Beneficial Effect of Proline Supplementation on Goat Spermatozoa Quality during Cryopreservation

Abstract

Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.

Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins

“…The recovery of cells cryopreserved in the freezing solution containing d -proline at 48 h after thawing was 87%, which was higher than that of cells cryopreserved in CELLBANKER 1 plus. The cryoprotective effect of l -proline has been reported in previous studies. ,,− The present results showed that d -proline had a superior effect on cryopreservation of 2D-cultured cells.…”

“…28,29 L-proline and trehalose are natural cryoprotectants that accumulate in organisms as a defense mechanism against freezing in low temperature. 14,15,30 L-proline has been studied as a cryoprotectant for various biological samples, including red blood cells, 3 spermatozoa, 22,31,32 oocytes, 33 and stem cells. 34 Recently, the mechanisms by which L-proline aids in cellular cryopreservation have been postulated.…”

“…Amphiphilic small molecules and polymers as xeno-free cryoprotectants have shown promising results in improving cell viability and reducing cryoinjury, namely amino acids, , block copolymers with 2-methacryloyloxyethyl phosphorylcholine (MPC) and di­(ethylene glycol) methyl ether methacrylate (DEGMA), poly­(vinyl alcohol), tertiary sulfonium, and hydroxyethyl starch (HES). , These compounds stabilize cellular structures and minimize osmotic stress, leading to improved post-thaw cell survival and functionality. Considering our background in the development of peptide-based biomaterials, we began to focus on amino acids. , l -proline and trehalose are natural cryoprotectants that accumulate in organisms as a defense mechanism against freezing in low temperature. ,, l -proline has been studied as a cryoprotectant for various biological samples, including red blood cells, spermatozoa, ,, oocytes, and stem cells . Recently, the mechanisms by which l -proline aids in cellular cryopreservation have been postulated.…”

Optimizing Bull Semen Cryopreservation Media Using Multivariate Statistics Approaches

“…We expected to see positive direct effects of antioxidants on motility but did not. Thus, removing antioxidants with lower loadings like melatonin and NGF may be advantageous, with the possibility of substituting more powerful antioxidants like proline [ 31 ] and humanin [ 1 ].…”

Advances in Cryopreservatives: Exploring Safer Alternatives

“…It is important to know that dehydration will occur if there is higher osmotic pressure outside the cell. Some dehydration is beneficial for cryopreservation, as it reduces the formation of penetrating CPAs reproductive cells or even embryos of different species: avian sperm, 13 Nile tilapia sperm, 28 eel sperm, 29 Glossogobius giuris spermatozoa, 30 Mytilus embryos, 31 human oocytes, 32,33 human embryonic stem cells, 34 ovarian carcinoma cell lines, 35 and neuron-like cells 36 sulfoxides and amides DMSO, acetamide, dimethylformamide, or dimethylacetamide penetrating CPAs synthesized from dimethyl sulfide sperm cryopreservation methods, 39−53 Trichogaster fasciata embryos, 40,54 and human ovarian carcinoma cell lines (SKOV-3, OVCAR-8) 35 DMSO: limit ice nucleation and promote viability after thawing 37,38 amino acids and amines proline, glutamine, betaine, L-carnitine, or tricine neutral amino acids (β-alanine, γ-aminobutyric acid (GABA), and ε-aminocaproic acid (ε-Ahx)) proline: great osmoregulatory capacity, as well as a strong capacity to prevent water crystallization 55 proline: sperm samples, oocytes or in human endothelial cells 58 (sRBCs); 56 GLC-82, LTEP-a-2, 3T3 and smooth muscle cells; 55 Jurkat cells; 26 and human spermatozoa 59 proline + trealose: preservation of cell morphology 56 glutamine: spermatozoa, including humans, 60 boar, 61 ram 62 and rooster, 63 and epididymal sperm in Egyptian spiny mice 20 glutamine: improves motility and protects membrane integrity against damage caused by the freeze−thaw process 20 neutral amino acid-based (β-alanine, γ-aminobutyric acid (GABA), and ε-aminocaproic acid (ε-Ahx)) CPA solutions: cryopreserve GLC-82 cells and SMCs 64 betaine: hydrophilic molecule that has a high capacity for hydration through the ionic solvation effect with its charged groups (inhibitor of ice formation) intracellular ice. However, if the dehydration is very high, then irreversible damage to the cell can occur because of cryopreservation.…”