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

Abstract

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.

Improved fertility of frozen‐thawed porcine spermatozoa with 3,3‐dimethylglutaric anhydride poly‐L‐lysine as a novel cryoprotectant

“…The addition of DMGA-PLL to the freezing extender may have increased the chance that ovulated oocytes would encounter fertile spermatozoa because the treatment improved the motility of spermatozoa at 12 h after thawing. Our previous study investigating the ability of spermatozoa cryopreserved with glycerol and CPLL to produce offspring after AI showed that the mean number of total piglets was significantly lower than that produced by sows inseminated with spermatozoa stored at 17 C. In addition, the mean number of live piglets tended to be lower (Jin et al, 2022). In contrast, in this study, there were no significant differences in the mean numbers of total and live piglets between spermatozoa cryopreserved with glycerol and DMGA-PLL and those stored For AI-based pig production on commercial farms, it is crucial to obtain as many live piglets as possible from inseminated sows.…”

“…Our previous study investigating the ability of spermatozoa cryopreserved with glycerol and CPLL to produce offspring after AI showed that the mean number of total piglets was significantly lower than that produced by sows inseminated with spermatozoa stored at 17°C. In addition, the mean number of live piglets tended to be lower (Jin et al, 2022). In contrast, in this study, there were no significant differences in the mean numbers of total and live piglets between spermatozoa cryopreserved with glycerol and DMGA‐PLL and those stored at 17°C.…”

“…A modified Niwa and Sasaki freezing extender (mNSF) (Jin et al, 2022), composed of 80% (v/v) of 0.31 M trehalose anhydrous (Hayashibara Co., Ltd., Okayama, Japan), 20% (v/v) of egg yolk, 0.075 mg/mL amikacin sulfate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), 0.025 mg/mL dibekacin sulfate (Meiji Seika), and 250 U/mL penicillin G potassium (Meiji Seika), was used as a basic freezing extender. Modified NSF‐1 (mNSF‐1) was prepared by adding 0.74% (v/v) Equex‐STM Paste (Reproduction Provisions LLC, Walworth, WI, USA) to the mNSF.…”

“…Carboxylated poly‐L‐lysine (CPLL) is an ampholytic polymer compound obtained by synthesizing an aqueous solution of ε‐poly‐L‐lysine and succinic anhydride, followed by the conversion of 65 mol% of the amino groups to carboxyl groups (Matsumura & Hyon, 2009; Matsumura et al, 2021). As CPLL has been shown to be effective for the cryopreservation of germ cells, such as porcine embryos at the pronuclear stage (Kamoshita et al, 2017) and bovine, buffalo, rabbit, and human spermatozoa (Fujikawa et al, 2018; Küçük et al, 2021; Takeuchi et al, 2021; Tariq et al, 2020), we investigated the efficacy of CPLL as a cryoprotectant for porcine spermatozoa in a previous study (Jin et al, 2022). The findings showed that the addition of CPLL to a glycerol‐containing freezing extender improved the motility of frozen‐thawed porcine spermatozoa and the in vitro development of embryos produced by in vitro fertilization.…”

“…in groups of 40 to 50 under paraffin oil (Nacalai) in a 35 mm polystyrene dish(Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and cultured for 36-38 h at 38.5 C in an atmosphere with 5% CO 2 .Following in vitro maturation, cumulus cells were removed from oocytes by vortexing the COCs in 2 mL of HEPES-TLP-PVA supplemented with 0.1% (w/v) hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) at 38.5 C for 90 s. Denuded oocytes were transferred to droplets (80 μL) of fertilization medium(Jin et al, 2022) in groups of 15 to 20 under paraffin oil in a 35 mm polystyrene dish. Immediately before insemination, the frozen spermatozoa were thawed as described above.…”