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Effect of Butylated Hydroxytoluene on Acrosome Integrity and Viability of Crossbred Bull Spermatozoa

Anoop Singh Mridula Sharma Shiv Prasad Yaqoob Bhat Anil Kumar Dinesh Pandey Santosh Shukla
Vol 7(7), 82-91

The present study was conducted to observe the effect of Butylated hydroxytoluene (BHT) on acrosomal integrity of crossbred bovine bull spermatozoa. Total eighteen ejaculates from two crossbred bulls were collected and extended with egg yolk tris dilutor along with addition of different concentrations of BHT i.e. 0.0mM (Control), 0.5mM (T1) and 1.0mM (T2), respectively. Acrosome integrity was assessed by Trypan blue giemsa staining during post dilution (Stage I), post equilibration (Stage II) and post thaw (Stage III). Mean percent of AIL spermatozoa were significantly (p<0.05) higher in group T2 compared to T1 and control at stage I and II. At stage III, T1 and T2 showed significantly (p<0.05) higher AIL sperms compared to control. Mean percent of AID spermatozoa were significantly (p<0.05) higher in control compared to T1 and T2 at Stage I, II and III. T2 showed significantly lower ALL and ALD spermatozoa compared to control at stage III. The study indicates that 1mM BHT increased the cryosurvival i.e., (higher acrosomal integrity and viability) i.e. higher AIL and lower AID, ALL and ALD spermatozoa.

Keywords : Semen Crossbred Bull BHT Trypan Blue/Giemsa Acrosomal Integrity and Viability


In all mammals, capacitation and acrosome reaction of spermatozoa are essential steps for successful fertilization and formation of a zygote (Chioham and Hunter, 2004). The process of acrosome reaction comprises vesiculation and fusion of outer acrosomal membrane to overlying plasma membrane and subsequent release of acrosomal contents and exhibition of inner acrosomal membrane (Kadirvel et al., 2011). Acrosomal integrity status can be used as a suitable marker for fertilization ability of spermatozoa. Thus, for in-vitro fertilization ability assessment, a reliable assay of acrosome reaction rate evaluation is needed (Boccia et al., 2007). Identification of a quick and reliable method for determining the fertilizing capability of sperm becomes imperative in selecting genetically superior bulls.

Cryopreservation ensures long-term storage of sperm and its subsequent use in the breeding of domestic as well as wild animals. In spite of lots of development in cryopreservation and cryopreservation protocols, the sperms suffer from irreversible damage both at structural and functional level leading to a reduction in viability, motility, and fertility (Thomas et al., 2006). Free radical generation is a major limitation during the process of sperm cryopreservation, which is generated from the non-viable sperms as well as from the extenders, which contain the molecular oxygen (Thomson et al., 2009). The free radicals target the sperm plasma membrane, and in specific the polyunsaturated fatty acids, membrane proteins, acrosome and sperm DNA causing lipid per oxidation of membrane lipids, chromatin cross-linking and fragmentation of sperm DNA (Tremellen, K., 2008). These changes induce irreversible cell damage and sperms undergo induced apoptosis causing a consequent reduction in the sperm quality. Generation of free radicals during cryopreservation cannot be inhibited, but can be reduced by the introduction of suitable antioxidants in the extenders used for sperm cryopreservation (Ball et al., 2001; Andreea and Stela, 2010). In the recent past, many of the laboratories are focusing on various antioxidants and their dose dependent incorporation in the semen extenders to reduce the generation of free radicals and their deleterious effect on sperm (Andreea and Stela, 2010; Khalifa et al., 2008; Andrabi et al., 2008). Butylated hydroxytoluene (BHT) is a phenolic antioxidant being supplemented in the semen extender as a measure to prevent the membrane permeability changes in the sperms during cryopreservation (Neagu et al., 2010). BHT also exhibits antiviral activity and has been associated with the inactivation of lipid-containing viruses (Farshad et al.,2010). Chemically BHT is a synthetic analog of vitamin E, and is involved in the auto-oxidation reaction, thereby converting the peroxy radicals to hydroxyperoxides (Fujisawa et al., 2004). These may be the best possible reasons for which, BHT has been used as a potential antioxidant additive in a number of species, but no literature is available in the Hariana bulls till date. Antioxidant addition has been emerged as one of the most powerful way to overcome the excessive generation of free radicals during the time of semen cryopreservation and hence we focused our study to observe the effect of BHT addition in the semen extenders for its antioxidant effect reflecting protection to sperm cells.

Ascorbic acid, a non-enzymatic antioxidant is potentially involved in protecting cells against oxidative stress (Anane and Creppy, 2001). The supplementation of ascorbic acid in the diluents exerted an antioxidant effect during freezing and thawing of bovine (Hu et al., 2010) and caprine sperm (Gangwar et al., 2015). Butylated hydroxytoluene (BHT) has been found to have positive effects in minimizing cold shock of spermatozoa during liquid preservation of semen of bulls (Shoae and Zamiri, 2008), buffaloes (Ijaz et al., 2009) and from freezing damage during cryopreservation (Iqbal et al., 2015; Naijian et al., 2013).The beneficial effects of vitamin E, vitamin C and various proteins which provide cryo-protection to the sperms during freeze thaw process (data unpublished) have been studied by Patel et al., 2016. However, literature is scanty regarding the incorporation of BHT in semen extender, as an antioxidant to protect sperm cells during cryopreservation.

Acrosome reaction status and viability of sperm was evaluated using Trypan-blue giemsa stain. The stain enables simultaneous detection of viability (trypan blue) and acrosome reaction (giemsa) status of spermatozoa. A double Trypan blue/giemsa staining method was first used for sperm evaluation by Kovács and Foote in 1992. The application of this staining technique is very simple, rapid and allows examining of results even after many years. This procedure can be used to assess sperm viability evaluating the membrane status of sperm cells, subdomains (head, acrosome, tail) through different staining of the viable and not viable segments. This technique was successfully employed to assess viability and acrosome status in water buffalo spermatozoa (Presicce et al., 2003). On the basis of staining characteristics (Kovacs and Foote, 1992), the spermatozoa were divided into four categories (viz. AIL, AID, ALL and AID). Pink acrosome and unstained post-nuclear cap was categorized as AIL. The AIL sperms were negative for trypan blue stain hence post-nuclear cap- region was unstained. Simultaneously, acrosome intact dead sperm (AID) was identified as pink acrosome and blue postnuclear cap as it was positive for both the stains.

The acrosome lost live sperm (ALL) was detected as unstained acrosome and post nuclear cap (Fig. a) as acrosomal contents were lost due to acrosomal membrane damage and viable sperms were impermeable to the dye. Acrosomes lost dead sperms (ALD) were detected as unstained acrosome but blue coloured postnuclear cap (Boccia et al., 2007).

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