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Effect of multiple lyophilization or freeze-thaw cycles on chromatin quality of stallion sperm
Animal Reproduction Science 94 (2006) 19–20
Abstract
Effect of multiple lyophilization or freeze–thaw cycles on chromatin quality of stallion sperm夽 D.D. Varner a,∗ , C.C. Love a , Y.H. Choi b , S. Teague c , J.A. Thompson a , K. Hinrichs b a
b
Department of Large Animal Clinical Sciences, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, College Station, TX 77843, USA Department of Veterinary Physiology and Pharmacology, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, College Station, TX 77843, USA c Veterinary Medical Teaching Hospital, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, College Station, TX 77843, USA Available online 25 April 2006
1. Introduction When using intracytoplasmic sperm injection (ICSI), many sperm traits are unnecessary, with chromatin integrity being the main feature of sperm required for fertilization. The chromatin of mature sperm is resistant to damage. Sperm lyophilization has not been reported for stallions and there are no reports regarding the effects of sperm lyophilization on chromatin integrity in any species. In this study, we compare chromatin integrity of lyophilized or with that of frozen/thawed stallion sperm. 2. Materials and methods Semen from three fertile stallions was subjected to one of five treatments: (1) freezing in raw form (RAW); (2) lyophilization (LYO); (3) a commercial cryopreservation protocol (FREEZE); (4) a commercial cryopreservation protocol, followed by thawing, washing and lyophilization (FREEZE-WASH-LYO); or (5) a commercial cryopreservation protocol, followed by thawing, gradient centrifugation, and lyophilization (FREEZE-GRAD-LYO). Aliquots of semen were sub-
夽 This paper is part of the special issue entitled Proceedings of the Ninth International Symposium on Equine Reproduction, Guest Edited by Margaret J. Evans. ∗ Corresponding author. Tel.: +1 979 845 3541; fax: +1 979 847 8863. E-mail address: [email protected] (D.D. Varner).
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.03.027
20
D.D. Varner et al. / Animal Reproduction Science 94 (2006) 19–20
jected to one to three cycles of freezing or lyophilization. Semen samples were evaluated for chromatin susceptibility to denaturation (%COMP; Evenson et al., 1980; Love and Kenney, 1998). 3. Results Mean %COMP was less for Group FREEZE-GRAD-LYO (2.2 ± 0.6; mean ± S.D.) than for all other treatment groups (P < 0.05). Mean %COMP was similar (P > 0.05) between Groups LYO (5.6 ± 1.4) and FREEZE-WASH-LYO (5.8 ± 1.6), and mean %COMP was similar (P > 0.05) between Groups FREEZE-WASH-LYO and FREEZE (7.6 ± 2.8). The mean %COMP for RAW (7.9 + 2.2) was similar to Group FREEZE (P > 0.05), but greater than the remainder of treatment groups (P < 0.05). Mean %COMP was unaffected (P > 0.05) by the number of freeze/thaw or lyophilization cycles. 4. Discussion The sperm chromatin in this trial was remarkably resilient to denaturation, as all processed semen yielded similar or enhanced %COMP than did the RAW control group. Semen in Group FREEZE-GRAD-LYO yielded the least %COMP, probably because centrifugation in a density gradient removed many abnormal sperm prior to lyophilization. Multiple freeze/thaw or lyophilization cycles did not have a detrimental effect on sperm chromatin susceptibility to denaturation. Pregnancies have resulted in our laboratory by ICSI of sperm lyophilized in the manner reported here (unpublished data). The present study indicates that stallion sperm chromatin is tolerant of lyophilization, thus offering a potential alternative for long-term storage of sperm without loss of genetic integrity. Acknowledgments Financial support for this study was provided by the Patsy Link Equine Research Fund, Texas A&M University, and the American Quarter Horse Association. References Evenson, D.P., Darzynkiewicz, Z., Melamed, M.R., 1980. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210, 1130–1133. Love, C.C., Kenney, R.M., 1998. The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. Theriogenology 57, 955–972.
Abstract
Effect of multiple lyophilization or freeze–thaw cycles on chromatin quality of stallion sperm夽 D.D. Varner a,∗ , C.C. Love a , Y.H. Choi b , S. Teague c , J.A. Thompson a , K. Hinrichs b a
b
Department of Large Animal Clinical Sciences, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, College Station, TX 77843, USA Department of Veterinary Physiology and Pharmacology, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, College Station, TX 77843, USA c Veterinary Medical Teaching Hospital, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, College Station, TX 77843, USA Available online 25 April 2006
1. Introduction When using intracytoplasmic sperm injection (ICSI), many sperm traits are unnecessary, with chromatin integrity being the main feature of sperm required for fertilization. The chromatin of mature sperm is resistant to damage. Sperm lyophilization has not been reported for stallions and there are no reports regarding the effects of sperm lyophilization on chromatin integrity in any species. In this study, we compare chromatin integrity of lyophilized or with that of frozen/thawed stallion sperm. 2. Materials and methods Semen from three fertile stallions was subjected to one of five treatments: (1) freezing in raw form (RAW); (2) lyophilization (LYO); (3) a commercial cryopreservation protocol (FREEZE); (4) a commercial cryopreservation protocol, followed by thawing, washing and lyophilization (FREEZE-WASH-LYO); or (5) a commercial cryopreservation protocol, followed by thawing, gradient centrifugation, and lyophilization (FREEZE-GRAD-LYO). Aliquots of semen were sub-
夽 This paper is part of the special issue entitled Proceedings of the Ninth International Symposium on Equine Reproduction, Guest Edited by Margaret J. Evans. ∗ Corresponding author. Tel.: +1 979 845 3541; fax: +1 979 847 8863. E-mail address: [email protected] (D.D. Varner).
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.03.027
20
D.D. Varner et al. / Animal Reproduction Science 94 (2006) 19–20
jected to one to three cycles of freezing or lyophilization. Semen samples were evaluated for chromatin susceptibility to denaturation (%COMP; Evenson et al., 1980; Love and Kenney, 1998). 3. Results Mean %COMP was less for Group FREEZE-GRAD-LYO (2.2 ± 0.6; mean ± S.D.) than for all other treatment groups (P < 0.05). Mean %COMP was similar (P > 0.05) between Groups LYO (5.6 ± 1.4) and FREEZE-WASH-LYO (5.8 ± 1.6), and mean %COMP was similar (P > 0.05) between Groups FREEZE-WASH-LYO and FREEZE (7.6 ± 2.8). The mean %COMP for RAW (7.9 + 2.2) was similar to Group FREEZE (P > 0.05), but greater than the remainder of treatment groups (P < 0.05). Mean %COMP was unaffected (P > 0.05) by the number of freeze/thaw or lyophilization cycles. 4. Discussion The sperm chromatin in this trial was remarkably resilient to denaturation, as all processed semen yielded similar or enhanced %COMP than did the RAW control group. Semen in Group FREEZE-GRAD-LYO yielded the least %COMP, probably because centrifugation in a density gradient removed many abnormal sperm prior to lyophilization. Multiple freeze/thaw or lyophilization cycles did not have a detrimental effect on sperm chromatin susceptibility to denaturation. Pregnancies have resulted in our laboratory by ICSI of sperm lyophilized in the manner reported here (unpublished data). The present study indicates that stallion sperm chromatin is tolerant of lyophilization, thus offering a potential alternative for long-term storage of sperm without loss of genetic integrity. Acknowledgments Financial support for this study was provided by the Patsy Link Equine Research Fund, Texas A&M University, and the American Quarter Horse Association. References Evenson, D.P., Darzynkiewicz, Z., Melamed, M.R., 1980. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210, 1130–1133. Love, C.C., Kenney, R.M., 1998. The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. Theriogenology 57, 955–972.