Effects of Bovine Serum Albumin and Trehalose in Semen Diluents for Improvement of Frozen-Thawed Ram Spermatozoa

Abstract

Abstract. In this study, two following experiments were performed to improve post-thaw motility and viability of frozen-thawed ram spermatozoa. We examined i) the effects of different concentrations of bovine serum albumin (0, 0.3, 1, 5, 10 and 15% BSA) in semen diluents lacking egg yolk and ii) the effects of four semen diluents, fructose (F: control) and trehalose (T) in semen diluents containing egg yolk, 15% BSA in semen diluents without egg yolk (BSA), and modified phosphate buffered saline (m-PBS). Frozen-thawed spermatozoa were examined for progressive sperm motility, viability, morphological abnormality, sperm tail swelling test, and sperm acrosome integrity. In Experiment 1, the rates of sperm motility immediately after thawing (0 h) were significantly (P<0.05) higher in the 10 and 15% BSA groups (55.0 ± 2.9 and 58.3 ± 6.7%, respectively) than in the positive control (F) group (41.7 ± 4.4%). The rate of sperm viability in the negative control (0% BSA) group (80.2 ± 3.3%) was significantly (P<0.05) lower than in the positive control (F) group (89.8 ± 1.5%), but when compared with the F group, no significant differences were found among the 0.3, 1, 5, 10 and 15% BSA groups at 0 h. The rates of sperm morphological abnormality of the 10 and 15% BSA groups (6.5 ± 1.3 and 6.3 ± 1.1%, respectively) were significantly (P<0.05) lower at 0 h than that in the 1% BSA group (16.3 ± 5.2%). In Experiment 2, T addition improved (P<0.05) the post-thaw motility compared with the F and BSA groups. Furthermore, at 3 and 6 h, the post-thaw motility of the T group (36.3 ± 2.4 and 25.0 ± 2.0%, respectively) was significantly (P<0.05) higher than in the BSA (26.3 ± 2.4 and 18.8 ± 1.3%, respectively) and F (28.8 ± 3.8 and 18.8 ± 2.4%, respectively) groups. The post-thaw sperm motility and viability in the m-PBS group were significantly (P<0.05) lower than those of the control (F), T, and BSA groups throughout all observation points. These results indicate that 10 and 15% BSA can be substituted for egg-yolk for ram semen diluent and that the addition of trehalose enhances motility and viability of ram spermatozoa after freezing and thawing. Key words: Bovine serum albumin (BSA), Semen diluent, Sheep, Spermatozoa, Trehalose (J. Reprod. Dev. 52: [675][676][677][678][679][680][681][682][683] 2006) t has been reported that frozen-thawed spermatozoa are greatly damaged during the freezing and thawing processes [1][2][3][4][5]. This damage may decrease sperm motility and the fertilization rate after artificial insemination (AI). Several semen analysis methods have been developed to evaluate the functional parameters of spermatozoa more objectively. The hypo-osmotic swelling test (HOS-test) has been applied to various species, such as dogs [6,7], cattle [8,9], horses [10][11][12], rams [13], minke whales (Balaenoptera bonarensis) [13] and humans [14,15]. It has been recognized that the HOS-Test is a simple and useful method with good reliability and repeatability for examination of the sperm plasma membrane.

Fertility of Ewes Inseminated Intrauterinally with Frozen Semen Using Extender Containing Bovine Serum Albumin

“…Bovine serum albumin (BSA) is one of proteins available for replacement of egg yolk [11,12]. Matsuoka et al [13] reported that the rates of postthaw sperm progressive motility were significantly higher in 10% and 15% BSA than in control extender (Tris-fructose-egg yolk) and concluded that 10 or 15% BSA could be substituted in place of egg yolk in a semi-defined semen extender. The present field trial confirmed these laboratory results [13].…”

“…Semen was collected from a Suffolk ram (3 years old) using an artificial vagina and was diluted (semen : extender=1:4) in a water bath (30 C) with one of the two Tris-based extenders containing 297.58 mM Tris, 96.32 mM citric acid, 82.66 mM fructose, 5% (v/v) glycerol, and either 15% (v/v) egg yolk [14] or 10% (w/v) BSA [13]. The diluted semen was gradually cooled to 4 C for 2-3 h. The diluted semen was frozen in 0.25 ml straws according to the methods described previously [13].…”

“…The diluted semen was gradually cooled to 4 C for 2-3 h. The diluted semen was frozen in 0.25 ml straws according to the methods described previously [13]. In brief, the semen samples were packed in straws and kept at 4 C before freezing.…”

“…Our previous study [13] showed that 10% or 15% BSA could be substituted for egg yolk in a semen extender for freezing of ram spermatozoa. Therefore, in the present study, we examined the fertility of ewes inseminated intrauterinally with frozen semen extender containing 10% BSA in comparison with an extender containing 15% egg yolk.…”

Pure Egyptian Cattle Bulls Show both Individual Variation and Different Interaction with Extender in the Post-Thawing Sperm Parameters

“…Semen cryopreservation is a well-developed technique commonly used worldwide; it extends the life of spermatozoa by decreasing sperm metabolism and toxin production, but induces partially irreversible injury to sperm membranes, which may reduce sperm motility, viability and the fertilization rate after artificial insemination [1][2][3][4][5]. Sperm damage during preservation in liquid nitrogen at -196°C has been attributed by many authors to cold shock, ice crystal formation, oxidative stress, osmotic changes, cryoprotectant toxicity and reorganizations of lipid-protein and phospholipid layers within the cell membranes [6][7][8].…”