Survival and Fertility of Boar Spermatozoa After Freeze‐Thawing in Extender Supplemented With Butylated Hydroxytoluene

Abstract

This study evaluated the protective effect of butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, against cryopreservation injuries to boar spermatozoa. In experiment 1, the lowest BHT concentrations able to reduce lipid peroxidation in boar spermatozoa were determined. Nine BHT concentrations (ranging from 0.025 to 3.2 mM) were evaluated, and the lowest (P <.05) production of malondialdehyde (MDA), as an indicator of lipid peroxidation, was obtained when BHT ranged from 0.2 to 1.6 mM. In experiment 2, sperm survivability was evaluated when BHT was added to a postthaw freezing extender by measuring the degree of sperm lipid peroxidation (using MDA production) and by measuring parameter such as motility, plasma membrane and acrosome integrity, and cell apoptosis. The ability of thawed spermatozoa to fertilize in vitro-matured oocytes and of embryos to develop to the blastocyst stage in vitro was also assessed. Pooled sperm-rich fractions collected from 3 mature Pietrain boars were frozen in 0.5-mL straws after dilution with lactose-egg yolk-glycerol-Orvus ES Paste extender supplemented with 0, 0.2, 0.4, 0.8, and 1.6 mM BHT. Postthaw sperm survival, evaluated 30 and 150 minutes after thawing, was higher in BHT-treated spermatozoa, being significant (P <.05) when the freezing extender was supplemented with 0.2, 0.4, and 0.8 mM BHT. The addition of BHT to the freezing extender resulted in a significant (P <.05) decrease in the MDA concentration in thawed spermatozoa, irrespective of the level of BHT used. BHT had no effect on oocyte cleavage rates, but the development to blastocyst was improved for embryos derived from spermatozoa frozen in extender supplemented with 0.4 mM BHT (16% vs 29% of blastocysts per total oocytes; P <.05). In conclusion, under the conditions tested in the present study, the addition of BHT to the freezing extender improved the overall efficiency of thawed boar spermatozoa.

Integration of future biotechnologies into the equine industry

“…Butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, also was shown to increase post-thaw sperm survival of boar sperm when added to the freezing extender. Furthermore, BHT improved the development of rate to blastocyst of embryos derived from sperm frozen in extender supplemented with BHT (Roca et al, 2004). Almeida and Ball (2005) compared the ability of ␣-tocopherol and a vitamin E ester, ␣-tocopherol succinate, to prevent lipid peroxidation and maintain motility of equine sperm during storage at 5 • C for 48 h. During short-term incubation in TALP-PVA, ␣-tocopherol succinate reduced membrane lipid peroxidation as determined by using the fluorescent probe C11 bodipy.…”

Quality of boar spermatozoa from the sperm-peak portion of the ejaculate after simplified freezing in MiniFlatpacks compared to the remaining spermatozoa of the sperm-rich fraction

“…Thus it is not surprising that certain amounts of SP present in slightly cooled or frozen semen can improve sperm motility (Rodriguez-Martinez, 1991), maintain acrosome integrity (Maxwell et al, 1997), delay capacitation-like changes (Pursel et al, 1973), and increase the resistance to cold shock (Pursel et al, 1973;Watson, 2000) or oxidative stress (Roca et al, 2004;. Maintenance of these sperm attributes obviously lead to higher sperm longevity, cryosurvival and, subsequently, fertility (Zhu et al, 2000;Hernández et al, 2007;Okazaki et al, 2009;Garcia et al, 2010).…”

“…Cryopreservation of boar semen has advanced over the past decade, with inclusion of various additives in the semen extender (Peña et al, 2003a(Peña et al, , 2004Roca et al, 2004Roca et al, , 2005, novel packaging systems (Eriksson & Rodriguez-Martinez, 2000;Saravia et al, 2009), and simplified freezing protocols (Saravia 4 et al, 2010). The latter was possible by findings in this laboratory, which demonstrated that the spermpeak portion of the ejaculate (the first 10 mL of the SRF, P1) can equally sustain conventional or simplified cryopreservation and -when this is done-more than 50% of the time allotted for conventional freezing is saved, avoiding extension or centrifugation.…”

Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen

“…vitamin E, glutathione, taurine) in the freezing extenders of frozen boar semen in an attempt to minimize the detrimental effect of ROS, which occurs during the freezing process [11][12][13][14]. Funahashi and Sano [15] demonstrated that a supplement of L-cysteine (5 mmol L −1 ) could improve the viability and progressive motility of fresh extended boar semen.…”