Factors affecting chromatin stability of bovine spermatozoa

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Abstract

The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertilization process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on incidence of sperm nuclear chromatin instability (NCI), and (2) effects of the interaction between variation of NCI within a frozen ejaculate and variation of oocytes quality due to maturation time and/or season on the efficiency of in vitro embryo production (IVEP). Semen samples were collected once a week from six bulls using an AV and only ejaculates (n = 220) of >0.30 × 109 sperm/ml and ≥60% motility were used. NCI was measured by: (1) detection of lysine-rich histones in sperm chromatin using aniline blue staining, (2) sperm susceptibility to acid-induced nuclear DNA denaturation in situ using acridine orange test, and (3) sperm susceptibility to nuclear chromatin decondensation (NCD). Bovine oocytes (n = 695) were matured in vitro for 18 or 24 h, fertilized after sperm selection through a swim-up procedure and cultured for 72 h. The results showed that the 2nd ejaculates were superior to the 1st ones with respect to chromatin stability. Dilution of semen to 49.67 ± 8.56 × 106 sperm/ml (1:19) decreased resistance of sperm to NCD. Cooling of semen had no significant effect on chromatin stability. Cryopreservation of semen augmented sperm vulnerability to DNA denaturation. Improvement of SQC (semen volume, sperm motility, velocity, viability and morphological normalcy) was generally concomitant with increase of sperm resistance to NCI. While Blonde d’Aquitaine bulls had a resistance to NCD higher than Limousine bulls in fresh semen, the former showed a greater susceptibility to DNA denaturation than the latter in cooled semen. Individuality significantly influenced NCI. The variability of NCI within a frozen ejaculate affected efficiency of IVEP. Significant negative correlations were observed between incidence of NCI and both fertilization rate and developmental capacity of embryos after maturation of oocytes for 18 h. The significant variation in IVEP traits due to season was independent of the effect of sperm chromatin instability.

Introduction

Failure of fertilization and embryonic mortality, particularly after AI, have long been recognized as potential sources of loss in breeding cows and numerous studies have reported on them (Gordon, 1996). The potential sire effects on bovine embryonic development have been shown to occur as early as the initiation of S-phase in the zygote and after expression of the embryonic genome at the four- to eight-cell stage (Eid et al., 1994). Therefore, laboratory assessment of semen must include testing of most sperm attributes relevant for fertilization and embryo development, such as evaluation of sperm genomic integrity (Evenson and Wixon, 2006).

Mammalian sperm genome is composed of nuclear DNA (Gledhill, 1970), mitochondrial DNA (Sutovsky et al., 2003) and cytoplasmic messenger RNAs (Miller, 2000). The DNA in sperm nuclei is bound to basic nuclear proteins to form the deoxyribonucleoprotein (DNP) complex (Livolant, 1984). The only essential component of the sperm needed to form normal embryos is a nucleus with an intact nuclear matrix (Ward et al., 1999). A high level of sperm nuclear chromatin instability (NCI) in semen is associated with reduced breeding efficiency of bulls (Ballachey et al., 1988, Karabinus et al., 1990, Dobrinski et al., 1994, Anzar et al., 2002, Madrid-Bury et al., 2005).

The presence of sperm with NCI in freshly ejaculated semen is considered an uncompensable defect, which cannot be tolerated at levels greater than 15–20% of spermatozoa (Barth and Oko, 1989). Fresh semen from bulls classified as questionable or unsatisfactory potential breeders contains a higher percentage (13.50%) of spermatozoa with abnormal DNA condensation than does semen from bulls classified as satisfactory potential breeders (7.10%) (Dobrinski et al., 1994). Freshly diluted semen of mature dairy bulls contains 0.53–8% sperm with NCI (Krzyzosiak et al., 2000). The factors that may affect the incidence of NCI in fresh semen are individuality and semen quality characteristics (Dobrinski et al., 1994) as well as variation between and within ejaculates of the same individual (Duty et al., 2002). However, the effects of breed, successive ejaculations and dilution rates on the incidence of NCI in fresh semen of beef bulls have not yet been investigated.

Previous studies on cooled bull semen (Salisbury et al., 1961, Hanada et al., 1965) did not show any changes in sperm DNP complex for storage up to 24 h. Moreover, the literature contains conflicting results regarding the impact of cryopreservation on sperm genome. Freezing and/or thawing of semen altered DNP complex of bull sperm (Salisbury et al., 1964). On the contrary, overcondensation of sperm chromatin has been observed in frozen-thawed semen of bulls (Dobrinski et al., 1994, Anzar et al., 2002). Other authors did not find any effect for cryopreservation of bull semen on sperm chromatin stability or DNA integrity (Martin et al., 2004).

Concerning the variation range (0–15%) of NCI in frozen semen, many factors have been studied, such as individuality and sperm traits (Dobrinski et al., 1994, Januskauskas et al., 2003), variation between ejaculates of the same bull (Bochenek et al., 2001), bull age and post-thawing incubation time and temperature (Karabinus et al., 1990). However, the effect of breed on the incidence of NCI in frozen semen of beef bulls has not yet been studied. Moreover, scarce information is available regarding the relationship between sperm kinematic characteristics and chromatin stability.

Incomplete nuclear condensation may appear in the form of nuclear vacuoles, which have been described in bull spermatozoa (Walters et al., 2004). The percentage of sperm with single and multiple vacuoles in frozen semen of bulls is positively correlated with the percentage of sperm with NCI (Dobrinski et al., 1994). Vacuolated sperm cells bind to the zona pellucida, penetrate oocytes at a low rate, reduce formation of male pronuclei and decrease the rate of bovine in vitro embryo production (IVEP) (Walters et al., 2004).

IVEP represents a desirable option in strategies to enhance reproductive and genetic advances in cattle (Trounson, 1992). Sperm DNA damage or alteration of protamine packing around sperm DNA may contribute to a delay in initiation of zygotic S-phase (Eid et al., 1994), an increase in length of zygotic G2-phase (Eid and Parrish, 1995), a block in blastocyst formation (Fatehi et al., 2006) and, thus, to a decrease in numbers of morphologically normal bovine embryos (Smorag et al., 2000). The developmental block that arises in bovine embryos at the eight-cell stage may be correlated with the cytoplasmic quality of oocytes (Meirelles et al., 2004). Oocytes and early embryos have been shown to repair sperm DNA damage (Ashwood-Smith and Edwards, 1996). Consequently, the biological effect of abnormal sperm chromatin structure depends on the combined effects of the level of chromatin damage in the spermatozoa and the capacity of the oocyte to repair that pre-existing damage (Evenson et al., 2002). Therefore, if spermatozoa are selected from samples with extensively damaged DNA and used for IVF, the oocyte's repair capacities may be inadequate, leading to a low rate of embryonic development (Ahmadi and Ng, 1999).

The objectives of the present study were to investigate: (1) effects of breed, individuality, successive ejaculations, semen quality characteristics, semen dilution rates and hypothermic preservation of semen on the incidence of NCI in spermatozoa of beef bulls, and (2) effects of the interaction between variation of sperm NCI within a frozen semen ejaculate of a single dairy bull and variation of oocytes quality due to maturation time and/or season on the efficiency of bovine IVEP.

Section snippets

Location

The study was carried out (February to August 2005) at the AI center and IVEP laboratory, NAGREF, Veterinary Research Institute, Ionia, Thessaloniki, Northern Greece.

Chemical reagents and semen extender

Unless otherwise stated, all chemicals were purchased from Sigma–Aldrich Co., Greece. Milli-Q purified water was utilized in all experiments. Media, buffers and staining solutions were filtered before use through a 0.22 μm filter. A home-made Tris-based extender (pH 6.8, 300 mOsm/kg) was used for two-step dilution and hypothermic

Experiment 1

The percentage of AB-positive sperm (Fig. 1) ranged from 0% in the second ejaculate of bull 6 to 19% in the first ejaculate of bull 3. The intra-assay CV of AB staining test ranged from 3.44 to 8.13%. The repeatability of AB test showed CV ranging from 6.99 to 11.90%. The percentages of AB-positive sperm were not significantly influenced by individuality, breed or successive ejaculations (Table 1). The incidence of AB-positive sperm was significantly influenced by sperm viability (r = −0.40, P < 

Discussion

During the past decade, evaluation of sperm genomic integrity brought us closer to a complete and accurate assessment of male reproductive competence. In the present study, we used three assays to evaluate stability of sperm DNP complex. The positive correlations between AB-reactive cells and reduced resistance of sperm to DNA denaturation and NCD indicate that removal of lysine-rich histones from sperm DNA is necessary for chromatin compaction (Balhorn, 1982). Moreover, the low CVs obtained

Conclusion

Individuality, bull breed, successive ejaculations and semen quality traits can be considered as factors affecting stability of the transcriptionally silent sperm chromatin. Dilution of semen to low cell numbers increases susceptibility of spermatozoa to chromatin decondensation. Cooling and equilibration of semen in a Tris-egg yolk extender have no significant effect on stability of sperm chromatin. Freezing and/or thawing of semen augment vulnerability of sperm cells to DNA denaturation.

Acknowledgements

This study was technically and financially supported by NAGREF, Veterinary Research Institute, Ionia, Thessaloniki, Greece. The authors wish to thank the members of AI center and IVEP laboratory for their support. Appreciation is expressed to Dr. S. Belibasaki (NAGREF), Dr. E. Vainas (NAGREF) and E.A. Rekka (Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Greece) for their valuable advice and technical assistance. The authors would like to

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