best results (70% fertility with frozen-thawed sperm) were obtained by the use of the permeant cryoprotectant agents dimethyl formamide combined with a freezing rate of 30°C/min. The initial insemination frequency also affected the fertility results: 2 consecutive days of inseminations were needed in the first week to ensure enough filling of the utero-vaginal glands of the guinea fowl hen and thus to get successive fertile eggs. Thereafter, a 2-wk insemination frequency was sufficient. This new method, combining biophysical (cryoprotectant agents, freeze/thaw rate) and zootechnical (artificial insemination frequency) features, is the first cryopreservation method successfully developed in secured packaging for guinea fowl sperm. This method is now available for the practice of gene bank conservation and male reproductive management.

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.

Semen cryopreservation for ex situ management of genetic diversity in chicken: creation of the French avian cryobank.

The need for semen preservation in domestic birds is a result of the reduction in genetic variability of domestic bird livestock and of the increasing risk of line extinction for health and safety reasons. Cryopreservation of embryos and primordial germ cells (PGC) is not routinely feasible in birds. The project therefore involved semen frozen in optimal safety and traceable conditions. Whole blood samples were also frozen to provide samples of analyses of genomes and health status. The feasibility of using ex situ conservation, i.e., collecting biological material to be stored outside the usual production area of the species (ex situ genetic stock), to preserve and manage rare breeds was tested with 4 subfertile populations: 3 rare experimental lines used for research into energy metabolism (R+), growth (Y33), and immunity (B4/B4), reared under known health status and the oldest endangered patrimonial French breed, the Gauloise dorée with an unknown health status. A general infrastructure was set up for the health screening and remediation of diseases, collection and storage of frozen cells and 2 sites were created for the storage of frozen samples. The screening and remediation of diseases of the Gauloise dorée, which was contaminated with various Salmonella and Mycoplasma strains, was achieved by successive treatment of parents, incubated eggs and young chicks with Baytril followed by Tiamulin. For each line, 474 to 994 semen straws have been frozen, thawed, and the semen evaluated. Insemination of frozen-thawed semen into females of the same genetic origin or of an egg-type commercial breed produced chicks in every case. For the most subfertile lines, insemination with egg-type females significantly increased the reproductive success. In conclusion, we report on the benefits of a semen and blood cryobanking complex for the management of endangered lines and strains of domestic birds. Current stocks made possible the restoration of more than 96% of the initial genome. This project also provided technical solutions to resolve some of the health problems frequently encountered for gene preservation in poultry.

Membrane fluidity and the ability of domestic bird spermatozoa to survive cryopreservation.

The ability to survive cryopreservation varies in spermatozoa from different bird species. Among the biological factors potentially responsible for such differences, species variations in membrane fluidity have a role in the restoration of the physiological state after freezing. Membrane fluidity may be assessed by measuring fluorescence polarization anisotropy with a fluorescent dye. Anistropy values are proportional to membrane rigidity and consequently inversely proportional to membrane fluidity. In the present study, polarization anisotropy of spermatozoa originating from species differing in the freezability of their semen (chicken, turkey and guinea fowl) was measured in addition to lipid composition (cholesterol/phospholipid ratio), sperm viability (membrane permeability to eosine) and morphological integrity before and after cryopreservation. The percentages of viable and normal spermatozoa in fresh sperm were highest in the chicken (87%), lowest in guinea fowl (64%), and intermediate in turkeys (69%). Anisotropy values were highest in guinea fowl (0.205), lowest in chickens (0.155), and intermediate in turkeys (0.180). As a consequence, membrane fluidity was highest in chickens and lowest in guinea fowl. Cryopreservation significantly decreased sperm viability and morphological integrity and increased anisotropy in all species but did not change the inter species hierarchy. Initial cholesterol/phospholipid ratios were lower in chickens than in guinea fowl, and intermediate in turkeys (0.25, 0.26 and 0.29, respectively). Cryopreservation induced a severe decrease in cholesterol/phospholipid ratios in turkeys and guinea fowl. Sperm membrane fluidity in chickens, turkeys and guinea fowl behaves as an indicator of sperm freezability in these species. Inter species differences for this parameter may be partly explained by differences in initial cholesterol/phospholipids content of spermatozoa. On the other hand, the rigidifying process induced by cryopreservation is not related to lipid damage by the same mechanisms.

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.

Phospholipid fatty acid composition, vitamin E content and susceptibility to lipid peroxidation of duck spermatozoa.

Recent studies on chicken semen have suggested that the lipid and fatty acid composition of spermatozoa may be important determinants of fertility. Phospholipid fatty acid composition, vitamin E content and in vitro susceptibility to lipid peroxidation of duck spermatozoa were investigated using GC-MS and HPLC based methods. The total phospholipid fraction of duck spermatozoa was characterized by high proportions of the n-6 polyunsaturated fatty acids arachidonic (20:4n-6), docosatetraenoic (22:4n-6) and docosapentaenoic (22:5n-6) acids but a substantial proportion of the n-3 fatty acid docosahexaenoic (22:6n-3) acid was also present. Palmitic (16:0) and stearic (18:0) fatty acids were the major saturates in sperm phospholipids. Among the phospholipid classes, phosphatidylserine (PS) had the highest degree of unsaturation due to very high proportions of 22:6n-3, 22:5n-6, 22:4n-6 and 20:4n-6, comprising together more than 75% of total fatty acids in this fraction. Phosphatidylethanolamine (PE) also contained high proportions of these four C(20-22) polyunsaturates, which together formed 60% of total fatty acids in this phospholipid. Spermatozoa and seminal plasma of duck semen were characterized by unexpectedly low content of vitamin E, being more than 4-fold lower than in chicken semen. In duck semen the major proportion of the vitamin E (>70%) was located in the spermatozoa. The very high proportion of 22:6n-3 in PS and PE fractions of duck sperm lipids and the comparatively low levels of vitamin E could predispose semen to lipid peroxidation. Nevertheless the in vitro susceptibilities to Fe2+-stimulated lipid peroxidation of duck and chicken spermatozoa were very similar. The results of the study suggest that increased superoxide dismutase and glutathione peroxidase activity and increased antioxidant activity of seminal plasma may compensate for the low levels of vitamin E to help protect the membranes of duck spermatozoa, which exhibit a high degree of unsaturation from oxidative stress.

In vivo assessment of the quantity of breast muscle by sonography in broilers.

Two experiments were conducted to estimate the amount and yield of breast meat in living chickens by sonography. Results from the first experiment showed that the use of some cross-sectional areas (CSAs) of the breast muscles (Pectoralis major and Pectoralis minor, PM) in combination with the body weight (BW) value could be used to predict breast muscle and yield. Values of the coefficient of determination (r(2)) were near 0.90 and 0.65 for the amount and yield of breast meat respectively. Accurate results were obtained by the combination of three chosen (among more than 40) CSAs of the PM muscle. In the second experiment, the use of the CSAs was limited to the three most explicative ones defined in the first experiment. This experiment was conducted with a larger number of animals and confirms that it is possible to predict in vivo the amount and yield of breast meat with a good accuracy in chickens.

Estimation of poultry breastmeat yield: magnetic resonance imaging as a tool to improve the positioning of ultrasonic scanners.

Magnetic resonance imaging (MRI) was used to reconstruct three-dimensional breast muscle volume of 30 broilers and to locate the most suitable cross-sections to estimate breast muscle yield by ultrasonic scanner. The high accuracy of the determination of the breast muscle yield (R(2)=0.92) from the volume calculated by the sum of 6 mm-spaced MRI transverse images justified the choice of MRI as a reference method. Treatment of the images showed that it was possible to obtain acceptable breast meat yield prediction by MRI from a combination of two or three muscle transverse cross-section area measurements. It also showed that the need to find interfaces reflecting ultrasound is a considerable handicap for optimizing the ultrasonic technique. The oblique echotomographic plane crossing the coracoid bone lengthwise and the fore part of the breast bone appears to be the most appropriate to improve significantly the determination provided by the transverse image situated at the fore part of the breast bone.

In vitro comparison of fowl sperm viability in ejaculates frozen by three different techniques and relationship with subsequent fertility in vivo.

A series of experiments was conducted to compare the viability of fresh fowl spermatozoa, samples suspended in three cryoprotectants (CPAs), frozen/thawed samples, and frozen/thawed samples maintained in vitro for up to 24 h. The CPAs used were glycerol (Glyc), dimethylacetamide (DMA), and dimethylformamide (DMF). Viability was assayed using two double stains, Eosin + Nigrosin or SYBR-14 + PI (propidium iodide). Semen samples examined with SYBR-14 + PI indicated significant differences in viability between fresh and ready-to-freeze preparations (fresh, 83%; Glyc, 73%; DMA, 74%; DMF, 72%; P < 0.05). In contrast, Eosin + Nigrosin did not detect any difference at this stage (fresh, 88%; Glyc, 86%; DMA, 87%; DMF, 88%; P > 0.05). The percentages of viable spermatozoa in frozen/thawed ejaculates stored in vitro for 0, 4, and 24 h were generally higher in samples treated with glycerol than in those treated with DMA or DMF, irrespective of the technique used to assess sperm viability (P < 0.05). Fertility in eggs obtained from hens inseminated with semen frozen in DMA reached levels comparable to those obtained from hens inseminated with fresh undiluted semen (88 and 93%, respectively; P > 0.05). In contrast, fertility of eggs from hens inseminated with semen frozen in DMF or glycerol was significantly lower, although still very good, than that observed in eggs from hens inseminated with semen frozen/thawed in DMA (79 and 76%, respectively; P < 0.05). Finally, the double stain SYBR-14 + PI was proven more effective than Eosin + Nigrosin to assess sperm viability in fresh, stored, and frozen fowl semen. However, additional tests (e.g., morphology, acrosomal status, motility) remain necessary to develop a working model of in vitro sperm analysis capable of revealing the fertilizing potential of fresh and frozen fowl spermatozoa.

Comparison of cryoprotectants and methods of cryopreservation of fowl spermatozoa.

The deleterious effects of three cryoprotectants, glycerol, dimethylsulfoxide (DMSO), and dimethylacetamide (DMA), were compared on fowl spermatozoa. The viability and integrity of spermatozoa were measured with eosin-nigrosin smears. Glycerol was the least deleterious cryoprotectant, followed by DMA, and DMSO was the most toxic. Methods employing either glycerol or DMA were then compared for the cryopreservation of semen in either straws or pellets. Fertility was measured following artificial insemination. The highest fertility rates were obtained with semen frozen with DMA in pellets directly plunged in liquid nitrogen, DMA being added at -6 C (92.7%) or 5 C (84.7%). When semen was frozen in straws, glycerol equilibrated for 1 or 30 min gave the highest fertility results, but the fertility rates were lower (53.7 and 63.9%) than with DMA in pellets. The lowest results (26.7%) were obtained when semen was frozen in straws with DMA. When semen was frozen in pellets at very high cooling rates, DMA was superior to glycerol as a cryoprotectant, as evidenced by fertility. In contrast, when straws and low freezing rates were used, glycerol gave better results; however these results were never as high as those obtained with DMA and pellets. In conclusion, under our experimental conditions, the highest fertility rates were achieved with DMA and pellets. However, for gene banking, which requires high levels of safety and clear identification, glycerol and straws are more convenient.

Induced moulting in cockerels: effects on sperm production, plasma concentrations of luteinising hormone, testosterone and thyroxine, and on pituitary sensitivity to luteinising hormone-releasing hormone.

1. The ability of a moult-inducing procedure to restore high levels of sperm production was assessed, in two experiments, using cockerels with reduced sperm production. The moulting procedure consisted of a period of food and light restriction for 6 weeks. The moulted birds were compared with control birds for 20 weeks. 2. Moult induction resulted in decreased daily sperm output (DSO) and plasma testosterone concentration, from weeks 3 to 7. In the first experiment, plasma luteinising hormone (LH) concentration in moulted birds was reduced on week 5. 3. No change in pituitary sensitivity to chicken luteinising hormone-releasing hormone-I (cLHRH-I) was detected at week 3 in moulted cockerels. In moulted birds, a transient increase in plasma thyroxine concentration was detected. 4. After the end of moult induction, testosterone concentrations increased, plasma LH showed a rebound at week 7 and the pituitary sensitivity to LHRH was increased at week 9. 5. This increased activity of the pituitary-testicular axis resulted for a short time in an increase in DSO of moulted birds compared with that of controls. Although amelioration was moderate, this result indicates the possibility of improving sperm production in the cockerel by using an induced moulting procedure.

Effect of thyroxine on testicular function, circulating luteinising hormone and pituitary sensitivity to luteinising hormone-releasing hormone in the cockerel (Gallus domesticus).

1. The effect of thyroxine (T4) on reproductive function in the adult cockerel was followed for 11 weeks. Broiler cockerels aged 96 weeks were fed on diets containing either 0, 2 or 5 mg T4/kg for 4 weeks. 2. Daily sperm output (DSO) was significantly reduced (P < 0.01) in the T4-treated groups compared with that of controls at weeks 5 and 7. In the group given 5 mg T4/kg, plasma testosterone concentration was significantly reduced (P < 0.01) compared with that in controls during the T4 treatment, in spite of the fact that there was a decrease in concentration in both control and experimental birds. Plasma luteinising hormone (LH) concentration was significantly decreased (P < 0.05) in both of the groups given T4 treatments after 3 weeks. 3. Plasma testosterone concentrations and DSO had returned to control values at weeks 5 and 11 respectively, while plasma LH showed a transient but significant (P < 0.001) rebound after removal of thyroxine from the food. 4. In contrast to other variables, the pituitary responsivity to cLHRH-I injections, was not decreased during the feeding of the T4 diet but was, on the contrary, significantly increased (P < 0.05) during treatment with 5 mg T4/kg diet, and after the end of the treatment with 2 mg T4/kg diet. 5. These results provide some evidence for an inhibitory effect of large doses of T4 on the reproductive function in the adult cockerel. Although the possibility of a direct effect of T4 on the testes cannot be excluded, T4 is likely to act, at least in part, at the hypothalamo-pituitary level, and not through a reduction in the pituitary sensitivity to LHRH.