Cryopreservation Of Boar Semen Research Articles

Pyrroloquinoline Quinone Improved Boar Sperm Quality via Maintaining Mitochondrial Function During Cryopreservation.

Due to oxidative damage and mitochondrial dysfunction, boar semen cryopreservation remains a significant challenge. This study investigates the effects of pyrroloquinoline quinone (PQQ), a mitochondrial-targeted antioxidant, on the post-thaw boar sperm quality during cryopreservation. Boar semen was diluted in a freezing extender containing different concentrations of PQQ (0, 10, 100, 1000, 10,000 nM). After freezing-thawing, the sperm motility, viability, acrosome integrity, mitochondrial activity, adenosine triphosphate (ATP) levels, DNA integrity, malondialdehyde (MDA) levels, reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, mitochondrial transcription proteins levels, and fertilization capacity were assessed. The results show that 1000 nM PQQ supplementation to the freezing extender significantly enhanced post-thaw sperm motility, viability, and acrosome integrity compared to the control (p < 0.05). Additionally, 1000 nM PQQ increased mitochondrial membrane potential (MMP) and ATP levels, while decreasing MDA and mitochondrial ROS levels, and reducing DNA damage (p < 0.05). Furthermore, the levels of mitochondrial-encoded proteins were significantly elevated in the 1000 nM PQQ group compared to the control (p < 0.05). Interestingly, sperm in the 1000 nM PQQ group showed a higher binding rate to oviductal epithelial cells and the zona pellucida (ZP), indicating higher fertilization potential. These findings suggest that the use of mitochondria-target antioxidant, PQQ, can improve post-thaw boar sperm quality and fertilization via its capacity to reduce oxidative stress and protect mitochondrial function.

Preliminary insights into Red LED Irradiation for thawing cryopreserved boar semen

Preliminary insights into Red LED Irradiation for thawing cryopreserved boar semen

Effects of Taurine and Vitamin E on Sperm Characteristics and Oxidative Stress in the Freezing Semen of Pigs

Background: We evaluated the effects of taurine and vitamin E on sperm characteristics (motility and viability) and oxidative stress (reactive oxygen species and nitric oxide) in the freezing semen of pigs. We determined sperm motility in fresh and freezing boar semen. Methods: Viability, reactive oxygen species, and nitric oxide determination in freezing sperm were determined by SYBR-14/PI kit, DCF-DA and DAF-2. Result: The motility of freezing sperm was not significantly different between taurine- and vitamin E-treated samples. We found that taurine and vitamin E increased the viability of sperm, and decreased reactive oxygen species and nitric oxide in the freezing semen of pigs. In conclusion, the use of taurine and vitamin E in boar semen cryopreservation can enhance sperm quality via protection from reactive oxygen species and nitric oxide damage.

Motility degradation rate, plasma membrane integrity, and kinematics during cryopreservation of boar (Sus scrofa domesticus) spermatozoa in different freezing extenders and thawing temperatures

Despite the very limited use of frozen-thawed semen (FTS) in pig artificial insemination, FTS in some instances can be truly beneficial as it is not constrained by time (shelf-life) and space (regional quarantine) restrictions unlike fresh-extended semen (FES). It also allows long-term banking of highly valuable genetics particularly during epidemics. This study compares existing and currently available freezing extenders used in boar semen cryopreservation aimed to optimize protocols useful for in-country local swine industry with special focus on the motility degradation rate (MDR), and plasma membrane structural (percent live) and functional (HOST reactive) integrity. Treatment samples from ten freezing runs using five different sperm-rich fractions were frozen using three different cooling/freezing extenders (CE/FE): A) LEYGE, B) BF5, and C) Cryoguard (~500 x 106 spz/mL) in liquid nitrogen (LN2) vapor, thawed either at ~38°C or ~50°C for 20 sec, and examined using the Sperm Class Analyzer® CASA system. LEYGE had significantly the highest MDR from about 50% reduction post-thawing to 70% one hour thereafter. Cryoguard consistently had the lowest MDR although closely similar to BF5. There was a minimal effect on the plasma membrane functional integrity and was primarily limited to LEYGE and BF5. A fertility trial is recommended to attest the performance of FTS vs FES in terms of conception rates and the litter size following post-cervical AI before full-scale production and potential adoption by the breeder swine industry.

Proteomic Analysis of Differences in the Freezability of Porcine Sperm Identifies α-Amylase As a Key Protein.

To investigate the mechanisms underlying the differences in the freezability of boar semen, Yorkshire boars with freezing-tolerant semen (YT, n = 3), Yorkshire boars with freezing-sensitive semen (YS, n = 3), Landrace boars with freezing-tolerant semen (LT, n = 3), and Landrace boars with freezing-sensitive semen (LS, n = 3) were selected for this study. Their sperm was subjected to protein extraction, followed by data-independent acquisition proteomics and functional bioinformatics analysis. A total of 3042 proteins were identified, of which 2810 were quantified. Some key KEGG pathways were enriched, such as starch and sucrose metabolism, carbohydrate digestion and absorption, mineral absorption, the HIF-1 signaling pathway, and the necroptosis pathways. Through PRM verification, we found that several proteins, such as α-amylase and epididymal sperm-binding protein 1, can be used as molecular markers of the freezing resistance of boar semen. Furthermore, we found that the addition of α-amylase to cryoprotective extender could significantly improve the post-thaw motility and quality of boar semen. In summary, this study revealed some molecular markers and potential molecular pathways contributing to the high or low freezability of boar sperm, identifying α-amylase as a key protein. This study is valuable for optimizing boar semen cryopreservation technology.

Enrichment of thawed boar spermatozoa with an intact membrane using Magnetic Activated Cell Sorting

Not all boar sperm samples survive cryopreservation well. A method of eliminating damaged sperm might enable more cryopreserved boar semen to be used for pig breeding. In this study we investigated the use of Magnetic Activated Cell sorting (MACS) to eliminate damaged sperm from thawed boar semen samples. The thawed samples were mixed with Dead cell removal particles and were applied to the column in a SuperMACS II. Different fractions were collected: Original sample (O), Flow-through (FT), and Eluate (E). Sperm membrane integrity, mitochondrial membrane potential and reactive oxygen species were evaluated by flow cytometry after staining with SYBR 14 and propidium iodide, or 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolylcarbocyanine iodide, or hydroethidine and dichlorodihydrofluorescein diacetate, respectively. The FT samples had increased membrane integrity, a greater proportion of sperm with high mitochondrial membrane potential and a greater proportion of sperm negative for hydrogen peroxide than O samples (P<0.0001), which in turn had increased membrane integrity than E samples (P <0.0001). However, differences were seen between boars. The FT samples had increased values of live, superoxide positive sperm than O samples (P <0.0001) and O samples had greater values than E samples (P <0.0001), while there was no effect of boar. Sperm quality was best in the FT fraction, comprising approximately 32% of the sperm sample. In conclusion, although there were differences between boars, MACS separation can improve sperm quality in thawed semen samples. It would be interesting to see if this improvement is reflected in fertility outcomes.

Camellia oil with its rich in fatty acids enhances post-thawed boar sperm quality

BackgroundBoar sperm are highly susceptible to specific conditions during cryopreservation, leading to a significant decrease in their fertilizing potential due to damage to their membranes. Camellia oil, known for its fatty acids with antioxidant and biological properties, has not been previously explored for the cryopreservation of boar semen. This study aimed to examine the effects of camellia oil on post-thawed boar sperm quality. Boar semen ejaculates (n = 9) were collected and divided into six equal aliquots based on camellia oil concentrations (0, 0.5, 1, 1.5, 2 and 2.5% v/v) in the freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm morphology by scanning electron microscope, sperm motility using a computer-assisted sperm analyzer, sperm viability, acrosome integrity, mitochondrial function, MDA level and total antioxidant capacity.ResultsThe results demonstrated that the supplementation of 1.5% (v/v) camellia oil showed superior post-thaw sperm qualities such as improved sperm morphology, motility, acrosome integrity and mitochondrial function by 14.3%, 14.3% and 11.7%, respectively, when compared to the control group. Camellia oil at a concentration of 1.5% (v/v) showed the lowest level of MDA (18.3 ± 2.1 µmol/L) compared to the other groups.ConclusionsIn conclusion, adding 1.5% (v/v) camellia oil in the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in a higher post-thawed sperm quality.

Post-Thaw Storage Temperature Influenced Boar Sperm Quality and Lifespan through Apoptosis and Lipid Peroxidation.

Cryopreservation deteriorates boar sperm quality and lifespan, which restricts the use of artificial insemination with frozen-thawed boar semen in field conditions. The objective of this study was to test the effects of post-thaw storage time and temperature on boar sperm survival. Semen ejaculates from five Landrace boars (one ejaculate per boar) were collected and frozen following a 0.5 mL-straw protocol. Straws from the five boars were thawed and diluted 1:1 (v:v) in BTS. The frozen-thawed semen samples were aliquoted into three parts and respectively stored at 5 °C, 17 °C, and 37 °C for up to 6 h. At 0.5, 2, and 6 h of storage, sperm motility, viability, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels and apoptotic changes were measured. Antioxidant and oxidant levels were tested in boar sperm (SPZ) and their surrounding environment (SN) at each timepoint. The results showed significant effects of post-thaw storage time and temperature and an impact on boar sperm quality (total and progressive motility, VCL, viability, acrosome integrity), early and late sperm apoptotic changes, and changes in MDA levels in SPZ and SN. Compared to storage at 5 °C and 37 °C, frozen-thawed semen samples stored at 17 °C displayed better sperm quality, less apoptotic levels, and lower levels of SPZ MDA and SN MDA. Notably, post-thaw storage at 17 °C extended boar sperm lifespan up to 6 h without obvious reduction in sperm quality. In conclusion, storage of frozen-thawed boar semen at 17 °C preserves sperm quality for up to 6 h, which facilitates the use of cryopreserved boar semen for field artificial insemination.

Effect of L-proline on sperm quality during cryopreservation of boar semen

L-proline has been reported to be useful in semen cryopreservation. However, its use has rarely been reported in the freezing of boar semen. The objective of this study was to evaluate the effects of different concentrations of L-proline (0, 10, 30, 50, and 90 mM) on the quality of boar semen after freezing and thawing. Semen samples from boars (n = 6) were frozen using freezing extenders with added concentrations of L-proline. Total sperm motility, progressive motility, survival time at 37 °C, acrosome integrity, mitochondrial activity, DNA integrity, the content of the lipid peroxidation product malondialdehyde (MDA), total antioxidant capacity (T-AOC) and, expression levels of apoptosis protein (cleaved caspase 3 and Bax) were evaluated after thawing. The results showed that total sperm viability (73.96% vs. 63.58%) and progressive motility (56.88% vs. 47.26%) after thawing were significantly higher in the 10 mM L-proline treatment group than in the control group. The survival time at 37 °C and the total motility of sperm in the 10 mM group within one hour after thawing were significantly higher than in the control group. Acrosome integrity and mitochondrial activity of sperm in the 10 mM group were significantly higher than those in the control, 50 mM, and 90 mM groups. The DNA integrity rate in the 10 mM group was significantly higher than in the control group. The L-proline treatment did not affect sperm MDA content or T-AOC. The expression levels of apoptosis protein (cleaved caspase 3 and Bax) in the 10 mM L-proline supplemented group were lower than those in the control group. In conclusion, the freezing extender containing 10 mM L-proline improved semen quality after freezing and thawing and thus would be a useful reagent for boar semen cryopreservation.

Palm Kernel Meal Protein Hydrolysates Enhance Post-Thawed Boar Sperm Quality.

Boar sperm is sensitive to particular conditions during cryopreservation, resulting in an extreme reduction in fertilizing ability due to damage to the sperm membranes. PKMPH contains bioactive peptides that have antioxidant and antimicrobial activities. There is no information on the use of palm-kernel-meal-derived bioactive peptides for boar semen cryopreservation. This study aimed to examine the effects of bioactive peptides from PKMPH on post-thawed boar sperm quality. Boar semen ejaculates (n = 17) were collected and divided into six equal aliquots based on PKMPH concentrations (0, 1.25, 2.5, 5, 10, and 15 µg/mL) in a freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, the frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm motility using a computer-assisted sperm analyzer and for sperm viability, acrosome integrity, mitochondrial function, and lipid peroxidation by measuring the level of malondialdehyde (MDA). The results demonstrate that the supplementation of PKMPH with 2.5 µg/mL afforded superior post-thawed sperm qualities, such as increased total motility, viability, acrosome integrity, and mitochondrial function by 10.7%, 12.3%, 18.3%, and 12.7%, respectively, when compared to the control group. PKMPH at a concentration of 2.5 µg/mL showed the lowest level of MDA (40.6 ± 2.0 µMol/L) compared to the other groups. In conclusion, adding PKMPH peptides at 2.5 µg/mL to the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in higher post-thawed sperm quality.

Antioxidant Effects of Myo-Inositol Improve the Function and Fertility of Cryopreserved Boar Semen.

During cryopreservation, sperm undergoes structural and molecular changes such as ice crystal formation, DNA fragmentation, and reactive oxygen species (ROS) production, leading to decreased sperm quality after thawing. Antioxidants play a crucial role in preventing these damages, both in vivo and in vitro. One potent antioxidant is myo-inositol, known for its protective effects on sperm against ROS. This study aimed to investigate the protective effect of myo-inositol on cryopreserved boar semen. The semen was diluted, cooled, and cryopreserved using a BF5 extender. It was then divided into five groups: control and different concentrations of myo-inositol (0.5, 1, 1.5, and 2 mg/mL). The post-thaw evaluation included assessments of motility, viability, acrosome integrity, mitochondrial membrane potential (MMP), caspase activity, gene expression, ROS levels, apoptosis, and IVF with treated semen. Results showed that myo-inositol at 0.5 mg/mL improved motility, acrosome integrity, and fertilization ability. It also reduced the expression of pro-apoptotic genes and increased SMCP expression. Lower concentrations also demonstrated improved viability and reduced apoptosis and ROS levels. In conclusion, myo-inositol treatment during cryopreservation improved sperm quality, reduced apoptosis and ROS levels, and enhanced fertility rates in boar semen.

Profiling of differentially expressed proteins between fresh and frozen-thawed Duroc boar semen using ProteinChip CM10.

Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.

Cryoprotectant effects of natural honey on spermatozoa quality of pre-freezing and frozen-thawed boar semen.

Natural honey has been successfully used in the preservation of mammalian gametes because of its beneficial properties. The objectives of this study were to determine the inclusion level of honey in extender for improving boar semen quality before freezing and to investigate the effects of honey inclusion in extender and freezing media on post-thaw quality of frozen-thawed boar semen samples. Ejaculates from six terminally crossbred boars were collected using the gloved-hand technique for two experiments. Experiment 1 was a randomized block design, evaluating four inclusion levels of honey in boar semen extender [Control (0H)-Androhep Plus or Androhep Plus with 0.25%, 0.50%, and 0.75% honey (0.25H, 0.50H, and 0.75H respectively)]. Ejaculates were pooled, aliquoted according to treatments, and cooled for 24 h at 17 ºC. The results of this experiment were used to determine inclusion levels in exp. 2. Experiment 2 was a 2 x ×3 factorial design, evaluating the inclusion of honey in boar semen extender and freezing media. Semen samples from individual boars were cooled in extender with or without honey (C0: Androhep Plus; C1: Androhep Plus + 0.25% honey). After 24 h, semen samples were evaluated, diluted in lactose-egg yolk (LEY) media, and one of three freezing media types; F0: 93% LEY + 6% glycerol + 1% Equex-STM Paste (ESP); F1: 93% LEY + (3% glycerol and 3% honey) + 1% ESP; and F2: 93% LEY + 6% glycerol + (0.5% ESP and 0.5% honey). Samples were frozen in 0.5 mL straws using a controlled-rate freezer and stored in liquid nitrogen. In exp. 1, 0.25H and 0.50H improved motility (P = 0.033) and progressive motility (P = 0.001) of cooled boar semen. Nevertheless, 0.25H was selected for exp. 2. In exp. 2, post-thaw motility and progressive motility were highest (P < 0.05) in C0F2 but not different from C1F2. Morphologically normal cells and acrosomes were higher with all inclusion levels of honey (P < 0.05). In conclusion, 0.25% and 0.50% inclusion of honey in Androhep Plus improves motility and progressive motility of cooled boar semen samples after 24 h. Supplementing Androhep Plus with 0.25% honey maintains higher normal sperm cells and acrosomes of cryopreserved boar semen. Replacing 50% Equex-STM paste with honey in freezing media improves post-thaw sperm motility, progressive motility, percentage of normal sperm, and acrosome of cryopreserved boar semen.

Effects of mitoquinone (MitoQ) supplementation during boar semen cryopreservation on sperm quality, antioxidant status and mitochondrial proteomics

Mitochondria are the most important organelles and the main reactive oxygen species producers in spermatozoa. The objective of this study was to investigate the effects of mitochondria-targeted antioxidant mitoquinone (MitoQ) supplementation in boar semen extender during cryopreservation on sperm quality, antioxidant status and the changes of sperm mitochondrial proteomic profile. Semen collected from 10 Large White boars was cryopreserved in lactose-egg yolk extender supplemented with various concentrations of MitoQ (0, 5, 10, 20 and 40 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The comprehensive mitochondrial proteomic profiling was performed on spermatozoa in the control and MitoQ10 groups. Supplementation with 10 μM of MitoQ resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed boar sperm were also elevated in the MitoQ10 group. Excessive MitoQ (40 μM) supplementation induced a reduction of sperm motility parameters, sperm quality and antioxidant status and the abundance of GLUT3 and 8 proteins. A total of 189 proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05, and 33 of them were dramatically (FC > 1.5) regulated by MitoQ. These DEPs are mainly involved in sperm motility, energy metabolism and oxidative phosphorylation. Our data suggest that the beneficial effect of MitoQ on cryopreserved boar semen is achieved by regulating sperm antioxidant capacity and the mitochondrial proteins related to motility and metabolism.

Impact of cryopreservation protocols (one- and two-step) on boar semen quality at 5 °C and post-thawing

The two-step protocol (2 S) is currently used for boar semen cryopreservation. In this method, the cryoprotectant penetrant agents (CPAs) are added at 5 °C to reduce the toxicity of CPAs. An alternative is the one-step protocol (1 S), which is easier, cheaper, and reduces the necessity of equipment, but could increase the toxicity of CPAs. Currently, there are no studies that compared both protocols for boar semen cryopreservation. This experiment aimed to study the effect of cryopreservation protocol (1 S vs 2 S) on boar spermatozoa. In the one-step protocol, after centrifugation, the spermatozoa pellet was resuspended at 17 °C in the extender containing CPAs to achieve a concentration of 1 × 109 spermatozoa/mL and then submitted to cryopreservation. For the two-step protocol, the sperm pellet was resuspended in fraction A at 17 °C to achieve a concentration of 1.5 × 109 spermatozoa/ mL, and then allowed to cool to 5º C before fraction B with CPA was added to the sample to achieve a final concentration of 1 × 109 spermatozoa/mL and followed by freezing. The cryopreservation protocol did not impact total motility at 5 °C (1 S: 78.5 % vs 2 S: 79 %, p > 0.05). After thawing, the two-step protocol improved (p < 0.05) total (1 S: 18.2 % vs 2 S: 29.5 %) and progressive motility (1 S: 9 % vs 2 S: 15%). Further, the 2 S protocol increased (p < 0.05) the percentage of rapid spermatozoa (1 S: 8.7 % vs 2 S: 14.6 %) and spermatozoa with intact plasma and acrosomal membrane (IAIP) (1 S: 40.5 % vs 2 S: 61.5 %), and increased (p < 0.05) live sperm cells with high mitochondrial potential (MHIP) (1 S: 42.9 % vs 2 S: 60 %). The boar semen cryopreservation method (TRT) did not (p > 0.05) alter membrane lipid disorder, lipid peroxidation, and superoxide anion. Thus, the best method for boar semen cryopreservation is the two-step protocol.

Boar semen cryopreserved with trehalose-containing liposomes: disaccharide determination and rheological behaviour.

This study aimed to detect intracellular trehalose in boar sperm that were cryopreserved with liposomes and conduct an analysis of its effects on some characteristics of thawed sperm, including rheological properties. First, soybean lecithin cholesterol-based liposomes were produced and characterized in the presence of 300 mM trehalose. Next, semen samples were frozen in two freezing media: a control medium with 300 mM trehalose and an experimental medium supplemented with 300 mM trehalose and 10% liposomes, both of which were thawed and then studied to ascertain their integrity, motility, rheological response, and trehalose quantities by testing two methods of spermatic lysis via high-performance liquid chromatography with an evaporative light-scattering detector (HPLC-ELSD). The results found spherical liposomes measuring 357 nm that were relatively stable in an aqueous medium and had an entrapment efficiency of 73%. An analysis of the cryopreserved ejaculates showed that their viability and motility did not significantly differ between groups (P > 0.05). The viscous response of the samples was influenced by the extracellular medium rather than by the freezing-thawing process, which resulted in a loss of interaction between the cells and cryoprotectants. Finally, intracellular trehalose levels were determined using HPLC-ELSD, with no differences observed (P > 0.05) when comparing both sperm lysis methods. The use of liposomes with trehalose appears to be a promising option for boar semen cryopreservation, with a marked effect on rheological properties. The proposed HPLC-ELSD method was effective for measuring trehalose in cryopreserved cell samples.

Investigation of the Efficacy of Dithiothreitol and Glutathione on In Vitro Fertilization of Cryopreserved Large White Boar Semen.

Simple SummaryBoar sperm has proven to be extremely susceptible to cold shock and sensitive to peroxidative damage due to the high membrane content of polyunsaturated fatty acids. As a result, free radicals and oxidative stress can have a significant impact on the outcomes of in vitro fertilization (IVF). The objectives of this study were to evaluate the properties of sperm motility, viability, and morphology under induced oxidative stress; compare the antioxidant capacity of dithiothreitol (DTT) and glutathione (GSH) following the cryopreservation of Large White boar semen; investigate the ability of the cryopreserved Large White boar semen to fertilize the matured gilts oocytes under the same conditions; and to compare the efficacy of DTT and GSH antioxidants in improving the oocyte fertilization by the cryopreserved Large White boar semen. In this study, hydrogen peroxide (H2O2) was used as a source of oxidative stress (control). The treatment of boar semen with H2O2 reduced the percentage of sperm progressive motility (PM; 86.70 ± 10.24) after 3 h of incubation period (p < 0.05). The combination of GSH + DTT treatment reduced the percentage of sperm total motility (TM; 22.45 ± 11.14), PM (10.41 ± 4.59) and rapid motility (RAP; 9.20 ± 3.55) following thawing (p < 0.05). For IVF, fresh semen (11.84%) and a combination of DTT + GSH (14.10%) recorded a high percentage of zygotes with >2 pronucleus. However, the DTT treatment recorded a high percentage of zygotes with two pronuclei (19.76%).The objectives of this study were to evaluate the properties of sperm motility and morphology under induced oxidative stress, compare the antioxidant capacity of dithiothreitol (DTT) and glutathione (GSH) following the cryopreservation of Large White boar semen, investigate the ability of cryopreserved Large White boar semen to fertilize the matured gilts oocytes and compare the efficacy of DTT and GSH antioxidants in improving the oocyte fertilization by cryopreserved Large White boar semen. The semen was collected from three Large White boars (ten ejaculates per boar) and transported (37 °C) to the laboratory. Semen freezing extenders were supplemented with 5 mM DTT, 5 mM GSH and a combination of 2.5 mM DTT + 2.5 mM GSH. A liquid nitrogen vapor method was used to freeze boar semen. Gilts’ ovaries were collected from the local abattoir and transported (37 °C) to the laboratory. The slicing method was used to retrieve the oocytes from the ovaries. Fresh semen and frozen-thawed semen were used for in vitro fertilization (IVF). For frozen-thawed semen, four treatments (control, 5 mM DTT, 5 mM GSH, and a combination of 2.5 mM DTT + 2.5 mM GSH) were used during IVF in order to evaluate the fertilizing ability of the antioxidants. The supplementation of 5 µM DTT to H2O2-treated semen significantly improved progressive motility (PM) by 14.82%. A combination of 2.5 mM DTT + 2.5 mM GSH treatment reduced percentage of sperm total motility (TM) and rapid motility (RAP) following thawing (p < 0.05). Fresh semen and a combination of 2.5 mM DTT + 2.5 mM GSH treatment recorded a higher percentage of zygotes with polyspermy (p < 0.05). The control treatment numerically recorded a high percentage of zygotes with 1 PN, while the 5 mM DTT treatment recorded a high percentage of zygotes with 2 PN.

Boar Sperm Cryopreservation Improvement Using Semen Extender Modification by Dextran and Pentaisomaltose.

Simple SummaryCryopreservation of sperm is an important process used in livestock breeding. It is used to facilitate artificial insemination and helps to maintain genetic diversity by utilizing storage in cryobanks. Boar sperm are extremely sensitive to the conditions present during cryopreservation. Glycerol is the most used permeable cryoprotectant, showing both protective activity and some level of sperm toxicity. To improve the insemination rates, it is necessary to develop new cryoprotective agents. The goal of this paper is to evaluate the effect of the highly biocompatible, non-toxic polysaccharides dextran and pentaisomaltose on the cryopreservation of boar sperm. Additionally, we used computational modeling for the prediction of their cryoprotective action through interaction with selected sperm surface proteins. Our results show a lower impact of cryopreservation on sperm qualitative parameters when the extender is modified by pentaisomaltose. This approach represents a promising option for the complete removal of toxic glycerol in cryopreservation.The long-term storage of boar sperm presents an ongoing challenge, and the modification of the cryoprotective compounds in semen extenders is crucial for improving cryopreservation’s success rate. The aim of our study was to reduce the percentage of glycerol in the extender by elimination or substitution with biocompatible, non-toxic polysaccharides. For boar semen extender improvement, we tested a novel modification with the polysaccharides dextran and pentaisomaltose in combination with unique in silico predictive modeling. We targeted the analysis of in vitro qualitative sperm parameters such as motility, viability, mitochondrial activity, acrosome integrity, and DNA integrity. Non-penetrating polysaccharide-based cryoprotective agents interact with sperm surface proteins such as spermadhesins, which are recognized as fertility markers of boar sperm quality. The in silico docking study showed a moderate binding affinity of dextran and pentaisomaltose toward one specific spermadhesin known as AWN, which is located in the sperm plasma membrane. Pentaisomaltose formed a hydrophobic pocket for the AWN protein, and the higher energy of this protein–ligand complex compared with dextran was calculated. In addition, the root mean square deviation (RMSD) analysis for the molecular dynamics (MD) of both polysaccharides and AWN simulation suggests their interaction was highly stable. The in silico results were supported by in vitro experiments. In the experimental groups where glycerol was partially or entirely substituted, the use of pentaisomaltose resulted in improved sperm mitochondrial activity and DNA integrity after thawing when compared with dextran. In this paper, we demonstrate that pentaisomaltose, previously used for cryopreservation in hematopoietic stem cells, represents a promising compound for the elimination or reduction of glycerol in extenders for boar semen cryopreservation. This novel approach, using in silico computer prediction and in vitro testing, represents a promising technique to help identify new cryoprotectants for use in animal breeding or genetic resource programs.

Recent development in freezing strategies of pig semen-A review

Of late studies on frozen thawed boar semen have dramatically improved boar semen cryopreservation technique, albeit the commercial application of cryopreserved boar semen has not yet been popular. Some studies claimed successful fertility/ fertilization with frozen boar semen. Multiple researches are being carried out to evolve a suitable freezing protocol for cryopreservation of boar semen. In general, freezing protocol adopts freezing rates of either 20°, 40° or 60°C/min in lactose egg yolk extender with 2–3% glycerol using medium straw (0.5 ml) for freezing of boar semen. The supplementation of vitamin E or its analogues Trolox, butylated hydroxytoluene, reduced glutathione, catalase, superoxide dismutase, ascorbic acid, and alpha-lipoic acid to the freezing media of boar semen increase the cryosurvival of frozen-thawed boar spermatozoa. Treating sperm with cholesterol-loaded methyl-β-cyclodextrin increases sperm cryosurvival rates and sperm quality after thawing by partly decreasing membrane damage induced during phase transition from fluid to the crystalline-gel state. High fertility rates with cooled, frozen-thawed or sex-sorted boar semen are feasible to achieve by using appropriate insemination procedures. Post-cervical intra-uterine insemination allowed a three-fold reduction of spermatozoa to be inseminated, whereas deep uterine insemination allowed a substantial reduction in the number of cooled (5–20 folds) or frozen-thawed (6-folds) spermatozoa. With combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved facilitating its use in routine and commercial application. This review depicts best ways possible to adopt suitable freezing strategies for cryopreservation of boar semen.

In vitro effects of two different commercial freezing and thawing extenders on boar sperm quality

This study was conducted to evaluate whether there were differences in viability of cryopreserved semen when using two different freezing (Minitube Cryoguard – F1 or Androstar® CryoPlus – F2) and thawing (Minitube Cryoguard Thawing solution – T1 or Androstar® Plus – T2) extenders. Ejaculates were collected, diluted (1:1), and cooled before shipping at 17 °C overnight. Samples were aliquoted in cryopreservation extender F1 or F2. Four straws from each treatment sample were thawed and diluted in T1 or T2, resulting in four treatments (F1-T1, F1-T2, F2-T1, and F2-T2). The sperm in diluted semen were evaluated for motility kinetics at 30, 180, and 360 min after thawing. The integrity assessments of the plasma and acrosomal membranes were performed at 30 and 360 min after thawing. There was no interaction between F × T × Time (P > 0.05), and no interaction between F × T (P > 0.05). The sperm progressive motility (PMOT) as time post-thawing increased was greater (P = 0.015) when dilutions occurred using F1 compared with F2 extender. Sperm thawed in T1 had a greater TMOT (P = 0.008) and PMOT (P = 0.033) at all times evaluated. The sperm plasma and acrosomal membrane integrity (AIMI) were greater (P = 0.009) when samples were preserved in F1 compared to F2 extender. The use of T2, as compared with T1 thawing extender, resulted in an enhanced integrity of the plasma and acrosomal membranes (P = 0.008). It is concluded different combinations of commercial freezing extenders and thawing solutions have effects on the quality of cryopreserved boar semen in vitro.