The Effects of Limonin, Myo-Inositol, and L-Proline on the Cryopreservation of Debao Boar Semen

Abstract

Semen cryopreservation is associated with sperm vulnerability to oxidative stress and ice crystal-induced damage, adversely affecting in vitro fertilization (IVF) success. This study aimed to investigate the effects of freezing diluent supplemented with antioxidant limonin (Lim), myo-inositol (MYO), and the ice crystal formation inhibitor L-proline (LP) through sperm motility, morphological integrity, and antioxidant capacity. The Lim (150 mM), MYO (90 mM), and LP (100 mM) significantly ameliorated the quality of post-thaw sperm in Debao boar, and combined treatment of these agents significantly enhanced sperm motility, structural integrity, and antioxidant capacity compared with individual agents (p < 0.05). Notably, the combined use of these agents reduced glycerol concentration in the freezing diluent from 3% to 2%. Meanwhile, the integrity of the sperm plasma membrane, acrosome membrane, and mitochondrial membrane potential was significantly improved (p < 0.05), and the result of IVF revealed the total cell count of the blastocysts was also greater in the 2% glycerol group (p < 0.05). In conclusion, the newly developed freezing diluent for semen, by adding Lim (150 mM), MYO (90 mM), and LP (100 mM), can enhance the quality of frozen-thawed Debao boar sperm and reduce the concentration of glycerol from 3% to 2% as high concentrations of glycerol can impair the quality of thawed sperm and affect in vitro fertilization outcomes. In conclusion, the improved dilution solution formulated demonstrated efficacy in enhancing the quality of porcine spermatozoa following cryopreservation and subsequent thawing.

Keywords: L-proline; freezing diluent; limonin; myo-inositol; sperm.

Figure 1

Effects of limonin (Lim; 0, 50, 100, 150, and 200 mM) supplementation on the freezing diluent on boar sperm cryo-survival. (A) Total motility (TM) of frozen–thawed sperm. (B) Morphology and structure of frozen–thawed sperm. (C) Mitochondrial membrane potential level of sperm of frozen–thawed sperm. (D) Total antioxidant capacity (T-AOC) level of frozen–thawed sperm. (E) Catalase (CAT) level of frozen–thawed sperm. (F) Glutathione peroxidase (GSH-PX) level of frozen–thawed sperm. (G) Superoxide dismutase (SOD) level of frozen–thawed sperm. (H) Malondialdehyde (MDA) level of frozen–thawed sperm. Different lowercase letters indicate statistical differences at p  <  0.05.

Figure 2

Effects of myo-inositol (MYO; 0, 30, 60, 90, and 120 mM) supplementation on the freezing diluent on boar sperm cryo-survival. (A) Total motility (TM) of frozen–thawed sperm. (B) Morphology and structure of frozen–thawed sperm. (C) Mitochondrial membrane potential level of sperm of frozen–thawed sperm. (D) Total antioxidant capacity (T-AOC) level of frozen–thawed sperm. (E) Catalase (CAT) level of frozen–thawed sperm. (F) Glutathione peroxidase (GSH-PX) level of frozen–thawed sperm. (G) Superoxide dismutase (SOD) level of frozen–thawed sperm. (H) Malondialdehyde (MDA) level of frozen–thawed sperm. Different lowercase letters indicate statistical differences at p  <  0.05.

Figure 3

Effects of L-proline (LP; 0, 50, 100, and 150 mM) supplementation on the freezing diluent on boar sperm cryo-survival. (A) Total motility (TM) of frozen–thawed sperm. (B) Morphology and structure of frozen–thawed sperm. (C) Mitochondrial membrane potential level of sperm of frozen–thawed sperm. (D) Total antioxidant capacity (T-AOC) level of frozen–thawed sperm. (E) Catalase (CAT) level of frozen–thawed sperm. (F) Glutathione peroxidase (GSH-PX) level of frozen–thawed sperm. (G) Superoxide dismutase (SOD) level of frozen–thawed sperm. (H) Malondialdehyde (MDA) level of frozen–thawed sperm. Different lowercase letters indicate statistical differences at p  <  0.05.

Figure 4

Effects of combined addition (Lim: 150 mM, MYO: 90 mM, and LP: 100 mM) on the freezing diluent on boar sperm cryo-survival. (A) Total motility (TM) of frozen–thawed sperm. (B) Morphology and structure of frozen–thawed sperm. (C) Mitochondrial membrane potential level of sperm of frozen–thawed sperm. (D) Total antioxidant capacity (T-AOC) level of frozen–thawed sperm. (E) Catalase (CAT) level of frozen–thawed sperm. (F) Glutathione peroxidase (GSH-PX) level of frozen–thawed sperm. (G) Superoxide dismutase (SOD) level of frozen–thawed sperm. (H) Malondialdehyde (MDA) level of frozen–thawed sperm. Different lowercase letters indicate statistical differences at p  <  0.05.

Figure 5

Effects of different concentrations of glycerol (0%, 1%, 2%, and 3%) on the freezing diluent on boar sperm cryo-survival. (A) Total motility (TM) of frozen–thawed sperm. (B) Morphology and structure of frozen–thawed sperm. (C) Mitochondrial membrane potential level of sperm of frozen–thawed sperm. (D) Total antioxidant capacity (T-AOC) level of frozen–thawed sperm. (E) Catalase (CAT) level of frozen–thawed sperm. (F) Glutathione peroxidase (GSH-PX) level of frozen–thawed sperm. (G) Superoxide dismutase (SOD) level of frozen–thawed sperm. (H) Malondialdehyde (MDA) level of frozen–thawed sperm. Different lowercase letters indicate statistical differences at p  <  0.05.

Figure 6

Effects of different concentrations of glycerol (0%, 2%, and 3%) on the freezing diluent on post-thaw boar sperm ultrastructure and fertilization ability. (A) SEM image of post-thawed boar sperm. Red circled areas in the images correspond to sites of structural defects in sperm. (B) TEM image of post-thawed boar sperm. PM, plasma membrane; C, acrosomal cap; M, mitochondrial sheath. Orange-boxed regions in the images indicate localized enlargements for detailed observation. (C) Cleavage rate after IVF with post-thawed boar sperm. (D) Blastocyst rate after IVF with post-thawed boar sperm. (E) Total number of cells per blastocyst after IVF with post-thawed boar sperm, scale bar: 200 μm. Different lowercase letters indicate statistical differences at p  <  0.05.