43 Cryopreservation of Bovine Sperm Using Antifreeze Polyamino-Acid

Abstract

Carboxylated poly-l-lysine (CPLL), an ampholytic polymer compound, is reported to have a cryoprotective property similar to that of antifreeze proteins. We previously reported the effectiveness of CPLL as cryoprotective material for bovine sperm (43rd Annual Conference of International Embryo Technology Society, Austin, TX, USA; http://www.iets.org/2017/IETS_2017_Program_Book_FINAL.pdf). In this research, we investigated additional aspects of CPLL for bovine sperm. The conventional cryopreservation medium used for Control group consisted of 6.5% (v/v) glycerin, and the cryopreservation medium used for the CPLL group consisted of 3.25% (v/v) glycerin and 0.5% CPLL (w/v). In experiment 1, sperm motility was measured 1, 3, and 6 h after thawing. The post-thaw motility was assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). The CPLL treatment yielded better motility rate at 6 h (Control v. CPLL; 23.7% v. 38.5%; P < 0.01), average path velocity (μm s−1) at 1 and 3 h (Control v. CPLL; 49.8 v. 57.7, 35.8 v. 42.8; P < 0.01), straight-line velocity (μm s−1) at 1 h (Control v. CPLL; 35.2 v. 45.7; P < 0.01), and curvilinear velocity (μm/s) at 1 and 3 h (Control v. CPLL; 93.7 v. 106.2, 59.9 v. 68.4; P < 0.01) than the Control. In experiment 2, sperm membrane integrity was assessed by using the LIVE/DEAD Sperm Viability Kit (Thermo Fisher Scientific K.K., Kanagawa, Japan). The CPLL group yielded greater sperm membrane integrity rate than control (Control v. CPLL; 49.6% v. 60.6%; P < 0.01). In experiment 3, AI was carried out on 111 cows (Control v. CPLL; 49 v. 62) and the conception rate of the CPLL group was significantly higher than that of the control group (53.1% v. 79.0%; P < 0.01). Previously, we reported the effectiveness of CPLL for bovine sperm. In this study, we clarified how CPLL works to improve the conception rate of AI: CPLL maintains post-thaw motility and protects the sperm membrane. These results suggest that CPLL has potential as a new cryoprotective material for bovine sperm.

Cryopreservation of pig spermatozoa using carboxylated poly-L-lysine as cryoprotectant

“…This phenomenon was not observed when using DMSO, indicating that the toxicity of CPLL is lower than that of DMSO [8]. In addition, the membrane integrity rate of cryopreserved bovine spermatozoa and conception rate after AI were improved by reducing the glycerol concentration in the freezing extender from 6.5% to 3.25% and adding 0.5% CPLL [11]. Consequently, the improvement in the motility of frozen-thawed pig spermatozoa and in vitro development of derived embryos in the present study could be attributed to the addition of CPLL to the freezing extender, which enhanced the cryoprotective effect without increasing the toxic effect.…”

“…Consequently, the improvement in the motility of frozen-thawed pig spermatozoa and in vitro development of derived embryos in the present study could be attributed to the addition of CPLL to the freezing extender, which enhanced the cryoprotective effect without increasing the toxic effect. The most suitable concentration of CPLL in the presence of 3% glycerol for pig spermatozoa was 0.25%, whereas the most suitable concentration of CPLL in the presence of 3.25% glycerol for bovine spermatozoa was 0.5% [11]. Moreover, the most suitable concentrations of CPLL have been reported to be 0.75% in the presence of 5% glycerol for buffalo spermatozoa [12], 0.3% in the presence of 7% glycerol for human spermatozoa [14] and 1% in the presence of 8% DMSO for rabbit spermatozoa [13].…”

“…Recent studies have shown that CPLL is also effective for cryopreservation of germ cells such as pig embryos at the pronuclear stage [10] and bovine, buffalo, rabbit, and human spermatozoa [11][12][13][14]. In the present study, we investigated the efficacy of CPLL as a cryoprotectant for pig spermatozoa.…”