Identification of sperm subpopulations with defined motility characteristics in ejaculates from Holstein bulls: effects of cryopreservation and between-bull variation.
The aims of the present study were: (1) to determine the existence of sperm subpopulations with specific motility characteristics in fresh ejaculates from Holstein bulls, (2) to investigate the effects of semen cryopreservation and post-thaw incubation on the distribution of spermatozoa within the different subpopulations, and (3) to evaluate the existence of between-bull variation in the sperm subpopulations structure of fresh and frozen-thawed semen. Six ejaculates were collected from each of 9 Holstein bulls and cryopreserved following a standard protocol. Overall sperm motility and the individual kinematic parameters of motile spermatozoa, determined using a CASA system, were evaluated before freezing and after 0, 2 and 4h of post-thaw incubation at 37 degrees C. Data from 16,740 motile spermatozoa, defined by VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF, were analysed using a multivariate clustering procedure to identify and quantify specific subpopulations within the semen samples. The statistical analysis clustered all the motile spermatozoa into four separate subpopulations with defined patters of movement: Subpopulation (Subp. 1) moderately slow but progressive spermatozoa (23.2%), (Subp. 2) highly active but non-progressive spermatozoa (16.0%), (Subp. 3) poorly motile non-progressive sperm (35.5%), and (Subp. 4) highly active and progressive sperm (25.3%). Subpopulations 2 and 4 significantly (P<0.01) decreased during cryopreservation and post-thaw incubation (Subp. 2: 21.1%, 18.1%, 8.7% and 5.9%; and Subp. 4: 34.1%, 20.6%, 15.2% and 7.3%, respectively, for fresh, 0, 2 and 4h post-thaw) whereas Subp. 3 significantly (P<0.01) increased (10.7%, 27.2%, 27.2% and 30.7%, respectively, for fresh, 0, 2 and 4h post-thaw). The frequency distribution of spermatozoa within subpopulations was quite similar for the 9 bulls, either in fresh or frozen-thawed semen, and differences among bulls were mainly due to differences in the Subp. 4. Significant correlations (P<0.01) were found between the proportions of spermatozoa assigned to Subp. 4 in the fresh ejaculates and those in frozen-thawed semen after 0 (r=0.473), 2 (r=0.513) and 4h post-thaw (r=0.450). This indicated that the ejaculates with the highest subpopulations of rapid and progressive sperm were also the most resistant to cryopreservation and showed the best post-thaw sperm longevity.
Departamento de Patología Animal, Facultad de Veterinaria, Unidad de Reproducción y Obstetricia, Universidad de Santiago de Compostela, Lugo, Spain.
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D000818:Animals; D001696:Biomechanical Phenomena / Q000502:physiology; D002417:Cattle / Q000235:genetics / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D004542:Ejaculation / Q000502:physiology*; D005615:Freezing; D007315:Insemination, Artificial / Q000379:methods; D008297:Male; D010781:Photography; D012661:Semen / Q000502:physiology*; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility / Q000502:physiology*; D013094:Spermatozoa / Q000145:classification* / Q000502:physiology*