The Seminology sector is mainly dedicated to guarantee an andrologic support to animal reproduction in livestock through the specific skills in the sperm quality evaluation and in statistic control of the quality including the training of the technicians. The lab activity is subdivided into three main areas related to sperm analysis, cryopreservation and flow cytofluorimetry, where modern tools such as computerized analysis, flow cytometry and programmable auto-freezers, are used.
The sperm quality evaluation conducted with the help of technologies aimed at ensuring the objectivity and repeatability of the analytical data becomes essential both from technical and managerial points of view.
Semen analysis
Standard Analysis
Sperm concentration and membrane integrity
The concentration, i.e. the evaluation of the total sperm numbers per unit volume, is one of the essential parameters for quality determination either for fresh, chilled or frozen semen.
The analysis of the membrane integrity represents one of the main requirements for the definition of the spermatozoon standard functionality.
Sperm concentration and membrane integrity are determined by an instrument equipped with a fluorescence microscope in which are integrated a sampler and an image analysis system.
Motility
The motility, assessed as the percentage of motile spermatozoa, allows to calculate the essential kinetic variables for the quality definition of the sperm, either fresh, chilled or frozen.
For the fresh semen, it contributes to the definition of “normal” ejaculates, both for the estimation of the stallion fertility that for the production of frozen or refrigerated semen doses. For the refrigerated and / or frozen sperm, it contributes to the definition of the usability of the fertilizing doses.
The motility is assessed through the computerised video micrographs using CASA systems (Computer Assisted Sperm Analysis) HTM-IVOS vs. 12.3 and HTM-IVOS vs. 14.0 (Hamilton –Thorne)
The equipment has an integrated microscope, with a strobe source of lighting and with two (negative phase contrast and fluorescence) 10x a lenses
The classification of the spermatozoa is based on their speed in movements as follows:
The total motility: the spermatozoa are considered mobile when the VAP exceeds an established number according to the set configurations and which may differ according to the species.
The progressive motility: this is evaluated excluding from the mobile number of spermatozoa those that have VSL/VAP under 0.75 : THRESHOLD STRAIGHTNESS (75%).
The average speed of the movement is the average of all VAP of all spermatozoa classified as mobile.
Cytomorphology
The morphological evaluation requires the cytomorphologic analysis of spermatozoa with the particular reference to the main components involved in the fertility process: the head, acrosome and the tail.
The analysis is carried out by Differential Interference Contrast (DIC) microscopy on a fixed semen sample in suspension.
Advanced functional analysis:
Advanced functional analyses are conducted by flow cytofluorimetry in combination with fluorescence dyes.
The cytofluorimeter EasyCyte Plus Millipore is basically composed of four main systems: fluidic, optical, electronic and a management software.
The parameters determined by cytofluorimetry are the following:
The membrane integrity
The method is based on the triple staining of the sperm by three different fluorophores. The triple staining allows the simultaneous evaluation of the membrane and acrosome integrity.
The membrane fluidity
The information regarding the membrane fluidity assessed before and after freezing may supply a useful marker aimed at the definition of the semen freezing capacity providing the information related to the membrane changes
Mitochondrial functionality
At a mitochondrial level, the spermatozoa are able to independently generate energy by ATP production through aerobic or anaerobic metabolism, which is fundamental for sustaining sperm motility.
The damage at the mitochondrial level may lead to the cell immobilization. Such an evaluation provides data related to metabolic status of the cell.
The status of the spermatic chromatin.
Advanced analytical techniques have allowed to note a strong relation between the fragmented chromatin and infertility, both in a human and zootechnical field.
For this reason, the definition of this parameter is fundamental for the definition of an advanced analytical protocol.
Freezing
The Cryobiology lab develops various protocols to establish the freezability of the sperm in order to provide an estimate of the yield of freezing of the ejaculates of individual sires, and to develop the best technological approach to get it.
The sperm freezing is carried out by programmable auto-freezers.
Cytogenetics
The main service offered by the Institute in this research area relates to the andrological evaluation of young bulls present in the Genetic Centres.
The aim of these analyses is to avoid the transmission of chromosomal abnormalities during breeding.
The cytogenetic screening has become of considerable importance on a practical level, making it possible to almost totally eliminate some chromosomal abnormalities, e.g. the translocation 1/29 which has strong implications on fertility, the freemartinism and the Klinefelter Syndrome, pointed out more than once by the Institute among research subjects of zootechnical and reproductive interest.
Calibration and Inspection of the measuring instruments
Calibration service and inspection of the instruments used in the semen production centres in order to optimize and standardize their efficiency.
• Weighing scales:
Control by means of certificated weights
• Automatic Micropipettes:
Control of the Taring on the basis of weights consisting of predefined volumes of twice distilled water at a predefined temperature using analytic scales calibrated by certified weights
• Spectrophotometers:
Calibration by means of a study of linear regression which aims at judging the relation between the significant spectrophotometric readings and the concentrations obtained using the Nucleocounter SP100 as the instrument of reference.
Verification of the already existing taring curves using the Nucleocounter SP100
• CASA systems
Standardisation and optimisation of the analytic methodologies, both in the preparation of the sample which is to be analysed and in the parametrisation settings of the machines which are necessary for their proper functioning
• Equipment like refrigerators, refrigerator-thermostats, thermostats and thermostatic plates, etc.
Control and monitoring of the work temperatures using certified digital thermometers equipped with diversified probes (both liquid and solid).
Support of the productive lines
• development of software applications created to simplify the controlling of the reproduction within the herd and the optimisation of the use of the breeders by identifying in advance possible situation of fertility risk.
• development, definition and optimisation of the Standard Operative Procedures (SOP) as a means of support of the entire analytic-productive process
• training and informing operating technicians in the productive centres by means of specific courses organised in the ISILS and/or the productive centres.
These services are, in some cases, to be linked to the research activities carried out in the Institute, in other cases (e.g. in the estimation of fertility) they are carried out directly in the companies according to the agreement.