47 Cryopreservation of Bovine Germ Cell Using Antifreeze Polyamino-Acid (Carboxylated Poly-L-Lysine)

Abstract

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.

Proximate Composition and Fatty Acid Profiles of Selected Fish Species From Pakistan

“…The addition of 3.25% (v/v) glycerol along with 0.5% (w/v) carboxylated poly L-Lysine (CPLL) in Japanese black bull semen improved sperm plasma membrane and fertility. The addition of CPLL in cryopreservation medium improves sperm cells survival due to its cryoprotective property and results in improved conception rates (Fujikawa et al, 2017) According to our knowledge, no work has been reported on addition of L-lysine in buffalo bull semen in terms of post-thaw quality. This study aimed to evaluate post-thaw semen quality parameters, antioxidant enzymatic activity and oxidative stress after addition of various concentrations of L-lysine in Nili-Ravi buffalo bull semen during cryopreservation.…”

Anti-Oxidative Effect of L-Lysine on Post Thaw Quality of Nili-Ravi Buffalo Bull Semen (Bubalus Bubalis)

“…The addition of 3.25% (v/v) glycerol along with 0.5% (w/v) carboxylated poly L-Lysine (CPLL) in Japanese black bull semen improved sperm plasma membrane and fertility. The addition of CPLL in cryopreservation medium improves sperm cells survival due to its cryoprotective property and results in improved conception rates (Fujikawa et al, 2017) According to our knowledge, no work has been reported on addition of L-lysine in buffalo bull semen in terms of post-thaw quality. This study aimed to evaluate post-thaw semen quality parameters, antioxidant enzymatic activity and oxidative stress after addition of various concentrations of L-lysine in Nili-Ravi buffalo bull semen during cryopreservation.…”