Production
– Genetics (sire selection, management, nutrition)
– Semen collection
– Semen evaluation
– Semen processing
– Cryopreservation
– Quality control
Sires selection
– Geneticly superior progeny proven sires:
– Semex Alliance (Canada)
– Alta Genetics (Canada)
– Preska (Slovenia)
– Krnjaca (Srbija)
Management of the sires
– Permanent quarantine system
– Regular health control
– Environment in compliance and according the regulations listed in Animal Protection and Welfare Law (Official Journal of Republic of Macedonia 113/2007)
Nutrition of the sires
– Quality and health control of the feedstuffs entering the facilities
– Quality and health control of the water entering the facilities
– Controled vitamine and mineral additives for stimulation of the spermatogenesis
– No additives of animal origin, listed as specific risk material, accordint the Reg. 1774/2002
Semen collection
– Mounting on natural phantom (other animal)
– Artificial vagina (Hannover type)
– Preheated at 42 ̊С
– Lubrificated with the extender
Semen evaluation
– Macroscopical evaluation
– Microscopical evaluation
Microscope evaluation
– Phase-contrast microscope (light field – Phase 1)
– Magnification 10 – 40 х
– Preheated stage at 39°С
– Microchamber for fresh semen evaluation
Macroscopical evaluation
– Volume
– Color
– Consistency
– Presence of foreign artifacts
Criteria
– Concentration: > 9 х 103/ml
– Proportion of motile spermatozoa: > 70%
– Absence of increased number of epithelial cells, leucocytes, erythrocytes
– Absence of foreign origin particles
Processing of the ejaculates
– Dilution (Suspension)
– Microscope evaluation after the dilution
– САЅА evaluation и and determination of the final concentration
– Packing and labeling
– Equilibration
Dilution
– Аndromed® (Minitub, Germany)
– Free of compounds from animal origin (according Reg. 1774/2002)
– Primary diludion ratio 1:1
– Dilution up to ratio 1 : 10
– Microscope evaluation
САЅА evaluation and determination of the final concentration
– HTM IVOS® (Hamilton Thorne Research, USA)
– Determination of the ratio of the final dilution
– Recording of the exach concentration of the ejaculate
– Recording of the proportion of motile spermatozoa
– Determination of the qualitative motility parameters of the spermatozoa
Packing and labeling
– MPP 133 Combo Jet (Minitub, Germanu)
– 0,5 ml medium-straws
– Labeling of the straws: sire’s name, ID No, designation of the AI centre, batch No.
Equilibration
– Cooling to +4°С
– Equlibration period 2 – 2,5 hours
Cryopreservation
– Automatic MT freezer for semen straws
Cryo-Curve
– from 20 to 4°С in 10 min, temperature slope -1,6°С/min
– at 4°С – equilibration in period of 2 hours
– from 4° to – 6°С in period of 2 min., temperature slope – 5°С/min
– at -6°С – stabilization in period of 1 min. crystallization – solidification
– from -6°С to -20°С in period of 1 min, temperature slope -14°С/min, to compensate the heath release during the crystallization
– from -20 to -140°С С in period of 4 min, temperature slope -30°С/min
– from -140°С to -196°С Direct submerging in LN2. (Mueller-Schlosser et al. 2000)
Control of produced AI doses
– Thawing (39°С, at least 30 sec.)
– Microscope evaluation (criteria > 50% motile spermatozoa)
– САЅА assessment and recording of the obtained results