Effect of Butylated Hydroxytoluene on Dog Sperm Longevity in Chilling Storage and Cryopreservation

Abstract

ABSTRACT. This study evaluated the effect of butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, on dog sperm in chilling storage and cryopreservation. In Experiment 1, 0.2, 0.4, 0.8 and 1.6 mM BHT were added to egg yolk Tris extender (EYT), and sperm were stored at 4C for 96 hr. Sperm motility, viability, acrosomal integrity and morphological abnormality in the BHT treatment groups were not different from those of the control (0 mM BHT). In Experiment 2, the effect of BHT in EYT containing 0.75% Equex STM paste and 5% glycerol on survivability of cryopreserved sperm was examined after culture at 39C for 3 hr. Sperm motility, viability and acrosomal integrity in the 0.2 to 0.8 mM BHT treatment groups were not different from those of the control. However, sperm motility, viability and acrosomal integrity decreased when 1.6 mM BHT was added to the extender (P<0.05). In conclusion, supplementation of the extender with 0.2 to 0.8 mM BHT did not affect characteristics of dog sperm in chilling storage and cryopreservation. Supplementation of 1.6 mM BHT did not affect characteristics of chilled sperm but impaired longevity of cryopreserved sperm in the dog.KEY WORDS: butylated hydroxytoluene, canine, freezing, sperm.J. Vet. Med. Sci. 73 (7): [895][896][897][898][899] 2011 Semen collection and artificial insemination (AI) play an important role for breeding of purebred dogs. In spite of the important potential applications of semen preservation, chilled and cryopreserved sperm are not generally used for AI in the dog [8,9,15,19,21]. For successful conception, the fertilizing capacity of sperm must be maintained until AI is performed. Many studies about chilled and cryopreservation techniques for dog sperm have been reported. Fertility of chilled dog sperm can be maintained for 4 days [15]. However, post-thaw motility of dog sperm decreases markedly within 1 hr [8,9,21], resulting in a lower whelping rate and litter size [22]. Therefore, improvement of sperm longevity is desirable for both chilling storage and cryopreservation of dog sperm.Injury of sperm by reactive oxygen species (ROS) during chilled storage and cryopreservation may result in a low success rate of AI. Supplementation of semen extenders with antioxidant compounds could overcome this problem. Butylated hydroxytoluene (BHT), a synthetic phenolic antioxidant, is not only commonly used as a food additive [26], but is also widely used for low-temperature preservation of mammalian [2, 3,11,13,14,17,18,24,25] and avian sperm [6]. It was reported that BHT suppressed sperm mitochondrial metabolism and protected the cell membranes from cold shock by decreasing oxygen toxicity in the bull [12].Protection of sperm by BHT against cold shock has been reported in the bull [11], ram [25] and boar [3]. Addition of BHT to semen extender also maintained the motility and viability of chilled turkey sperm [6] and frozen sperm of boars [18], goat bucks [14], cattle [24] and buffalo bulls [13]. Recently, addition of 1 mM BHT to the freezing medium reportedly in...

Effects of different concentrations of BHT on microscopic and oxidative parameters of Mahabadi goat semen following the freeze–thaw process

“…There are many factors which can contribute to the microscopic sperm parameters. In our study, CASA, progressive motilities and viability were influenced by the concentration of BHT as previously reported [51,29,47,24]. However, in our trial, subjective motility, HOST, acrosome integrity, sperm motion characteristics and acrosome reaction status were not influenced by the level of BHT which is in agreement with the findings of Bucak et al [17].…”

Optimal inclusion level of butylated hydroxytoluene in semen extender improves the quality of post-thawed canine sperm

“…These findings are in accordance with a recent work done, in which BHT at high concentrations of 1.6mM significantly decreased the sperm motility, viability, and acrosomal integrity in canine semen (Sahashi et al, 2011). The damage inflicted on the sperm membrane during cryopreservation renders the membrane more permeable to the BHT metabolites which, in turn, has a harmful effect (Shoae and Zamiri, 2008;Sahashi et al, 2011).…”

“…Sahashi et al (2011) have recently reported an optimal inclusion level of 0.4mM BHT in canine semen. The optimal inclusion level for BHT seems to be a species specific characteristics as indicated by previous works for boar (Roca et al, 2004), cattle bull (Shoae and Zamiri, 2008), and buffalo bull (Ijaz et al, 2009) semen recorded as 0.05−2.0, 0.5−1.0, and 1.0−2.0mM, respectively.…”

“…Semen collection was carried out once every ten days through digital manipulation (Sahashi et al, 2011) in a conical tube. A total of 100 ejaculates were collected with a mean ejaculate number of 10 per dog.…”

“…BHT has been used successfully for preservation of liquid semen in turkey tom (Donoghue and Donoghue, 1997) and to minimize cryoinjury in ram (Watson and Anderson, 1983), boar (Roca et al, 2004), cattle bull (Shoae and Zamiri, 2008), and goat spermatozoa (Khalifa et al, 2008). However, there are scanty reports (Neagu et al, 2010;Sahashi et al, 2011) regarding its cryoprotective potentiality and optimal inclusion level in canine semen. This study, therefore, was conducted to evaluate the cryoprotective potentiality of BHT on canine semen and its optimal Optimal inclusion level of butylated hydroxytoluene in semen extender improves the quality of post-thawed canine sperm …”