Elisabeth Blesbois

Differences in ability of jennies and mares to conceive with cooled and frozen semen containing glycerol or not.

A suitable method for the cryopreservation of donkey semen would be very valuable for the ex situ management of genetic diversity in this species. This report uses a variety of observation and trials to evaluate the effect of cryoprotectants in per-cycle pregnancy rates (PC) in equids females (jennies (donkey) and mares (horse)). This was explored by (1) comparing the results of insemination of jennies and mares with cooled or frozen donkey semen, (2) examining the possible toxic effect of the cryoprotectant (CPA) glycerol in these two species and (3) studying alternative solutions. Donkey and horse semen was either used immediately, or cooled according to some steps of the pre-freezing procedure or frozen and thawed. The pre-freezing procedure included semen dilution, centrifugation, resuspension in milk or in INRA82+2% egg yolk+various % CPA (expressed as final concentrations in extended semen (v/v)) and then cooling to 4 degrees C. PC was similar in mares and jennies inseminated with donkey semen cooled to 4 degrees C in milk. However, the PC was significantly higher in mares than in jennies when donkey semen was frozen with 2.2% glycerol (36%, n=50 cycles vs. 11%, n=38 cycles; P<0.01). Increasing the concentrations of glycerol (0, 2.2, 3.5, 4.8%) before cooling stallion semen resulted in a progressive decrease in mare PC (87, 53, 53, 13% (n=15 cycles for each concentration); P<0.0001). The addition of 2.2% glycerol before cooling donkey semen decreased the PC measured in jennies to 0. The replacement of glycerol by 2% dimethylformamide increased the fertility obtained in jennies with cooled donkey semen (PC: 67%, n=12 cycles) but did not increase the fertility obtained with frozen-thawed donkey semen (PC: 11%, n=28 cycles with dimethylformamide vs. 0%, n=16 cycles with glycerol). In conclusion, this study clearly shows that the ability of jennies to conceive after AI with donkey frozen semen is lower than that of mares. Glycerol affects the fertility of donkey and stallion spermatozoa as early as during the pre-freezing procedure. In consequence, the glycerol level must be low in frozen equine semen to provide good fertility. The toxic dose of glycerol for donkey spermatozoa seems to be almost half that for stallion spermatozoa. Whether this greater sensitivity of donkey spermatozoa to glycerol is responsible for the low success of semen cryopreservation in jennies is not so obvious because replacement of glycerol by dimethylformamide was not much more effective in terms of fertility.

Expression and biological effects of bone morphogenetic protein-15 in the hen ovary.

The bone morphogenetic protein 15 (Bmp15) and growth differentiation factor 9 (Gdf9) genes are two members of the transforming growth factor-beta superfamily. In mammals, these genes are known to be specifically expressed in oocytes and to be essential for female fertility. However, potential ovarian roles of BMPs remain unexplored in birds. The aim of the present work was to study for the first time the expression of Bmp15 in the hen ovary, to compare its expression pattern with that of Gdf9, and then to investigate the effects of BMP15 on granulosa cell (GC) proliferation and steroidogenesis. We found that chicken Bmp15 and Gdf9 genes were preferentially expressed in the ovary. We showed using in situ hybridization that Bmp15 and Gdf9 mRNAs were specifically localized in oocytes of all ovarian follicles examined. We also demonstrated using real-time quantitative RT-PCR that Bmp15 and Gdf9 expression was maintained during hierarchical follicular maturation in the gerrminal disc region and then progressively declined after ovulation. BMP15 was able to activate Smad1 (mothers against decapentaplegichomolog1) signaling pathway in hen GCs. Moreover, we showed a strong inhibitory effect of BMP15 on gonadotropin-induced progesterone production in hen GCs. This inhibitory effect was associated with a decrease in steroidogenic acute regulatory protein (STAR) level. Taken together, our results suggest that BMP15 may have a key role in the female fertility of birds.

Cryoprotectant and freezing-process alter the ability of chicken sperm to acrosome react.

Sperm surviving after freezing-thawing is usually 40-50% of the initial population. Damage during this process affects both fertilizing ability and its duration in avian species. However, the effect of cryopreservation on the sperm ability to undergo the acrosome reaction, the initial event of fertilization, is still in question in birds. In this paper, the influence of cryoprotectant (glycerol and dimethylacetamide-DMA) and of two different cryopreservation processes (pellets or straws) on the ability of rooster sperm to acrosome react (AR measured with PNA-FITC) was studied. Motility parameters (CASA) and plasma membrane integrity (propidium iodide exclusion) were also measured. The addition of cryoprotectants (CPA) immediately provoked a dramatic decrease in the ability of sperm to undergo the acrosome reaction, glycerol being more harmful than DMA. The cryoprotectant removal was also harmful. The other parts of the freezing process further decreased the ability to AR. Motility was affected to a lesser extent by CPA presence although plasma membrane integrity was not altered. The DMA/rapid freezing procedure was the most harmful for plasma membrane integrity. Taken together, these results show that AR is more dramatically altered by CPA presence than motility and membrane integrity and CPA provokes a more pronounced effect than the freezing-thawing process especially in the case of using glycerol/slow freezing process.

Temperatures from 4 to 15 °C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h or more in INRA96 extender

This study tested whether variable temperatures (from -0.5 to 15 °C) and air exposure could be used under laboratory and under field conditions to store stallion sperm diluted in extender INRA96 without loss of fertility. Experiment 1 (laboratory conditions) measured the effects of two 72 h storage conditions (5 °C with air vs. 15 °C without air). Experiment 2 (fixed field conditions) measured the effects of 22 h of storage without air in disposable containers maintained at four ambient temperatures (7 °C, 17 °C, 27 °C, 39 °C with semen at -0.5 °C to 3 °C, 4 °C to 7 °C, 8 °C to 10 °C, 12 °C to 15 °C, respectively). Per cycle pregnancy rate (PC) was measured after one artificial insemination (AI) in uterine body of 200×10(6) total spermatozoa, 7 h (Experiment 1) or 17 h (Experiment 2) before ovulation. In Experiment 1, PC was similar for both conditions (60% (n=40 cycles) vs. 63% (n=40), respectively, 5 stallions×8 cycles). Only velocity VCL and ALH were slightly higher at 15 °C. In Experiment 2, PC was reduced when ambient temperature was low (semen at -0.5 °C to 3 °C; PC=25%) rather than intermediate (semen at 4 °C to 7 °C; PC=53%) or high (semen at 8 °C to 10 °C; PC=50%) (4 stallions×8 cycles) (P=0.002). Sperm stored at -0.5 °C to 3 °C had lower acrosome integrity/responsiveness, similar membrane integrity (HOS test) and motilities, and higher VCL and ALH, than semen stored between 4 and 15 °C. These results demonstrate a wide tolerance of equine sperm to variable positive temperatures and air exposure for 22 h storage and more. However, temperatures close to 0 °C are detrimental for fertility.

Characterization of red-legged partridge (Alectoris rufa) sperm: Seasonal changes and influence of genetic purity

The general decline in wild Iberian populations of the red-legged partridge (Alectoris rufa) has been accompanied by an increase in game-farm facilities producing hybrids with chukar partridges (Alectoris chukar). Genetic introgression from chukar partridges is thought to modify male red-legged partridge reproductive indicators. The aim of the present study was to determine the effects of such genetic introgression on seasonal reproductive patterns by comparing the sperm and plasma testosterone concentrations of males from pure red-legged and hybrid red-legged/chukar populations. Semen was collected twice monthly over a 12-mo period using a massage technique. Both types of bird showed a clear seasonal pattern of spermatogenic activity. The proportion of males ejaculating sperm was higher (P<0.05) among the pure red-legged birds. The greatest sperm production was recorded in March to May among the pure birds and April to May among the hybrids. Reproductive activity in both groups decreased in June, to reach a minimum in August to December among the hybrids and in September to December among the pure birds. Spermatogenic activity resumed in January in both groups. The sperm concentration produced by the pure birds was smaller than that of the hybrids (P<0.001), but the percentage of motile sperm was higher in the pure birds (P<0.001). The sperm of the hybrids showed greater straight-line velocity (P<0.05), linearity (P<0.001), straightness (P<0.001), sperm wobble (P<0.05), and beat-cross frequency values (P<0.001). The length and area of the sperm head were smaller in the pure birds (P<0.05). The seasonal plasma testosterone concentration pattern followed a trend roughly parallel to the ejaculatory response. The present results suggest that genetic introgression influences the reproductive variables of the red-legged partridge.

Semen characteristics of the Indian Red Jungle Fowl (Gallus gallus murghi)

The Indian Red Jungle Fowl is a wild native gallus subspecies of Southern Asia. Semen has never been studied in this species. In order to better know the male reproductive capacities, experiments were conducted to study the semen characteristics, impact of ejaculate collection frequencies, and timing of collection on sperm quality parameters. Mean sperm concentration 800 million/mL, total sperm per ejaculate (0.015 billion), motility (63.5xa0%), live/total sperm (92.4xa0%), intact acrosome (75.5xa0%), and plasma membrane integrity (89.2xa0%) were recorded. Percentage of abnormal sperm (head, mid-piece, and tail) was 8.1xa0% and recovered mainly mid-piece abnormalities. The motile sperm percentage was positively correlated with intact acrosomes (ru2009=u20090.34) and plasma membrane integrity (ru2009=u20090.41). Total sperm per ejaculate (billion) was maximum at 72xa0h of collection followed by 24 and 48xa0h of collection. Daily and weekly sperm production (billion) was found maximum at 24xa0h of collection compared to 12, 48, and 72xa0h of collection. Sperm motility was higher at 24, 48, and 72xa0h of collection compared to 12xa0h of collection, but the number of live sperm were higher at 12xa0h of collection compared to 24, 48, and 72xa0h. Sperm concentration was better in the morning time, while the values for sperm viability and plasma membrane integrity were higher in the semen collected at evening time. In conclusion, the Indian Red Jungle Fowl shows a semen production quantitatively relatively low for the species as compared to domestic chicken and contrasted parameters of quality. The semen production is affected by the frequency of collection with an optimum for a daily collection preferentially held in the evening period. These results may now be used for artificial insemination and conservation program.

Identification of Germinal Disk Region Derived Genes Potentially Involved in Hen Fertility

Despite the regular decrease in fertility observed in hens, especially in “meat” lines, little is known about genes affecting fertility. We used the Affymetrix microarray to search for oocyte genes whose expression would vary in relation to fertility rate in both “laying” and “meat” line hens. We focused on oocyte genes because several of them have been found to be involved in fertility in other species. Based on microarray analysis, 54 and 84 genes were differentially expressed between germinal disc regions (GDR) of F1 maturation stage oocytes from hens exhibiting either high (100%) or low (from 22% to 80%) fertility rate from laying and meat lines respectively. Most of these differentially expressed genes were distributed between “laying” and “meat” lines indicating that mechanisms involved in the decrease in fertility rates in these two cases were independent. Real time RT‐PCR performed on the same samples which were used for microarray confirmed in several cases differences in gene expression levels detected by microarray. Moreover the correlations between gene expression levels and fertility rates were evaluated for the 10 most interesting genes at different stages of follicular maturation and early embryo development on individual GDR samples from hens exhibiting different fertility rates. In total, we identified five genes whose expression levels correlated with fertility rate in accordance with findings of microarray analysis and real time RT‐PCR: VWC2, CR407412, TAPA, FGL2, and TRAP6. The biological significance of these genes sheds light on potential mechanisms influencing fertility and could provide candidates for fertility markers in the hen. Mol. Reprod. Dev. 76: 1043–1055, 2009.

Successful use of artificial insemination in the production of red-legged partridges (Alectoris rufa)

This paper reports the first successful artificial insemination (AI) of red-legged partridges (Alectoris rufa), the fertility rate achieved, and the length of time sperm cells can survive inside the oviduct (i.e., the post-AI fertile period). Semen from 20 mature males was collected by massage and pooled for use in single intravaginal inseminations (20xa0μL of fresh, undiluted semen) of eight females (15u2009×u2009106 sperm/female). The latter’s eggs were then collected for 4xa0weeks, and the fertilizing capacity of the sperm used in the preceding AIs was determined by observing the development of the blastoderm. The duration of the post-AI fertile period was determined by subjecting fertilized eggs to the SP-holes assay. A second experiment was then performed to measure the percentage of viable embryos at 20xa0days of incubation (30xa0%) and hatchability (40xa0%). The mean fertility rate was 34.5u2009±u200911.7xa0% and the SP-holes value 17.3u2009±u20094.3. The mean duration of the post-AI fertile period was 3xa0weeks. In conclusion, the present work reports the first ever birth of partridge chicks following AI and shows that this procedure may be of use in the conservation of this species.

Successful chilling of red-legged partridge (Alectoris rufa) sperm for use in artificial insemination

ABSTRACT The fertilizing capacity of pure, fresh avian semen may disappear in just half an hour, hindering its successful use in artificial insemination (AI) projects. Longer storage requires the use of infra‐physiological temperatures and of semen diluents that help preserve the spermatozoa but that do not interfere with their fertilizing capacity. This study examines the effect on sperm quality of storing red‐legged partridge sperm for 3 h at 5°C with 2 different semen extenders: 1) a medium referred to as L&R‐84, composed of sodium glutamate, glucose, magnesium acetate, potassium acetate, and polyvinylpyrrolidone, and 2) Lake 7.1 medium, composed of sodium glutamate, glucose, magnesium acetate, potassium citrate, and N,N‐Bis(2‐hydroxyethyl)taurine (BES). Extending with L&R‐84 returned better curvilinear velocity (P < 0.01), straight‐line velocity (P < 0.01), average path velocity (P < 0.01), linearity (P < 0.05), straightness (P < 0.05), and wobble (P < 0.05) values, while extending with the Lake 7.1 medium was associated with higher percentages (P < 0.001) of motile sperm. The fertility rate was higher (P < 0.05) when birds were inseminated with L&R‐84‐extended sperm than with Lake 7.1‐extended sperm. The mean number of penetrations of perivitelline layer samples (taken from above the germinal disc) was also higher for the L&R‐84‐extended sperm (P < 0.05). These results show L&R‐84 can be recommended as an extender for red‐legged partridge semen to be stored for at least 3 h at 5°C.

Access to pasture in an outdoor housing system affects welfare indicators and improves rooster sperm quality in two native Mediterranean breeds

The aim of the present work was to examine the influence of access to pasture in an outdoor housing system on rooster sperm quality and response to cryopreservation and to examine the possible correlation between values for sperm quality variables and welfare indicators. Two groups of Black-barred Andaluza and Red-barred Vasca roosters were housed in an outdoor system, with one group given daily access to a grazing area containing plant species that typically grow on uncultivated Mediterranean land. Semen was collected once per week from each group, and the following sperm quality variables were assessed: sperm volume, appearance, concentration, motility, membrane integrity, acrosome integrity, and morphological abnormalities. In addition, two welfare indicators were examined: the heterophil/lymphocyte (H/L) ratio, and the duration of tonic immobility (TI). Ejaculates from the birds with access to pasture had higher percentages of sperm showing progressive motility (P = 0.019), and returned a higher motility index (P = 0.035). Unexpectedly, the H/L ratio was also higher in these birds. Virtually no differences were seen between the treatment groups with respect to sperm quality after freezing-thawing, although the semen of the Red-barred Vasca birds with access to pasture did show a higher percentage of progressive motility (P = 0.023) than the birds of the same breed with no such access. Significant correlations were detected between the H/L ratio and sperm motility (rxa0=xa00.420, P = 0.038), the sperm motility index (r = 0.526, P = 0.002), and progressive motility (rxa0=xa00.467, P = 0.003). No differences were seen between the treatment groups with respect to the duration of TI. In conclusion, access to pasture improved fresh sperm motility.Abstract The aim of the present work was to examine the influence of access to pasture in an outdoor housing system on rooster sperm quality and response to cryopreservation and to examine the possible correlation between values for sperm quality variables and welfare indicators. Two groups of Black‐barred Andaluza and Red‐barred Vasca roosters were housed in an outdoor system, with one group given daily access to a grazing area containing plant species that typically grow on uncultivated Mediterranean land. Semen was collected once per week from each group, and the following sperm quality variables were assessed: sperm volume, appearance, concentration, motility, membrane integrity, acrosome integrity, and morphological abnormalities. In addition, two welfare indicators were examined: the heterophil/lymphocyte (H/L) ratio, and the duration of tonic immobility (TI). Ejaculates from the birds with access to pasture had higher percentages of sperm showing progressive motility (P = 0.019), and returned a higher motility index (P = 0.035). Unexpectedly, the H/L ratio was also higher in these birds. Virtually no differences were seen between the treatment groups with respect to sperm quality after freezing‐thawing, although the semen of the Red‐barred Vasca birds with access to pasture did show a higher percentage of progressive motility (P = 0.023) than the birds of the same breed with no such access. Significant correlations were detected between the H/L ratio and sperm motility (r = 0.420, P = 0.038), the sperm motility index (r = 0.526, P = 0.002), and progressive motility (r = 0.467, P = 0.003). No differences were seen between the treatment groups with respect to the duration of TI. In conclusion, access to pasture improved fresh sperm motility.