Effect of Cryopreservation on Bovine Sperm Viability as Determined by Dual DNA Staining

Abstract

: Living and dead spermatozoa were examined for the effects of sperm concentration level on sperm viability. Semen was collected from two different bulls on each of four collection dates. A ninth bull was collected on all four collection dates as a control for effects of collection date. The ejaculates from these nine bulls were diluted to 30 × 106 spermatozoa/0.5 ml and then serially diluted to 20, 10, 5, or 1 × 106 spermatozoa/0.5 ml French straw. One‐half of the straws for each dilution series was stored 24 hours at 5° C, while the other half was cryopreserved. Spermatozoa were stained with SYBR‐14 and propidium iodide (PI) to assess viability. Flow cytometry yielded dot plots showing three distinct sperm populations: dead red‐stained spermatozoa (PI), viable green‐stained spermatozoa (SYBR‐14), and moribund spermatozoa that stained both red and green (doubly‐stained). Populations were expressed and analyzed in terms of mean percentage of viable spermatozoa and by actual numbers of viable spermatozoa per insemination dose. The mean percentage of living spermatozoa decreased linearly with decreasing sperm concentration; whereas the decrease was parabolic when those same samples were expressed as the mean number of living spermatozoa per insemination dose. The percentage of SYBR‐14‐stained spermatozoa differed among concentration levels and among bulls (P < 0.01). There were no differences among straws from the same ejaculate. The total volume of ejaculated semen and the concentration of spermatozoa in that ejaculate were both significantly positively correlated with the percentage of SYBR‐14‐stained spermatozoa in that semen when it was cryopreserved and diluted to <10 × 106 spermatozoa/0.5 ml. In contrast, there were no significant correlations between the initial ejaculate characteristics and the proportion of SYBR‐14‐stained spermatozoa in the 24‐hour‐stored samples at any concentration. In conclusion, the percentage of viable spermatozoa in an ejaculate significantly decreased with increasing dilution. Further, in cryopreserved samples, the percentage of living spermatozoa <10 × 106 spermatozoa/0.5 ml depended on the original volume and the sperm concentration of that particular ejaculate.

Effect of Cryopreservation on Bovine Sperm Organelle Function and Viability As Determined by Flow Cytometry1

“…Although both studies used twelve bulls, the mean percentages of JC-l-stained sperm + SEM and post-thaw motility + SEM in the present study were 47. 9 3.3 and 71.7 + 1.3, respectively; the same values in the previous study were 59.0 + 7.4 and 65.8 + 5.8, respectively. The reason for the difference was obvious when one compared the two scatter plots (not shown).…”

“…Dot plots of SYBR-14/PI-stained spermatozoa showed three populations [9], as did JC-1/PI dot plots (Fig. 1).…”

“…The fluorometric staining combination, SYBR-14 and propidium iodide (PI), has been shown to be a rapid and reliable means for determining the proportions of living and dead spermatozoa in semen [9][10][11][12][13][14][15][16][17][18][19]. These nucleic acid-specific probes stain living spermatozoa green while dead, membrane-damaged spermatozoa stain red.…”

Inter- and intra-individual variability of total antioxidant capacity of bovine seminal plasma and relationships with sperm quality before and after cryopreservation

“…For example, the dilution of the ejaculates reduces the concentration of seminal plasma antioxidants. Previous studies (Garner et al, 1997 and2001) showed that the volume and sperm concentration of the ejaculates and the final concentration of the seminal plasma in the extended semen have important roles in plasma membrane and acrosome intact sperm after thawing of the cryopreserved sperm. Garner et al (2001) suggested that the source of the effect was seminal plasma.…”

Transcriptome Analysis of Bull Semen with Extreme Nonreturn Rate: Use of Suppression-Subtractive Hybridization to Identify Functional Markers for Fertility1

“…Studies regarding the pathophysiology of human sperm function have indicated that oxidative stress generated by mitochondrial activity in human sperm may be a potential cause of reduced human male fertility [26,34]. In addition, a correlation between sperm motility and mitochondrial activity has been reported in studies with equine [35], human [36], and bovine spermatozoa [37]. Nonetheless, a correlation with NRRs could not be established in bull spermatozoa [38], possibly because mtDNA is maternally inherited and selective pressure would not affect the male lineage [39].…”