Sperm cryopreservation is a vital tool for long-term preservation of genetic material, enabling the maintenance and transfer of genetic traits through assisted reproductive technologies.
Objectives:
This study aimed to assess the effects of incorporating Lactobacillus plantarum secretions (LS) into the cryopreservation protocol of goat semen.
Materials and Methods:
LS was added to semen extenders at concentrations of 20, 40, 60, 80, and 100 µL/mL. The control group received no additive. After freezing and thawing, various sperm quality parameters were evaluated.
Results:
The LS20 group showed significantly higher (p <0.05) total sperm motility compared with LS100, LS80, and the control. Progressive motility and straight-line velocity (VSL) were also improved in LS20 relative to LS100, though not significantly different from the control. LS20 demonstrated significantly higher amplitude of lateral head displacement (ALH) than the control, LS60, LS80, and LS100. LS40 also outperformed LS60–LS100 in ALH. Sperm viability was significantly increased in LS20 and LS40 compared with the control, LS80, and LS100. The sperm chromatin dispersion assay revealed significantly greater halo-to-core ratios in LS20 and LS40. Additionally, malondialdehyde levels, as a marker of oxidative stress, were markedly reduced in LS20 and LS40 compared with all other groups.
Conclusions:
Lower concentrations of LS, particularly 20 and 40 µL/mL, significantly improve sperm motility, viability, chromatin integrity, and oxidative status after thawing. These findings support the potential application of LS as an effective additive to enhance goat semen cryopreservation outcomes.
RezaeiA, BahmaniHR, MafakheriS, FarshadA, NazariP, MasoudiR. Protective effects of different doses of MitoQ separately and combined with trehalose on oxidative stress and sperm function of cryopreserved Markhoz goat semen. Cryobiology, 2023; 110:36–43; doi: 10.1016/j.cryobiol.2022.12.019
2.
BaghshahiH, RiasiA, MahdaviAH, ShiraziA. Antioxidant effects of clove bud (Syzygium aromaticum) extract used with different extenders on ram spermatozoa during cryopreservation. Cryobiology, 2014; 69(3):482–487; doi: 10.1016/j.cryobiol.2014.10.009
3.
YüceA, TürkG, ÇeribaşiS, SönmezM, ÇiftçiM, GüvençM. Effects of cinnamon (Cinnamomum zeylanicum) bark oil on testicular antioxidant values, apoptotic germ cell and sperm quality. Andrologia, 2013; 45(4):248–255; doi: 10.1111/and.12000
4.
MakkerK, AgarwalA, SharmaR. Oxidative stress & male infertility. Indian J Med Res, 2009; 129(4):357–367.
5.
MankeA, WangL, RojanasakulY. Mechanisms of nanoparticle-induced oxidative stress and toxicity. Biomed Res Int, 2013; 2013:942916; doi: 10.1155/2013/942916
6.
NickavarB, AlinaghiA, KamalinejadM, et al.Evaluation of the antioxidant properties of five Mentha species. Iranian Journal of Pharmaceutical Research, 2008; 7(3):203–209.
7.
SanockaD, KurpiszM. Reactive oxygen species and sperm cells. Reprod Biol Endocrinol, 2004; 2(1):1–7.
8.
BucakMN, SarıözkanS, TuncerPB, et al.The effect of antioxidants on post-thawed Angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Ruminant Research, 2010; 89(1):24–30; doi: 10.1016/j.smallrumres.2009.11.015
9.
BansalAK, BilaspuriGS. Impacts of oxidative stress and antioxidants on semen functions. Vet Med Int, 2010; 2010:686137; doi: 10.4061/2011/686137
10.
KhaliliB, JafaroghliM, FarshadA, Paresh-KhiaviM. The effects of different concentrations of Glycine and cysteine on the freezability of Moghani ram spermatozoa. Asian Australas J Anim Sci, 2010; 23(3):318–325; doi: 10.5713/ajas.2010.90387
11.
MaloC, GilL, CanoR, MartínezF, GaléI. Antioxidant effect of rosemary (Rosmarinus officinalis) on boar epididymal spermatozoa during cryopreservation. Theriogenology, 2011; 75(9):1735–1741; doi: 10.1016/j.theriogenology.2011.01.013
12.
NakibogluM, UrekRO, KayaliHA, TarhanL. Antioxidant capacities of endemic Sideritis sipylea and Origanum sipyleum from Turkey. Food Chem, 2007; 104(2):630–635; doi: 10.1016/j.foodchem.2006.12.012
13.
BaumberJ, BallBA, GravanceCG, MedinaV, Davies‐morelMC. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J Androl, 2000; 21(6):895–902; doi: 10.1002/j.1939-4640.2000.tb03420.x
14.
PanchawatS, RathoreKS, SisodiaSS, et al.A review on herbal antioxidants. Int J Pharmtech Res, 2010; 2(1):232–239.
15.
GadeaJ, MollaM, SellesE, MarcoMA, Garcia-VazquezFA, GardonJC. Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology, 2011; 62(1):40–46; doi: 10.1016/j.cryobiol.2010.12.001
16.
AzimiG, FarshadA, FarzinpourA, RostamzadehJ, SharafiM. Evaluation of used Purslane extracts in Tris extenders on cryopreserved goat sperm. Cryobiology, 2020; 94:40–48; doi: 10.1016/j.cryobiol.2020.05.001
17.
AriyanF, FarshadA, RostamzadehJ. Protective effects of Tribulus terrestris and Cinnamomum zeylanicum extracts and trehalose added to diluents on goat epididymal sperm freezability. Cryobiology, 2021; 98:172–180; doi: 10.1016/j.cryobiol.2020.11.005
18.
LiS, ZhaoY, ZhangL, et al.Antioxidant activity of Lactobacillus plantarum strains isolated from traditional Chinese fermented foods. Food Chem, 2012; 135(3):1914–1919; doi: 10.1016/j.foodchem.2012.06.048
19.
EvanovichE, de Souza Mendonça MattosPJ, GuerreiroJF. Comparative genomic analysis of Lactobacillus plantarum: An overview. Int J Genomics, 2019; 2019:4973214; doi: 10.1155/2019/4973214
WatterlotL, RochatT, SokolH, et al.Intragastric administration of a superoxide dismutase-producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice. Int J Food Microbiol, 2010; 144(1):35–41; doi: 10.1016/j.ijfoodmicro.2010.03.037
22.
Aguilar-ToaláJE, Estrada-MontoyaMC, LiceagaAM, et al.An insight on antioxidant properties of the intracellular content of Lactobacillus casei CRL-431. Lwt, 2019; 102:58–63; doi: 10.1016/j.lwt.2018.12.015
23.
TangW, LiC, HeZ, PanF, PanS, WangY. Probiotic properties and cellular antioxidant activity of Lactobacillus plantarum MA2 isolated from Tibetan kefir grains. Probiotics Antimicrob Proteins, 2018; 10(3):523–533.
24.
LiW, JiJ, ChenX, JiangM, RuiX, DongM. Structural elucidation and antioxidant activities of exopolysaccharides from Lactobacillus helveticus MB2-1. Carbohydr Polym, 2014; 102:351–359; doi: 10.1016/j.carbpol.2013.11.053
25.
ElfahriKR, VasiljevicT, YeagerT, DonkorON. Anti-colon cancer and antioxidant activities of bovine skim milk fermented by selected Lactobacillus helveticus strains. J Dairy Sci, 2016; 99(1):31–40; doi: 10.3168/jds.2015-10160
26.
SentürkM, ErcanF, YalcinS. The secondary metabolites produced by Lactobacillus plantarum downregulate BCL-2 and BUFFY genes on breast cancer cell line and model organism Drosophila melanogaster: Molecular docking approach. Cancer Chemother Pharmacol, 2020; 85(1):33–45.
27.
KimSH, SinghD, SonSY, et al.Characterization and temporal dynamics of the intra-and extracellular environments of Lactiplantibacillus plantarum using multi-platform metabolomics. Lwt, 2023; 175:114376; doi: 10.1016/j.lwt.2022.114376
28.
KannoT, KudaT, AnC, TakahashiH, KimuraB. Radical scavenging capacities of saba-narezushi, Japanese fermented chub mackerel, and its lactic acid bacteria. LWT-Food Science and Technology, 2012; 47(1):25–30; doi: 10.1016/j.lwt.2012.01.007
29.
ZhaiQ, WangG, ZhaoJ, et al.Protective effects of Lactobacillus plantarum CCFM8610 against acute cadmium toxicity in mice. Appl Environ Microbiol, 2013; 79(5):1508–1515; doi: 10.1128/AEM.03417-12
30.
RaadG, FakihF, BazziM, et al.Lactobacillus plantarum secretions may exert a cryoprotective effect on human sperm motility: A prospective in vitro study. Andrology, 2023; 11(7):1437–1450; doi: 10.1111/andr.13433
31.
Garrido-FernándezJ, Maldonado-BarragánA, Caballero-GuerreroB, Hornero-MéndezD, Ruiz-BarbaJL. Carotenoid production in Lactobacillus plantarum. Int J Food Microbiol, 2010; 140(1):34–39; doi: 10.1016/j.ijfoodmicro.2010.02.015
32.
TianX, YuZ, FengP, et al.Lactobacillus plantarum TW1-1 alleviates diethylhexylphthalate-induced testicular damage in mice by modulating gut microbiota and decreasing inflammation. Front Cell Infect Microbiol, 2019; 9:221; doi: 10.3389/fcimb.2019.00221
33.
AliSH, FarshadA, VaziryA, RostamzadehJ, AriyanF. Evaluation of ethanolic extract of prosopis farcta and trehalose on cryopreserved goat epididymal spermatozoa. Biopreserv Biobank, 2025; 23(3):214–224; doi: 10.1089/bio.2023.0132
34.
EvansG, MaxwellWC, et al.Salamons’ artificial insemination of sheep and goats. Tropical Animal Health and Production. Springer; 1987.
35.
RevellSG, MrodeRA. An osmotic resistance test for bovine semen. Anim Reprod Sci, 1994; 36(1–2):77–86; doi: 10.1016/0378-4320(94)90055-8
36.
WeitzKF. Unter suchungen zur Tiefgefriekonservierung von Kaninchensperma. Habil-Schr., Tieraerzh. Hochs. Honnover, Germany. 1977.
37.
EsterbauerH, CheesemanKH. Determination of aldehydic lipid peroxidation products: Malonaldehyde and 4-hydroxynonenal. Methods Enzymol, 1990; 186:407–421; doi: 10.1016/0076-6879(90)86134-H
38.
FernándezJL, MurielL, RiveroMT, GoyanesV, VazquezR, AlvarezJG. The sperm chromatin dispersion test: A simple method for the determination of sperm DNA fragmentation. J Androl, 2003; 24(1):59–66; doi: 10.1002/j.1939-4640.2003.tb02641.x
39.
SangeetaS, ArangasamyA, KulkarniS, SelvarajuS. Role of amino acids as additives on sperm motility, plasma membrane integrity and lipid peroxidation levels at pre-freeze and post-thawed ram semen. Anim Reprod Sci, 2015; 161:82–88; doi: 10.1016/j.anireprosci.2015.08.008
40.
DavoodianN, KadivarA, AhmadiE, MohebbiA. Effects of two amino acids on motion parameters and enzymatic antioxidant activity of freeze-thawed stallion spermatozoa. J Equine Vet Sci, 2017; 59:49–56; doi: 10.1016/j.jevs.2017.09.007
41.
BucakMN, TuncerPB, SarıözkanS, UlutaşPA. Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters, lipid peroxidation and anti-oxidant activities. Small Ruminant Research, 2009; 81(1):13–17; doi: 10.1016/j.smallrumres.2008.10.003
42.
KunduCN, DasK, MajumderGC. Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system. Cryobiology, 2001; 42(1):21–27; doi: 10.1006/cryo.2001.2296
43.
Ali Al AhmadMZ, ChatagnonG, Amirat‐BriandL, et al.Use of glutamine and low density lipoproteins isolated from egg yolk to improve buck semen freezing. Reprod Domest Anim, 2008; 43(4):429–436; doi: 10.1111/j.1439-0531.2007.00930.x
44.
Amirat-BriandL, BencharifD, Vera-MunozO, et al.Effect of glutamine on post-thaw motility of bull spermatozoa after association with LDL (low density lipoproteins) extender: Preliminary results. Theriogenology, 2009; 71(8):1209–1214; doi: 10.1016/j.theriogenology.2008.10.002
45.
DasD, GoyalA. Antioxidant activity and γ-aminobutyric acid (GABA) producing ability of probiotic Lactobacillus plantarum DM5 isolated from Marcha of Sikkim. LWT-Food Science and Technology, 2015; 61(1):263–268; doi: 10.1016/j.lwt.2014.11.013
46.
BarbonettiA, VassalloMRC, CinqueB, et al.Soluble products of Escherichia coli induce mitochondrial dysfunction-related sperm membrane lipid peroxidation which is prevented by lactobacilli. PLoS One, 2013; 8(12):e83136; doi: 10.1371/journal.pone.0083136
47.
DimCE, EkereSO, UgwuokeJI, Ndofor-FolengHM, OnyimonyiAE. Bio-appraisal of three strains of Lactobacillus based probiotics on the growth traits and semen characteristics of local toms. Afr J Biotechnol, 2020; 19(5):276–281; doi: 10.5897/AJB2020.17101
48.
Pourghassem GargariB, DehghanP, AliasgharzadehA, Asghari Jafar-AbadiM. Effects of high performance inulin supplementation on glycemic control and antioxidant status in women with type 2 diabetes. Diabetes Metab J, 2013; 37(2):140–148; doi: 10.4093/dmj.2013.37.2.140
49.
ZhangY, DuR, WangL, ZhangH. The antioxidative effects of probiotic Lactobacillus casei Zhang on the hyperlipidemic rats. Eur Food Res Technol, 2010; 231(1):151–158.
50.
FraserL, StrzeżekJ, KordanW. Effect of freezing on sperm nuclear DNA. Reprod Domest Anim, 2011; 46(Suppl 2):14–17; doi: 10.1111/j.1439-0531.2011.01815.x
51.
BaumberJ, BallBA, LinforJJ, MeyersSA. Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa. J Androl, 2003; 24(4):621–628; doi: 10.1002/j.1939-4640.2003.tb02714.x
52.
LewisSEM, AitkenRJ. DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res, 2005; 322(1):33–41.
53.
KudaT, KanekoN, YanoT, MoriM. Induction of superoxide anion radical scavenging capacity in Japanese white radish juice and milk by Lactobacillus plantarum isolated from aji-narezushi and kaburazushi. Food Chem, 2010; 120(2):517–522; doi: 10.1016/j.foodchem.2009.10.046
54.
LeeBJ, KimJS, KangYM, et al.Antioxidant activity and γ-aminobutyric acid (GABA) content in sea tangle fermented by Lactobacillus brevis BJ20 isolated from traditional fermented foods. Food Chem, 2010; 122(1):271–276; doi: 10.1016/j.foodchem.2010.02.071
55.
KobatakeE, NakagawaH, SekiT, MiyazakiT. Protective effects and functional mechanisms of Lactobacillus gasseri SBT2055 against oxidative stress. PLoS One, 2017; 12(5):e0177106; doi: 10.1371/journal.pone.0177106
56.
GaoD, GaoZ, ZhuG. Antioxidant effects of Lactobacillus plantarum via activation of transcription factor Nrf2. Food Funct, 2013; 4(6):982–989.
57.
HardingSV, FraserKG, WykesLJ. Probiotics stimulate liver and plasma protein synthesis in piglets with dextran sulfate-induced colitis and macronutrient restriction. J Nutr, 2008; 138(11):2129–2135.
58.
Rahimiyan-HeravanM, RoshangarL, KarimiP, et al.The potential therapeutic effects of Lactobacillus plantarum and inulin on serum and testicular reproductive markers in diabetic male rats. Diabetol Metab Syndr, 2020; 12(1):53; doi: 10.1186/s13098-020-00560-0
59.
HalttunenT, ColladoMC, El-NezamiH, MeriluotoJ, SalminenS. Combining strains of lactic acid bacteria may reduce their toxin and heavy metal removal efficiency from aqueous solution. Lett Appl Microbiol, 2008; 46(2):160–165; doi: 10.1111/j.1472-765X.2007.02276.x
ArasuMV, Al-DhabiNA, IlavenilS, ChoiKC, SrigopalramS. In vitro importance of probiotic Lactobacillus plantarum related to medical field. Saudi J Biol Sci, 2016; 23(1):S6–S10.
