Digit cool

Mature Holstein-Friesian bulls with high (HF; n = 10) or low (LF; n = 10) fertility were selected from a population of 1,665 AI bulls. Bull fertility was based on adjusted fertility scores (26 (link)), calculated from a record of at least 500 inseminations (mean = 13,292, min = 519, max = 100,288). HF bulls showed an average adjusted fertility score of +6.5%, whereas LF bulls showed an average fertility score of −6.6%. Sire fertility was defined as pregnancy to a given service identified retrospectively either from a calving event or where a repeat service (or a pregnancy scan) deemed the animal not to be pregnant. These raw data were then adjusted for factors including semen type (frozen, fresh), cow parity, month of service, day of the week when serviced, service number, cow genotype, herd, AI technician, and bull breed. The adjusted sire fertility index given for each bull was then weighted for the number of service records, resulting in an adjusted calving rate. The mean of the population was zero. Semen was collected at two AI centers in Ireland via an artificial vagina, frozen in 0.25 ml straws using a programmable freezer (Digitcool, IMV Technologies, L'Aigle, France) and stored in liquid nitrogen pending further analysis. All ejaculates passed quality control checks for motility (post thaw motility of >50% as assessed subjectively) and morphology (>70% normal sperm).

Initially, samples were equilibrated in a commercial extender at 22 °C (Bioxcell; IMV Technologies, L’Aigle, France), adjusting the concentration to 92 × 10⁶ sperm/mL. Cryopreservation was conducted following a standard procedure described previously [31 (link)]. The mixture was then cooled to 4 °C at a rate of − 0.2 °C/min, and equilibrated at this temperature for 3 h. At this point, sperm were packed into 0.25-mL straws, and the following cooling rates were applied using a controlled-rate freezer (Digit-Cool; IMV Technologies): 5 °C/min from 4 °C to − 10 °C; 40 °C/min from − 10 °C to − 100 °C; and 20 °C/min from − 100 °C to − 140 °C. Finally, straws were stored in liquid nitrogen until use. Thawing was performed through immersion of straws at 38 °C for 20 s in a water bath.

Five separate samples from five healthy and sexually mature Holstein bulls were used. All animals were kept in an artificial insemination center (Cenero-Asturias, Gijón, Spain), and ejaculates were collected through an artificial vagina. Collected semen was diluted in Bioxcell extender reaching a concentration of 92 × 10⁶ sperm/mL. Then, samples were cooled down to 4°C at a rate of −0.2°C/min, and after 3 h of equilibration, extended semen was packaged into 0.25-mL straws containing 23 × 10⁶ sperm/straw.
Cryopreservation was performed in a programmable freezer (Digit-cool; IMV Technologies, L’Aigle, France) following the standard curve for bovine sperm: −5°C/min from 4 to −10°C, −40°C/min from −10 to −100°C, and −20°C/min from −100 to −140°C. Thawing was performed by submerging straws in a water bath at 38°C for 40 s, with shaking.