Semen Cryopreservation Research Articles

Mitigating effect of chitosan-coated zinc oxide nanoparticles on cryopreservation-induced DNA damage in human spermatozoa

Zinc, an essential micronutrient, is an important mediator of sperm function. Supplementation of zinc oxide nanoparticle (ZnONPs) to semen cryopreservation medium has shown beneficial effects on sperm function, mainly due to its antioxidant and membrane stabilization properties. However, the ZnONPs are known to be membrane impermeable. In the current study, we explored the beneficial properties of membrane permeable zinc oxide nanoparticles on semen cryopreservation outcome. For the study, 52 liquefied human ejaculates were cryopreserved with chitosan-coated ZnONPs and stored in liquid nitrogen for one week. Samples were thawed and assessed for the sperm functional characters and DNA integrity. Significantly higher motility (P < 0.05), prolonged survival (P < 0.05) and lower DNA damage (P < 0.05) was observed in spermatozoa of semen samples cryopreserved with chitosan-coated ZnONPs. In conclusion, the presence of chitosan-coated ZnONPs in the ejaculate prior to cryopreservation increases the survival and decreases DNA damage without compromising with the functional competence of spermatozoa.

A comparative study on semen quality and cryopreservation ability in Italian native chicken breeds

This study provides a comparative assessment of fresh semen quality and sensitivity to cryopreservation in several Italian chicken breeds. The research involved 145 roosters from 13 breeds. The results showed a wide variability in all the parameters considered among the different breeds, especially in the quantitative variables of fresh semen, such as volume and concentration. For the qualitative characteristics (sperm membrane integrity and motility parameters), the variability across breeds was more pronounced for frozen than fresh semen. Interestingly, apart from total motility in fresh semen, breed had a significant effect on all semen quality parameters in both fresh and thawed ejaculates. Considering the overall qualitative characteristics, the Robusta maculata, Siciliana, and Mericanel della Brianza breeds produced ejaculates with better semen quality compared to other Italian breeds. By evaluating the main parameters of semen quality, our results underline the potential of these traits to influence the reproductive success and genetic conservation. The Bionda piemontese, Bianca di Saluzzo, Livorno bianca, Pepoi, and Siciliana breeds showed better resilience to cryopreservation, suggesting the need for breed-specific protocols to optimise semen quality after thawing. Importantly, the research highlights the central role of semen quality for both immediate fertilisation success and long-term conservation efforts. Future studies integrating OMICS technologies could elucidate molecular markers influencing breed-specific differences, helping to refine cryopreservation techniques and improve conservation strategies for indigenous Italian chicken breeds. This work contributes valuable insights to global efforts aimed at safeguarding poultry genetic diversity and sustainability.

Effect of Different Semen Extenders on Cat Epididymal Semen Cryopreservation

BACKGROUND: Cryopreservation of spermatozoa is a biotechnology used for fertilization purposes and preservation of genetic material in various domestic species. OBJECTIVE: To determine the efficacy of two different commercial semen diluents in the cryopreservation of epididymal semen of domestic cats. MATERIALS AND METHODS: Twenty male cats aged between 1 - 3 years and weighing 2.5 - 4.5 kg were used in the study. The testicular tissues removed from the cats were immediately brought to the laboratory in physiological saline and the epididymal parts were trimmed in commercial semen extenders (INRA 96, Group I; OPTIXCELL, Group II). Diluted semen samples were cooled to 4°C and filled into 0.25 mL straws. Semen samples were frozen in a programmable semen freezing device and then placed in a liquid nitrogen container at -196 C. Semen samples were thawed at 38°C for 25 s. Thawed semen samples were evaluated in terms of motility and kinematic parameters using CASA. RESULTS: No statistical difference was found between the groups in terms of total motility, progressive motility, and velocity parameters at 4 °C. The rate of spermatozoa at slow speeds was found to be lower in group II. In addition, after freezing and thawing process, no statistical difference was observed between the groups in terms of motility, kinematics, and velocity parameters. CONCLUSION: Both commercial semen extenders can be used for cryopreservation of cat epididymal semen.

Improvement of Post-Thaw Quality and In Vivo Fertility of Simmental Bull Spermatozoa Using Ferulic Acid.

Artificial insemination and semen cryopreservation have significantly improved the quality and quantity of cattle production. Through cryopreserved semen and artificial insemination, top-breeding bull sperm can be used to inseminate thousands of cows worldwide. Our study aimed to determine the effect of adding ferulic acid (FA) to a Tris-based semen extender on frozen and thawed Simmental bull sperm. Semen samples were collected from three Simmental bulls. Pooled Simmental semen (n=34 ejaculations) were diluted with a Tris-base extender containing varying FA concentrations (0.1, 0.15, 0.25, 0.35, and 0.45mM). After the samples were frozen and thawed, the samples were tested for malondialdehyde (MDA), total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GPx), total motility, progressive motility, motility characteristics, and plasma membrane functionality. The control and the groups with the best FA concentrations, 0.25 and 0.35, were compared for in vivo fertility. Fifty-one cows were inseminated 24h after the onset of oestrus. A rectal examination was used to diagnose pregnancies at least 60 days after fertilization. Results showed that adding FA-0.45, FA-0.35, FA-0.25, and FA-0.15 to the semen of Simmental bulls improved total and progressive motility, motility characteristics, and plasma membrane functionality. It also increased GPx and TAC levels, reducing MDA and DNA damage after freezing. The addition of FA did not affect SOD values. The fertility rate in the FA-0.25 and FA-0.35 groups was higher than in the control group, 35.29%, with rates of 76.47% and 70.58%, respectively. In conclusion, adding FA (0.15, 0.25, 0.35, and 0.45mM) to Tris-based semen extenders can improve the quality parameters of cryopreserved Simmental bull semen and increase in vivo fertility using 0.25 and 0.35 concentrations of FA.

Anethole improves mitochondrial activity and quality parameters in fresh and frozen-thawed ovine semen

Anethole, an antioxidant found in plants, appears to improve the survival of spermatozoa during semen cryopreservation. This study assessed the effects of commercial trans-anethole in ram semen cryopreservation. Thirty ejaculates from six rams were diluted in media containing anethole at the following concentrations: CONT (0 μM), AN10 (10 μM), AN50 (50 μM), and AN100 (100 μM). Semen was slow-frozen, preserved in liquid nitrogen, and thawed. Anethole at 10 μM or 50 μM did not compromise any studied sperm quality parameter but increased pre-freezing functionality of membrane and mitochondrial activity. At 10 μM, anethole reduced post-thawing spermatozoa lipoperoxidation. At 50 μM, anethole sustained higher mitochondrial activity after thawing, reduced minor defects in sperm, and increased the number of sperm binding to perivitelline membrane, while keeping lipoperoxidation levels as in control. Anethole at 100 μM promoted higher pre- and post-freezing mitochondrial activity and higher number of sperm binding to perivitelline membrane, in comparison to control. Additionally, some post-thawing kinematic parameters were enhanced by anethole at 100 μM. Of note, mitochondrial activity and lipoperoxidation were higher with anethole at 100 μM in comparison to 50 μM, not differing from control. At the hypoosmotic test, the highest concentration (100 μM) tested reduced sperm osmotic resistance. The results of this study indicate that using anethole in cryopreservation media promoted mostly positive effects on the fresh and post-thawed ram semen, and the advantages vary according to its concentration.

LC-MS metabolomics uncovers potential biomarkers of semen cryo-injury in goats.

Semen cryopreservation acts a crucial role in enhancing breed improvement and conserving genetic resources. However, it often leads to decreased sperm activity and reduced pregnancy rates. Despite significant advancements in semen freezing techniques for goats, the precise factors and mechanisms causing cryo-injury remain unclear. In this study, we examined the motility characteristics of fresh semen versus frozen-thawed semen and investigated changes in the metabolite profiles of seminal plasma using liquid chromatograph-mass spectrometry (LC-MS). A total of 364 differentially expressed metabolites (DEMs) were identified between fresh and frozen-thawed semen samples. Among these, 185 metabolites were significantly up-regulated, while 179 were down-regulated (p<0.05). The majority of these DEMs belonged to lipids and lipid-like molecules, as well as organic acids and derivatives. The Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that these DEMs were primarily involved in pathways related to amino acid synthesis and metabolism. Additionally, metabolite set enrichment analysis (MSEA) underscored the critical role of amino acid synthesis and metabolic pathways in semen cryopreservation. Specific metabolites such as alanine, proline, phenylalanine, tryptophan, tyrosine, adenosine, citric acid, flavin adenine dinucleotide (FAD), and choline emerged as potential biomarkers for sperm cryo-injury in goats. These findings provide valuable insights into enhancing the quality of semen cryopreservation in goats, contributing to improved breeding and genetic resource conservation efforts.

Using of Cottage Cheese Whey with or without Egg Yolk as a New Dilution for Cryopreservation of Ram Semen

Using of Cottage Cheese Whey with or without Egg Yolk as a New Dilution for Cryopreservation of Ram Semen

Piceatannol Protects Sperm from Cryopreservation Damage by Modulating the Keap1-Nrf2/ARE Signaling Pathway.

The purpose of this study was to explore the mechanism of action of Piceatannol (PIC) in attenuating oxidative damage to sperm during cryopreservation. Semen samples were collected and homogenized into six equal parts for freeze-thawing experiments. Four different concentrations of PIC were utilized as cryoprotectants during the freeze-thawing process, maintaing a semen to PIC ratio of 1:1, while sperm motility after freezing and thawing was analyzed using computer-assisted sperm analysis (CASA). Sperm plasma membrane integrity was assessed via the hypo-osmotic swelling (HOS) test. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities, long with the ability to scavenge sperm malondialdehyde (MDA), were examined in sperm following the addition of PIC. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression levels of Keap1, Nrf2, GCLC, GCLM, and HMOX1 in sperm. The mechanism by which PIC protects sperm during cryopreservation from oxidative stress damage was further verified. Treatment with PIC at a dose of 5.0 μmol/L significantly improved both sperm motility and viability while effectively reducing ROS levels in frozen sperm. Additionally, the integrity of the sperm plasma membrane was significantly enhanced. Furthermore, the expression level of Keap1 was significantly reduced, whereas the expression levels of GCLC, GCLM, HMOX1, and Nrf2 were significantly increased (p < 0.05) after the addition of PIC. Notably, a significant attenuation of sperm motility and viability was observed in this treatment group when PIC treatment was accompanied by the addition of an Nrf2 inhibitor, resulting in a significant elevation of ROS levels. The finding that PIC modulates ROS in frozen sperm via the Keap1-Nrf2/ARE pathway thereby enhancing sperm viability levels after freezing and thawing provides a novel approach to optimize semen cryopreservation.

Comparing pharmacological and manual collection for semen cryopreservation in dogs

Comparing pharmacological and manual collection for semen cryopreservation in dogs

Morphological characterization of spermatozoa in Apis mellifera and the effect of processing and semen storage

The aim of this study was to establish a standardized approach for evaluating sperm morphology in the honey bee and to investigate the impact of processing techniques and semen storage on the incidence of sperm morphoanomalies. The first experiment was designed to characterize the sperm morphoanomalies in unaltered honey bee semen, examining if they are compatible with sperm motility and viability and determining their occurrence in the ejaculates of mature and healthy honey bee drones. The different forms of sperm morphoanomalies were described in detail. In the morphological analysis of fresh drone ejaculates, 7.77% of spermatozoa showed abnormal morphology on average, with 2.20%, 4.75% and 0.81% of head, tail and multiple defects, respectively. The method also allowed us to determine the status of acrosome integrity. The second experiment was designed to assess the impact of smearing and air-drying on the occurrence of sperm morphoanomalies, showing a significant increase in the incidence of both head and tail sperm defects when comparing to wet samples. In the third and fourth experiments, the effect of semen storage at room temperature and semen cryopreservation, respectively, on the occurrence of sperm morphoanomalies were investigated. The preservation of semen at 22 °C led to a significant increase in spermatozoa with coiled tails after day 1, whereas the remaining anomalies did not exhibit significant variations over time. The freezing-thawing process showed a more pronounced effect on the incidence of morphological defects, with an increase in the percentage of spermatozoa with deformed heads and coiled tails.

Effects of Supplementation of Different Antioxidants to Cryopreservation Extender on the Post-Thaw Quality of Rooster Semen-A Meta-Analysis.

The standardization of the semen cryopreservation technique could be an effective tool in poultry for the conservation of genetic resources. During this process, the production of reactive oxygen species increases, leading to oxidative stress that causes damage to the spermatozoa. To reduce this effect, the addition of exogenous antioxidants in the cryopreservation diluent has been reported to be effective. Multiple antioxidants such as catalase, vitamin E, cysteamine, ergothioneine, and serine have been studied in roosters. Therefore, the present investigation aims to perform a meta-analysis to determine if the use of the aforementioned antioxidants added to the cryopreservation extender produces an improvement in semen quality parameters in roosters after thawing. After collecting the data, a discriminant canonical analysis was performed to determine which of the selected semen quality traits provided the most information, with hypo-osmotic swelling test (HOST), viability, and total motility variables showing the highest discriminatory power. However, according to the descriptive statistics, catalase and serine are the antioxidants that improve a greater number of seminal quality parameters, and since catalase gives the most favorable results for most of the discriminating variables, it could be the antioxidant of choice.

Damage to Mitochondria During the Cryopreservation, Causing ROS Leakage, Leading to Oxidative Stress and Decreased Quality of Ram Sperm.

Semen cryopreservation can achieve long-term preservation of sperm. Ice crystal damage, as well as oxidative stress, result in mitochondrial dysfunction and a reduction in sperm motility after thawing. However, limited information exists regarding the impact of reactive oxygen species (ROS) and mitochondria on the cryopreservation of ram sperm. The primary objective of this study was to investigate the relationship between ROS and mitochondria concerning sperm quality during the cryopreservation of ram sperm. This investigation assessed sperm motility, kinematic characteristics, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, expression of mitochondrial respiratory genes (NDUFV2, SDHA, CYC1, and COXIV), ROS levels, malondialdehyde (MDA) content, phosphatidylserine externalisation rate, sperm ultrastructure, mtDNA copy number, expression of apoptosis-related genes (Bax, Caspase-3, and Caspase-8), Cytochrome C, and Caspase-3 content. The results showed the cryopreservation significantly (p < 0.05) decreased motility, kinetic parameters, membrane integrity, acrosome integrity, MMP, ATP, mRNA expression levels of mitochondrial respiratory-related genes, and significantly (p < 0.05) increased ROS levels, MDA content, phosphatidylserine externalisation rate, damage of sperm ultrastructure, mtDNA copy number, mRNA expression levels of apoptosis-related genes, Cytochrome C and Caspase-3 content compared to the fresh semen group. In conclusion, the cryopreservation causes damage to mitochondria, leading to increased ROS and subsequent oxidative stress. This process also initiates mitochondrial dysfunction and interferes with the electron transport chain, ultimately resulting in decreased MMP and ATP production. Furthermore, the liberation of Cytochrome C prompted the increase in Caspase-3 expression and subsequent sperm apoptosis occurred, ultimately leading to a deterioration in sperm quality after thawing.

Seasonality analysis on cryopreserved doses from the autochthonous cattle breeds Asturiana de la Montaña and Asturiana de los Valles.

Germplasm banking is a fundamental tool for the preservation of autochthonous breeds. Semen cryopreservation is effective for this task, but protocols are adapted to commercial species, and post-thawing sperm quality could be sensitive to environmental cues. We compared the post-thawing sperm quality in doses from the CBA-SERIDA bank in northern Spain for the Asturiana de la Montaña (AM) and Asturiana de los Valles (AV) autochthonous cattle breeds. Doses from 23 AM and 16 AV bulls (ejaculates from at least three different seasons) were assessed for motility (computer-assisted sperm analysis), physiology and chromatin status (flow cytometry) after thawing and after 5 h at 38°C. Data were analysed using linear mixed-effects and cosinor models for seasonal and breed effects and by correlations with the association of sperm quality with temperature-humidity index (THI), considering the interval of spermatogenesis plus maturation. The breed affected sperm quality, with higher motility for AV and higher apoptotic ratio, mitochondrial activity, reactive oxygen species, DNA fragmentation and chromatin immaturity for AM. However, seasonality effects were minimal, and THI was not associated with sperm quality. In summary, the season seems to be a minor factor in the post-thawing quality of the AM and AV autochthonous breeds, well-adapted to their local environment.

High cooling rate of 60°C/min around ice nucleation during cryopreservation compromises chicken sperm viability.

The present study compares two protocols for the cryopreservation of chicken semen. Both protocols had an initial low cooling rate in the first step, followed by higher cooling rates around ice nucleation (Protocol 1) or following the dissipation of the latent heat of fusion (Protocol 2) in the second step. Semen ejaculates obtained from 12 roosters were diluted with Rootex with 6% dimethylformamide and frozen following either Protocol 1 (from +5°C to -10°C at 5°C/min and from -10°C to -130°C at 60°C/min) or Protocol 2 (from +5°C to -35°C at 7°C/min and from -35°C to -140°C at 60°C/min). Compared with fresh semen, following both protocols, cryopreservation resulted in reduced post-thaw sperm quality (p < .001). Post-thaw percentage of sperm with an intact plasma membrane was greater using Protocol 2 than Protocol 1 (p < .05). The results suggest that high cooling rates around the time of ice nucleation are not recommendable.

Advanced assisted reproduction technologies in endangered mammalian species.

A new synergistic approach of classical conservation strategies combined with advanced assisted reproduction technologies (aART) allows for protection and rescue of endangered keystone species at the brink of extinction, which can help to safeguard complex ecosystems. Reproduction biology and management in mammal species is not only challenging in regards to their diverging sizes, anatomy, and often unknown physiology; it also requires customized training or chemical restraint protocols for safe handling. Besides these general challenges, there are several new assisted reproduction techniques (ART) specifically tailored to critically endangered mammals. The current portfolio of ART in these mammalian taxa is ranging from sexual cycle characterization and manipulation, semen collection and cryopreservation, artificial insemination, biobanking of living cells, oocyte collection, invitro fertilization (IVF), and embryo production, embryo transfer as well as stem cell-derived invitro gametogenesis for generating gametes in culture. The article covers advanced assisted reproduction technologies (aART), success and challenges, as well as ethical implications.

Impact of semen cryopreservation season on invitro embryo production of prepubertal goat oocytes.

Goat production is affected by reproductive seasonality. Invitro embryo production (IVEP) could overcome this effect. This study aimed to evaluate the impact of the season of semen collection/freezing on IVEP of prepubertal goat oocytes and on sperm quality and functionality concerning capacitation. Semen from six fertile bucks was collected, pooled and cryopreserved in spring and autumn and used for IVEP of oocytes recovered during the breeding season. Oocytes were IVM in TCM-199 with hormones, EGF and cysteamine; fertilized and cultured in BO-IVF and BO-IVC media (IVF Bioscience, UK). Semen samples were assessed at 0 and 3 h after culture in capacitating (BO-IVF, CAP) and non-capacitating conditions for sperm plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), intracellular calcium and plasma membrane lipid disorder. Blastocyst production was higher with spring sperm compared to autumn (12.0% vs. 2.1%, respectively; p < .05). After CAP, acrosome reaction and intracellular calcium were higher (p < .05) in spring than autumn sperm. No differences were found in other sperm parameters. In conclusion, seasonal variations in the IVEP of prepubertal goats could be linked to differences in sperm ability to undergo invitro capacitation.

Effects of Butylated Hydroxytoluene and Sorbitol as Diluent Components on Structural and Surface Ultrastructural Changes of Gaga Chicken Sperm During Cryopreservation

The Gaga chicken is an indigenous Indonesian breed that is important to preserve using semen cryopreservation technology. The study was conducted to determine the effect of adding sorbitol and butylated hydroxytoluene (BHT) in the diluent on the structural and surface ultrastructure of cryopreserved Gaga chicken sperm during cryopreservation /frozen storage. The study aimed to assess how adding sorbitol and butylated hydroxytoluene (BHT) to the diluent affects the structure and surface ultrastructure of cryopreserved Gaga chicken sperm. A completely randomized design was employed with four treatments and 10 replications including egg yolk-lactate ringer diluent (EYLR) as the control group, EYLR diluent with 3 mM BHT, EYLR diluent with 2% sorbitol, and EYLR diluent with both 3 mM BHT and 2% sorbitol. Semen was collected using a massage technique from 4 male chickens aged approximately 10 months, pooled semen was diluted, packaged in 0.25 mL straws, equilibrated for 2 hours at 5 °C, pre-freeze for 10 minutes, frozen for 24 hours, and thawed for 30 seconds at 37 °C. The parameters evaluated were sperm plasma membrane integrity, acrosome integrity, DNA damage, mitochondrial functionality, and surface ultrastructure. The results showed that the treatment had a significant effect on plasma membrane integrity and post-thawing mitochondrial functionality compared to the control, but no effect was observed on acrosome integrity or DNA damage. The results showed that the combination treatment of BHT with sorbitol had a significant effect on plasma membrane integrity and post-thawing mitochondrial function, but did not affect acrosome integrity or DNA damage when compared to the control group. Ultrastructural observations indicated that cryopreservation caused damage to the head, middle, and tail of the sperm in the control groups. However, these changes were prevented by the diluent containing a combination of BHT and sorbitol. The addition of both components (BHT 3 mM + sorbitol 2%) effectively maintained plasma membrane integrity, mitochondrial functionality, and surface ultrastructure of Gaga chicken sperm during cryopreservation. Keywords: Butylated hydroxytoluene, Chicken sperm, Cryopreservation, Sorbito, Structure, Sperm ultrastructure

Long-term cryopreservation of bull semen as a main strategy for the ex situ conservation of endangered Polish Red cattle

Abstract Regular verification of the quality of cryopreserved semen derived from native cattle is one of the tasks performed at the bank as recommended by the Food and Agriculture Organization. The purpose of the present study was to evaluate the quality of semen from PR bulls stored for 40, 50 and 60 years in the BMB using standard evaluation parameters such as sperm motility as well as structural-functional parameters such as plasma membrane integrity, transmembrane mitochondrial potential and sperm chromatin damage. Semen pellets from 27 PR bulls (3 ejaculates/bull) were tested. The data were analysed by one-way and two-way ANOVA, and the significance of the difference (P≤ 0.01) between the means was determined using Duncan’s test. Our study results revealed that the long-term storage of semen had no effect on sperm characteristics after thawing. However, statistically significant differences (P≤0.01) in sperm plasma membrane integrity and transmembrane mitochondrial potential, following storage in liquid nitrogen were noted between bulls at all time points. However, there were no significant differences (P&gt;0.01) in sperm chromatin damage between breeds or between different storage times, and the degree of DNA fragmentation ranged from 0.4 to 0.8%.

Investigation of chia seed (Salvia hispanica L.) extract supplementation to extender on post-thawing ram sperm parameters and genes-related antioxidant capacity

Ram semen cryopreservation is not so widely used compared to bull semen. This study was carried out to explore the potential of methanolic chia seed extract (MCSE) in ram semen extender on kinematics, acrosome, apoptosis, antioxidant capacity, genes-related antioxidants, and caspase-3 in post-thawing ram semen. Semen from Rahmani rams (n=5) was collected, pooled, and diluted with Tris-extender (15 % egg yolk, 1 % soybean lecithin and 5 % glycerol) supplemented with MCSE (0, 125, 250, 375, and 500 µg/mL). MCSE had 67.5 % linolenic acid, 17.99 % linoleic acid, total phenolic, and flavonoid compounds. MCSE had higher antioxidant activity by DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and FRAP (Ferric-reducing antioxidant power) assays. MCSE (500 µg/mL) significantly improved sperm motility (43.6 vs. 30.0 %), vitality (44.0 vs. 30.7 %), and membrane integrity (43.1 vs. 31.3 %) as well as sperm velocity parameters and acrosome integrity (53.4 vs. 36.2 %) after thawing. MCSE at a level of 500 µg/mL increased viability, total antioxidant capacity level, and decreased apoptotic spermatozoa and malondialdehyde levels in post-thawed semen. MCSE at levels of 250, 375, and 500 µg/mL decreased caspase-3 level to 42.8, 35.6, and 30.0 % compared to control (54.6 %) after thawing, and resulted in a clear up-regulation of antioxidant-related genes of superoxide dismutase 1, catalase, glutathione peroxidase 1, and GA-binding protein transcription factor subunit beta-1 (GAPP1). The highest impact was observed for MCSE on GAPP1 as compared to other profiles as well as for MCSE at 500 µg/mL as compared to with other levels. All MCSE levels decreased Caspase-3 mRNA by about 44, 51, 61, and 61 %, respectively. In conclusion, MCSE had higher anti-oxidative activity. Tris-soya bean lecithin extender supplemented with MCSE at a level of 500 µg/mL, as a natural antioxidant, has a vital role in maintaining the freezing ability of ram spermatozoa after cryopreservation.

Effect of the addition of exogenous progesterone and the progesterone receptor inhibitor (RU 486) on boar cryopreservation semen extenders

Cryopreservation of porcine spermatozoa is detrimental due to their high sensitivity to cold shock, leading to changes akin to capacitation, known as cryocapacitation. These changes, including the acrosomal reaction, hypermotility induction, and protein phosphorylation, might be influenced by the presence of progesterone in seminal plasma and egg yolk, used in most freezing extenders. We tested the effect of various progesterone concentrations added to the freezing extenders (1, 10, and 100 μg/ml). At 100 μg/mL, progesterone decreased the proportion of straightness and tended to reduce viability and the proportion of progressive motility (p < 0.1). At 10 μg/mL, it increased reacted acrosomes in dead sperm (p < 0.05), protein phosphorylation rate (p < 0.05), and tended (p < 0.1) to enhance linear movement compared to the control. To counteract the capacitating effect of progesterone, we examined the effect of antiprogesterone mifepristone (RU 486) at concentrations of 5, 10, 20, 50, 100, and 200 μM, and co-incubated 10 μM of RU 486 with 10 μg/mL of progesterone. RU 486 maintained capacitation levels and motility parameters similar to the control, although high concentrations (100 μM) tended (p = 0.152) to increase protein phosphorylation. Co-incubation reduced the acrosome reaction in dead sperm, and RU 486 appeared to prevent hypermotility stabilizing motility and viability parameters compared to samples with progesterone alone. Protein phosphorylation increased and RU 486 could not restore capacitation to control levels due to its competitive antagonism for progesterone receptors, having less affinity than progesterone, which displaces RU 486 at high concentrations, allowing normal sperm capacitation.