Flavia Pizzi

The combined use of embryos and semen for cryogenic conservation of mammalian livestock genetic resources

The objective of this empirical simulation study was to evaluate the use of a combination of semen and embryos in the creation of gene banks for reconstruction of an extinct breed. Such an approach was compared for banks with varying proportions of embryos on the basis of the amount of the material to be stored, time for reconstruction, maintenance of genetic variability, and probability of failure during reconstruction. Four types of populations were simulated, based on reproductive rate: single offspring, twinning, enhanced reproduction, and litter bearing. Reconstruction was simulated for banks consisting of different combinations of semen and reduced numbers of embryos (expressed as a percentage of the material needed for a bank containing exclusively embryos and ranging from 10 to 90%). The use of a combination of semen and embryos increased the number of insemination cycles needed for reconstruction and the level of genetic relatedness in the reconstructed population. The risk for extinction was unacceptably high when a very low proportion of embryos (< 20%) was used. However, combining semen with embryos could decrease costs, allowing for the conservation of more breeds, and specific strategies for semen use could decrease the level of relationships in the reconstructed breed.

The costs of breed reconstruction from cryopreserved material in mammalian livestock species

The aim of this work was to compare costs, in the horse, cattle, sheep, swine, and rabbit species, for the creation of gene banks for reconstruction of an extinct breed, using different strategies: embryos-only, embryos in combination with semen, and semen-only. Three cost measures were used: time required for population reconstruction, cost for creation of the gene bank, number of years-keeping-female to reach reconstruction. Semen costs were estimated across four scenarios: the presence or absence of a commercial market for semen, purchase of semen donors, and semen extracted from the epididymus. The number of cells were doubled to take into account the creation of two storage sites. The strategy embryos-only required the shortest time to reach reconstruction. With the strategy embryos + semen, time increased with decreasing proportions of embryos. With semen-only, reconstruction time varied from 2 to 21 years. A high variation of costs was observed across species and strategies, from 360 Euros in the rabbit to 1 092 300 in the horse. In all species, the embryos-only strategy was about 10% more expensive than using 90% embryos + semen. Decreasing the percentage of embryos further diminished costs. The number of years-keeping-female ranged across strategies, from 2 in the rabbit, to a maximum of 12 878 in the horse.

In vitro toxicity of mercuric chloride on rabbit spermatozoa motility and cell membrane integrity

In this in vitro study the effects of mercuric chloride on the motility and structural integrity of rabbit spermatozoa were investigated. The spermatozoa motility was evaluated using CASA method and Annexin analysis was used for detection of structural changes. The concentration of mercury in the medium varied from 5.0 to 83.3 μ g HgCl2/mL. At Time 0 the highest motility was detected in the control group (67.09 ± 8.72%). Motility in groups with mercury administration was lower in comparison with control. Significant differences were detected in groups with 50.0–83.3 μ g HgCl2/mL (P < 0.001) at Time 0. After 60 and 120 minutes of incubation with mercuric chloride the motility significantly decreased almost in all experimental groups. Progressive motility had a decreasing trend in all experimental groups. At time 60 and 120 significant differences were noted in the group receiving 6.25–83.3 μ g HgCl2/mL. Significant differences were detected in all experimental groups, except the group with the lowest mercuric chloride administration. The concentration-dependent decrease of spermatozoa progressive motility up to 50% of control was detected for groups receiving 50.0 – 83.3 μ g HgCl2/mL at Time 0, for groups receiving 12.5–83.3 μ g HgCl2/mL at Time 60 and 120, decreasing from 36.46 ± 18.73% to 1.03 ± 2.50%. Detailed evaluation of spermatozoa distance (DAP, DCL, and DSL) and velocity (VAP, VCL, and VSL) parameters as well as straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) of spermatozoa revealed decrease in groups with the highest mercury concentration in comparison with the control group at all time periods. Detection of spermatozoa with disordered membrane was carried out for groups with higher mercury concentrations and control, using Annexin analysis. Analysis showed higher occurrence of positive spermatozoa in the mercury exposed groups. Some Annexin positive reactions from all spermatozoa were detected in the control group. In mercury-exposed groups positive reaction proved alteration in anterial part of head (acrosome), connection part (connection piece) and in mitochondrial segment. Detected data evidently confirm adverse effects of high mercuric chloride concentrations in rabbit semen on spermatozoa motility parameters.

Effect of testicle postmortem storage on goat frozen-thawed epididymal sperm quality as a tool to improve genebanking in local breeds.

The interest to develop assisted reproductive technologies and cryobanking for farm animal genetic resource conservation has recently increased. However, cryopreservation for ex-situ management of genetic diversity sometimes is not routinely feasible, owing to the lack of facilities (AI centres, laboratories) and expertise near the local breed farming area. In these cases, epididymal sperm obtained from slaughtered or castrated animals, associated with the possibility of managing rather long periods between animal death, sperm recovery and freezing, would increase the opportunities to create semen storages. This investigation addresses the pre-freeze/post-thaw quality of goat epididymal sperm as a function of testicle storage temperature (environment or +5°C) and time elapsed between animals death and sperm recovery (0, 24, 48, 72 h) to establish the optimal protocols for the recovery and cryopreservation of epididymal sperm in this species. Testicles of 50 mature bucks collected at the abattoir were divided in two groups: half of the testicles (n=50) were transported to the laboratory at environment temperature (E), whereas the remaining half (n=50) at a refrigeration temperature (R) of +5°C. In the two groups (E) and (R), one testicle from each pair was processed after slaughter forming the time 0 groups (0E and 0R). The contralateral testicle was processed after 24, 48 or 72 h of storage, at the corresponding temperature. Sperm motility and kinetic parameters, viability and morphology were assessed in pre-freeze and post-thaw samples. Until 48 h postmortem, both E and R temperatures are able to maintain good pre-freeze epididymal sperm quality. After 48 h postmortem, R temperature is fundamental to reduce epididymal sperm quality decay in pre-freeze samples. Moreover, testicle refrigeration also has a positive impact on post-thaw samples, allowing a lower decline through time considering total motility, kinetics parameters, sperm viability and sperm abnormalities. Therefore, when sperm cryopreservation is not immediately practicable, goat testicles should be transported and stored at 5°C up to a maximum of 48 h postmortem to ensure an acceptable sperm quality.

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.

Involvement of tyrosinase-related protein 1 gene in the light brown plumage phenotype of Falco cherrug.

Mammoth Jack and Poitou) and two mules from various studs in the USA and Australia, including 54 solid, 34 spotted and three cream-white animals (Fig. S1). Genotypes for the c.1978+2T>A variant were determined by PCR and direct sequencing of PCR products as previously described. All 54 solid-coloured donkeys, as well as the two solidcoloured mules, were homozygous for the wild-type T allele. Of the 34 spotted animals, 30 were heterozygous and four individuals were homozygous wild-type. Reviewing the phenotype of those four individuals indicated that three exhibited a dappled roan coat colour phenotype (Fig. S1d). This result indicates that dappled roan needs to be distinguished from spotting and that other genetic factors are responsible for this colour phenotype. The fourth animal had a white spot only on the face. Although white facial markings are believed to be the minimal expression of spotting in donkeys, this indicates that more than one type of spotting caused by differing genetic mechanisms is apparent in donkeys. All three cream-white animals were heterozygous. Cream-white could be an extreme form of white spotting, although it is also possible that it is the result of a dominant colour-masking phenotype caused by an as yet unknown genetic variant. In conclusion, the results of this study enhance the evidence that the previously identified variant is associated with white spotting in donkeys. Because the splice site variant was not observed in the homozygous state, this result also supports the hypothesis that homozygosity for this genetic variant is incompatible with life. The presence of the variant in donkeys of different breeds (Miniature Donkey and Mammoth Jack) suggests that the variant probably arose prior to breed separation.

Implementation and cost analysis of a regional farm animal cryobank: an Italian case study

Abstract Scientific and technical reports on farm animal genetic resources (AnGR) cryoconservation activities, and related costs, are needed to optimise conservation programmes. In this paper, we presented the recent Italian AnGR gene banking development, including the creation of the first regional animal cryobank, the ‘Lombardia Farm Animal Genetic Resources Cryobank’ (LABank). In order to provide indications on cryobanking costs, a detailed analysis of the expenses incurred during the creation of LABank was carried out. Currently, in LABank genetic material (spermatozoa, blood and hair bulbs) of Italian cattle, sheep and goat local breeds is cryopreserved, for a total amount of approximately 2500 semen doses collected from 46 donors of five local breeds. The costs incurred by creating the semen storage showed differences among species and semen collection procedures, providing indications to enhance the setting up of regional cryobanks.