Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden Seasonal Variation in Nuclear DNA Integrity of Frozen–Thawed Spermatozoa from Thai AI Swamp Buffaloes (Bubalus bubalis) S. Koonjaenak 1,2 , A. Johannisson 3 , P. Pongpeng 4 , S. Wirojwuthikul 4 , A. Kunavongkrit 5 and H. Rodriguez- Martinez 1,6 Addresses of authors: 1 Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Box 7054, SE-75 007, Uppsala, Sweden; 2 Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand; 3 Department of Anatomy and Physiology, Swedish University of Agricultural Sciences, Box 7011, SE-75 007, Uppsala, Sweden; 4 Artificial Insemination Centre (LamphayaKlang), Department of Livestock Development, Lopburi 15190, Thailand; 5 Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand; 6 Corresponding author: Tel.: +46 18672172; fax: +46 18673545; E-mail: heriberto.rodriguez@kv.slu.se With 1 figure and 4 tables Received for publication October 16, 2006 Summary In this study, we investigated the susceptibility of frozen– thawed swamp buffalo sperm nuclear DNA to undergo con- trolled acid-induced denaturation in situ, as analysed by flow cytometry, and aimed to correlate the results with sperm head morphology over three seasons in tropical Thailand. Artificial insemination (AI) doses (n = 218) from 18 AI buffalo sires, prepared between 1980 and 1989 and 2003 and 2005, were tested and compared among three seasons, the rainy season, July–October; winter, November–February; and summer, March–June. The overall mean of DNA fragmentation index (DFI) (± SD) was 1.84 ± 1.68%, range from 0.19 to 7.92%, with 0.221 ± 0.021 of the x-DFI ranging from 0.190 to 0.350 and 0.023 ± 0.009 of the SD-DFI ranging from 0.010 to 0.070. The DFI was consistently low (range 1.40 ± 0.21% to 2.16 ± 0.21%; LSM ± SEM), with x-DFI ranging from 0.216 ± 0.003 to 0.225 ± 0.003 and SD-DFI ranging from 0.022 ± 0.001 to 0.024 ± 0.001 across the seasons. The DFI was low enough to be related to high fertility potential. However, DFI values varied statistically among seasons, being lower in the rainy season (1.40 ± 0.21%, P < 0.05) than in winter (2.16 ± 0.21%) or summer (2.00 ± 0.20%), and were also affected by the year of semen collection and processing (P < 0.001). The proportion of morphologically abnormal sperm head shapes was low, with no significant differences between seasons. However, DFI was significantly related to the proportion of loose abnormal sperm heads (r = 0.27, P < 0.01). In conclusion, frozen–thawed swamp buffalo sperm chromatin integrity is not seriously damaged by cryo- preservation or affected by the seasonal variations in tem- perature and humidity seen in tropical Thailand. Introduction A sexually mature bull is able to produce about 1 billion spermatozoa per day, with sperm maturation taking place in the testes and the sperm being stored in the epididymal ducts until ejaculation occurs. Many environmental cues such as season, nutrition, management and differentiation events are able to affect the kinetics of cell division and differentiation and consequently also affect total sperm output, as well as sperm normality. Semen quality of the collected ejaculate, including the ejaculate volume, sperm motility and propor- tions of morphologically and physiologically normal sperma- tozoa, determines the quality of the processed (often frozen) semen and, ultimately, the potential fertility level achieved when artificial insemination (AI) is used (Rodriguez-Martinez, 2000). Buffalo semen has been studied for some variables such as semen volume, sperm concentration, motility, sperm viab- ility and sperm morphology (Kushwaha et al., 1955; Kapoor, 1973; Jainudeen et al., 1982; Sukhato et al., 1988; Nordin et al., 1990; Pant et al., 2003; Koonjaenak et al., 2006, 2007 a,b), but few studies have focused on cryopreservation or AI results (Heuer et al., 1987; Rasul et al., 2001; Sukhato et al., 2001). Over the past decades, our ability to study sperm structure and function has increased with the use of novel markers for membrane integrity (Garner et al., 1994; Garner and Johnson, 1995; Alm et al., 2001; Januskauskas et al., 2001; Hallap et al., 2004), membrane stability (Anzar et al., 2002; Januskauskas et al., 2003) and the structure and function of several organ- elles such as mitochondria (Graham et al., 1990; Hallap et al., 2005b) or the acrosome (Graham et al., 1990; Nagy et al., 2004). Moreover, the fertilizing capacity of spermatozoa can today also be evaluated in vitro (Zhang et al., 1999; Janusk- auskas et al., 2000a,b). A large number of attributes are required in order for spermatozoa to be fertile and there is a battery of tests to assess these. One requirement of importance for embryonic development is the ability of the chromatin to decondense after fertilization (Spano et al., 2000), with the nuclear DNA remaining intact, a prerequisite to normal embryonic development (Aravindan et al., 1997; Anzar et al., 2002). These variables in sperm structure and function are today determined using a flow cytometry (FCM), an instru- ment that makes it possible to evaluate thousands of sperma- tozoa per minute, enhancing the objectivity of semen evaluation (Graham, 2001) and allowing for correlations with in vitro (Maxwell et al., 1998) and in vivo fertility (Ericsson www.blackwell-synergy.com J. Vet. Med. A 54, 377–383 (2007) Ó 2007 The Authors Journal compilation Ó 2007 Blackwell Verlag ISSN 0931–184X