Identification of sperm subpopulations with defined motility characteristics in ejaculates from Holstein bulls: Effects of cryopreservation and between-bull variation

Abstract

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 4 h of post-thaw incubation at 37• 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 4 h 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 4 h 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. Muiño et al. / Animal Reproduction Science 109 (2008) [27][28][29][30][31][32][33][34][35][36][37][38][39] 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 4 h 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.

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls

“…The kinematic properties of these four subpopulations, as well as the frequency distribution of spermatozoa within them, were nearly the same as those previously found in ejaculates from Holstein bulls [18]. Moreover, for the two bull breeds, the overall sperm subpopulation structure showed little variation among individuals.…”

“…This suggests that such a specific subpopulational structure, depicted by spermatozoa with different physiologic and metabolic characteristics, may be a constant finding in bovine ejaculates. The presence of three or four well-defined motile sperm subpopulations has been demonstrated in numerous species [8,9,11,14], and, as indicated by our current and previous [18] results, the bovine species does not seem to be an exception.…”

“…The existence of four motile sperm subpopulations within a single ejaculate has recently been described in fresh and frozen-thawed semen from Holstein bulls [18]. The physiologic significance of such subpopulational structure is not known.…”

Protein biomarkers for male artificial insemination subfertility in bovine spermatozoa

“…The ATP is indispensable for the initiation, regulation, and maintenance of the progressive motility of bull spermatozoa, which is required for successful AI 42, 43, 44, 45, 46. Thus, it is important to conduct an objective investigation of the motility of bull cryopreserved spermatozoa before use in AI with a microscopic image recorder or computer‐associated sperm analyzer 30, 31, 47…”

Relationships between the results of hypo-osmotic swelling tests, sperm motility, and fertility in Estonian Holstein dairy bulls

“…Post-thaw viability and fertility of cryopreserved sperm are often reduced due to accumulated cellular damage during the cryopreservation phases (Muiño et al, 2008). Decrease in temperature, cold shock, and intracellular ice formation can affect sperm plasma membrane, acrosomal and mitochondrial membrane integrity (Thomas et al, 1998;Defoin et al, 2008), and cause the loss of intracellular components (Graham and Mocé, 2005), which can initiate cell death.…”