In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro . However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.
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.
In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive medicine, human serum albumin is frequently used because of low risks of zoonoses and allergic reactions. However, the risk of allogeneic infectious diseases exists, and the supply may be unstable because human serum albumin is derived from human blood. Therefore, the development of xeno-free human sperm cryopreservative reagents that could overcome the aforementioned problems is warranted. We succeeded in developing a new xeno-free and defined sperm cryopreservation reagent containing glycerol, carboxylated poly-l-lysine, and raffinose. The cryopreservation reagent was not significantly different in terms of sperm motility, viability, and DNA fragmentation and was comparable in performance to a commercial cryopreservation reagent containing human serum albumin. Moreover, the addition of saccharides was essential for its long-term storage. These results may help elucidate the unknown function of macromolecular-weight permeating cryoprotective agents.
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