The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull semen.

Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R 2=0.47, P<0.05) of the variation in the conception rate and included nine variables: five kinetic parameters measured by CASA (total motility, active cells, beat cross frequency, curvilinear velocity and amplitude of lateral head displacement) and four parameters related to DNA integrity evaluated by FCM (degree of chromatin structure abnormality Alpha-T, extent of chromatin structure abnormality (Alpha-T standard deviation), percentage of DNA fragmented sperm and percentage of sperm with high green fluorescence representative of immature cells). A significant relationship (R 2=0.84, P<0.05) was observed between real and predicted fertility. Once the accuracy of fertility prediction has been confirmed, the model developed in the present study could be used by artificial insemination centers for bull selection or for elimination of poor fertility ejaculates

DNA fragmentation in chicken spermatozoa during cryopreservation

Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at -196 \ub0C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 \ub0C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 \ub0C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 \ub0C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation

changes in sperm quality and lipid composition during cryopreservation of boar semen

The effect of cryopreservation on boar sperm viability, motility, lipid content and antioxidant enzymatic activities was studied. Three classes of semen were determined according to a cluster analysis on the basis of the proportion of live and dead cells after freezing and thawing. The classes identified were: high (H, n = 4), average (A, n = 12) and low (L, n = 3) viability. The concentration of sperm cells decreased from class H to A to L. Fresh semen samples with higher viability and a higher proportion of motile cells also maintained better quality after the freezing and thawing procedure. Sperm viability and motility in both fresh and thawed samples were similar in classes H and A, while significantly lower values were measured in class L. The relative decrease in sperm viability and motility after cryopreservation increased from class H to A to L. The lipid content of spermatozoa (\u3bcg per 109 cells) increased significantly after freezing and thawing in classes H and A but not in class L. This result indicated that active sperm lipid metabolism might be responsible for the increase in lipid content. Phospholipid and triacylglycerol contents increased whereas free cholesterol content decreased after thawing. The fatty acid composition of fresh spermatozoa was similar in all three classes. The proportion of polyunsaturated fatty acids decreased significantly after freezing and thawing, indicating contamination from the diluent or peroxidation. After freezing and thawing, superoxide dismutase activity in spermatozoa was significantly higher in class L than in classes H and A, which did not differ from each other

Spermatozoa DNA and plasma membrane integrity after pellet optimized processing for cryopreservation in meat type chicken breeders

1. Aim of this study was the development of an optimised cryopreservation pellet procedure for chicken semen and the assessment of DNA and membrane integrity in frozen/thawed spermatozoa in a Hubbard F15 meat type selected strain.2. The following semen processing conditions were studied: spermatozoa working concentration (SWC), 1.5 vs 2x10(9) cells/ml in pre-freezing extender; equilibration of diluted semen at 5 degrees C, 20 vs 40min; dimethylacetamide concentration, 6% vs 9%; dimethylacetamide equilibration time at 5 degrees C, 1 vs 30min; thawing at 60 degrees C for 10 vs 50 degrees C for 30sec. Spermatozoa viability (EtBr exclusion procedure - stress test), mobility (Accudenz (R) swim-down test) and subjective motility were assessed in fresh and frozen-thawed semen.3. The lower SWC (1.5 x 10(9) cells/ml) and the higher dimethylacetamide concentration (9%) had positive significant effects on the recovery rate of motile (22% vs 16%) and viable spermatozoa (39 vs 34%), respectively.4.Membrane (SYBR14-PI staining) and DNA integrity (comet assay) were assessed before and after freezing/thawing according to the optimised protocol.5. Recovery rates of spermatozoa with undamaged plasma membrane and DNA were 41% and 76%, respectively. The distribution of spermatozoa in classes of DNA damage was also analysed and discussed.6. It was concluded that pellet cryopreservation was a damaging process mainly for plasma membrane rather than nuclear DNA in chicken spermatozoa

Influence of recovery methods and extenders on bull epididymal spermatozoa quality

The aim of this study was to compare the effects of two extraction methods in combination with two different extenders in bull epididymal sperm collection. Testes from 23 sexually mature Limousine bulls were collected at the abattoir. Epididymal sperm recovery was performed using both the float-up (FL) and the retrograde flushing (RF) technique. Within extraction methods, half testes were processed with a Tris egg yolk extender and half with a Tris egg yolk-free extender. Sperm concentration, motility, viability and morphology were evaluated. Sperm concentration was not significantly different between methods. Flushing technique was significantly better than the FL method in terms of sperm quality, considering total motility (80.3 \ub1 2.3% vs 71.6 \ub1 2.0%, p < 0.001, respectively) and viability (84.5 \ub1 1.5% vs 77.2 \ub1 1.3%, p < 0.001, respectively). Egg yolk influenced positively motility and morphology in the FL method, whereas decreased viability in flushed samples. Results suggest the use of the RF technique to collect cattle epididymal sperm