Cryopreservation of Ram Semen Facilitates Sperm DNA Damage: Relationship Between Sperm Andrological Parameters and the Sperm Chromatin Structure Assay

Abstract

ABSTRACT:We hypothesized that cryopreservation and incubation in conditions that mimic the female genital tract following insemination increases the susceptibility of ram sperm DNA to denaturation. Ram sperm samples (n ϭ 12) underwent the sperm chromatin structure assay (SCSA) and semen quality tests, including motility parameters, viability, and chlortetracycline fluorescence (CTC) patterns. We also assessed correlations between SCSA variables and semen quality parameters. Analyses were performed for both fresh and cryopreserved samples at 0, 3, and 20 hours of incubation in synthetic oviductal fluid (SOF; 39ЊC, 5% CO 2 ). The SCSA variables, mean alpha t (X␣ t ) and standard deviation of alpha t (SD␣ t ), were higher because of cryopreservation (P Ͻ .05, P Ͻ .001, respectively) after 20 hours in SOF. For both fresh and frozen spermatozoa, SCSA values (X␣ t , SD␣ t , and the percentage of cells outside the main population of ␣ t [%COMP␣ t ]) increased during incubation in SOF. Motility was negatively correlated with both SD␣ t and %COM-P␣ t , ranging from Ϫ0.39 (P Ͻ .01) to Ϫ0.59 (P Ͻ .001) for both fresh and cryopreserved semen; viability also was negatively correlated with X␣ t , SD␣ t , or %COMP␣ t (Ϫ0.36; P Ͻ .05, Ϫ.40 and -.46; P Ͻ .01, respectively) in fresh semen. The %COMP␣ t was positively correlated to the percentage of CTC pattern AR (P Ͻ .001) and negatively correlated to the percentages of patterns F and B (Ϫ0.33 to Ϫ0.60, P Ͻ .05 to P Ͻ .001). Variation among ejaculates within ram was observed (P Ͻ .01). Cryopreservation clearly facilitates DNA damage in physiological conditions. The low to moderate correlations between SCSA variables and classical semen quality parameters indicate that the SCSA provides additional information to standard tests for evaluating ram sperm quality.

Sperm DNA integrity assays: diagnostic and prognostic challenges and implications in management of infertility

“…2 Comet image of sperm with fragmented and non fragmented DNA damage as quantified by TUNEL assay. The DNA fragmentation Index, evaluated by SCSA assay, provides a weak or a moderate correlation with conventional sperm parameters (Giwercman A et al, 2003 andPeris SI et al 2004 [103,104]). …”

Cryopreservation of Sheep Produced Embryos – Current and Future Perspectives

“…Frozen semen is easily available for routine use, and some authors argued that early embryo development is not influenced by the method of semen preservation [132], whereas others demonstrated that fresh semen could improve embryo production rates and quality [50]. Eventually, freezing and thawing disrupt the stability of ram sperm chromatin, reducing its fertilization efficiency [133,134]. On the contrary, cryopreserved semen is also advantageous, as it can be stored in seasonal propitious period.…”

Semen quality assessments and their significance in reproductive technology

“…The SCSA predicts infertility when the DFI exceeds 30% in natural conception (Evenson et al 2002, Evenson andWixon 2008), justifying its clinical application. The SCSA has identified compromised chromatin integrity of cryopreserved spermatozoa from different farm animals, such as the boar (Evenson et al 1994), bull (Janaskauska et al 2001) and ram (Peris et al 2004).…”