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.
Effects of the dose of heparin on the a, b: Values with diffe rent superscript letters were significantly different at the 5% level within c, d: Values with different superscript letters were significantly different at the 1% level within number of metaphase plates and on the sexing rates. for 2 h. SD). same stage. same stage. 713-717. 1972. 19) LARSON, R. C., G. G. IGNOTZ and W. B. CURRIE, Development of in vitro fertilized bovine embryos beyond the 8-cell block using completely defined medium containing fibronectin.
Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34 v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P = 0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48 h and hatched rate until 72 h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v. CPLL: 31 v. 50), and recipients were diagnosed for pregnancy ultrasonically 50 days after embryo transfer. Conception rate of CPLL group was higher than control group (50.0% v. 29.0%; P = 0.063). In both experiments, the significant differences between control group and CPLL group were determined by chi-squared test. The effectiveness of CPLL in cells and embryos has been reported; however, there is no report using CPLL in bovine germ cells. In this research, CPLL improved the conception rate of AI and ET, probably due to its low toxicity and protection of the cell membrane. These results suggest that CPLL is available as a new cryoprotective material for bovine sperm and embryo in slow freezing methods.
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