Boar Sperm Cryopreservation Research Articles

Combination of Exogenous Spermidine and Phosphocreatine Efficiently Improved the Quality and Antioxidant Capacity of Cryopreserved Boar Sperm and Reduced Apoptosis-Like Changes.

The low resistance of boar sperm to cryopreservation dictates that addition antioxidants and energetic substances to the diluent to improve sperm quality is necessary. This study evaluated the effect of spermidine and phosphocreatine in combination on the quality, antioxidant capacity, and antiapoptotic-like changes capacity of cryopreserved boar sperm based on previous reports. The results showed that the combined application of spermidine and phosphocreatine significantly enhanced the motility, average path velocity, straight-line velocity, curvilinear velocity, beat cross frequency, acrosome integrity, plasma membrane integrity, mitochondrial activity, and DNA integrity compared with the control group (p < 0.05). In addition, the combined application of spermidine and phosphocreatine significantly enhanced the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity, and catalase activity while significantly decreasing malondialdehyde content and hydrogen peroxide content (p < 0.05). Western Blot analysis further showed that spermidine and phosphocreatine significantly decreased the expression of CASP3 and BAX and significantly enhanced the expression of BCL2 (p < 0.05); therefore, the combination of spermidine and phosphocreatine has potentially positive implications for improving the quality of cryopreserved boar sperm.

Effect of cholesterol-loaded cyclodextrin treatment on boar sperm cryopreservation.

This study investigated the efficacy of different concentrations of cholesterolloaded cyclodextrin (CLC) on cryopreservation in boar sperm quality. In this study, we treated boar sperm with different concentrations of CLC before freezing and analyzed the sperm cholesterol concentration, plasma membrane, acrosome integrity rate and total motility rate before and after freeze-thawing. We also investigated the levels of reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), and structural- and oxidative-damage related proteins in all groups after thawing. The results revealed that the cholesterol concentration of the CLC-treated groups was higher than that of the control group, both before freezing and after thawing (p<0.05). The plasma membrane integrity rate, acrosome integrity rate, and total motility rate of sperm were also enhanced after thawing in the CLC-treated group (all p<0.05). Moreover, ROS and MDA production and ATP loss were reduced in CLC-treated sperm during freezing and thawing (p<0.05). Finally, CLC pretreatment partially prevented the consumption of various proteins involved in metabolism including capping actin protein of muscle Z-line subunit beta (CAPZB), heat shock protein 90 alpha family class A member 1 (HSP90AA1) and phosphoglycerate mutase 2 (PGAM2) (p<0.05). The CLC treatment increased cholesterol concentration and decreased structural injury and oxidative damage during boar sperm freezing and thawing, improving the efficacy of sperm cryopreservation in boar.

Predicting the cryotolerance of boar sperm through antioxidant stress.

High sperm cryotolerance is crucial to the successful cryopreservation of boar sperm. Evaluating the cryotolerance of boar sperm by using a rapid and convenient technique can enhance the commercial viability of these sperm. This study investigated the correlation between sperm parameters for three sample subsets-fresh sperm, sperm with H2O2-induced oxidative damage (hereinafter referred to as H2O2-induced sperm), and frozen-thawed sperm-to identify the potential of these correlations to predict cryotolerance. A total of 64 sperm samples were obtained from 64 Duroc boars. The sperm parameters of the three subsets, where the frozen-thawed sperm were analysed at 30 or 180 min after thawing, were determined, and the coefficients of correlation between these parameters were calculated. The results indicated that H2O2-induced oxidative stress resulted in decreases in various sperm parameters-including total motility (TM), viability (VIA), mitochondrial membrane potential (MMP), and live sperm with MMP (LMP)-but increased their coefficients of variation. Receiver operating characteristic (ROC) curve analysis revealed that the kinematic parameters of the H2O2-induced sperm effectively predicted those of the frozen-thawed boar sperm at 30 min after thawing; the corresponding area under the ROC curve (AUC) was 0.8667 for TM and 0.8733 for progressive motility in the H2O2-induced sperm. For measurement at 180 min after thawing, the sperm membrane and mitochondrial parameters of the H2O2-induced sperm effectively predicted the LMP of the frozen-thawed boar sperm; the corresponding AUC was 0.8489 for VIA, 0.8289 for MMP, and 0.8444 for LMP. To our knowledge, this is the first study to directly establish a strong correlation between post-thaw boar sperm quality and H2O2-induced oxidative stress before freezing. Our proposed technique can serve as a valuable reference for the development of practical applications aimed at enhancing techniques for cryopreserving boar sperm.

Cryopreservation, in addition to protein tyrosine phosphorylation, alters the distribution of phosphatidyl inositol bisphosphate and the localization of cytoskeletal and signaling proteins (gelsolin, tyrosine kinase c-SRC and phospholipase C-ζ) in the perinuclear theca of boar sperm

Cryopreservation of boar spermatozoa affects the perinuclear theca (PT) and involves several proteins and molecules that play important roles during capacitation and the acrosomal reaction. The objective of the present study was to evaluate whether the deleterious effects of cryopreservation in addition to protein tyrosine phosphorylation are accompanied by changes in the distribution of phosphatidyl inositol bisphosphate (PIP2) and the localization of cytoskeletal and signaling proteins in the perinuclear theca of cryopreserved boar spermatozoa. For this purpose, by immunocytochemistry (IC) the changes in localization of phosphorylated proteins in tyrosine residues, gelsolin, c-SRC kinase and PLC-ζ, as well as in the distribution of phosphatidyl inositol bisphosphate were analyzed in thawed spermatozoa (T) non capacitated (NC), capacitated (C) and in those with acrosomal reaction (AR) and compared with fresh spermatozoa (F) under the same physiological status. Western blotting (WB) and co-immunoprecipitation were performed to confirm the presence of these proteins in PT and to determine the interaction between these molecules. IC showed that immunostaining for phosphorylated proteins significantly increased in the acrosomal region and flagellum in TNC spermatozoa (p < 0.05). The proportion of cells displaying immunolabeling for gelsolin in the acrosomal region decreased after capacitation in cryopreserved spermatozoa; the same change was found (p < 0.05) in the proportion of spermatozoa immunoreactive to PIP2 in the sperm head. c-SRC was observed in the equatorial segment and acrosomal region, subdomains that coincide with the site where phosphorylated proteins were detected. PLC-ζ immunolocalization in fresh spermatozoa underwent changes after capacitation and acrosomal reaction, with a significant increase in the equatorial segment and post-acrosomal region in cryopreserved spermatozoa (p < 0.05). WB analysis indicated the presence of gelsolin, c-SRC and PLC-ζ in PT; besides, we confirmed that gelsolin co-immunoprecipitated with c-SRC and PLC-ζ, which changes according to the physiological state of spermatozoa. As a conclusion, cryopreservation together with increased immunodetection of tyrosine phosphorylated proteins decreases the detection of PIP2 and alters the immunolocalization patterns of gelsolin, c-SRC and PLC-ζ in the PT in boar spermatozoa.

The Beneficial Effect of Resveratrol on the Quality of Frozen-Thawed Boar Sperm.

This study aimed to determine the effect of resveratrol and its optimal concentration on the quality of frozen-thawed (FT) boar sperm. Semen ejaculates were obtained from 13 Duroc boars aged between 1.5 and 3 years. The sperm sample was separated into 7 groups based on the concentrations of resveratrol in the freezing extender, which were 0 (control), 25, 50, 75, 100, 125, and 250 µM, respectively. The sperm was frozen using liquid nitrogen vapor and thawed at 50 °C for 12 s. After thawing, total motility, progressive motility, viability, intact acrosomes, mitochondrial membrane potential and level of MDA were assessed. The supplementation of 50-100 µM resveratrol improved the sperm motility and viability of FT sperm in comparison to the control group (p < 0.05). Furthermore, the 50 µM resveratrol group was significantly more protective than the control group in terms of intact acrosome, mitochondrial membrane potential, and level of MDA (p < 0.05). Nonetheless, the detrimental effect of resveratrol was found at a concentration of 250 µM. In conclusion, the addition of 50-100 µM resveratrol to a freezing extender is the optimal concentration for enhancing the quality of cryopreserved boar sperm.

Successful recovery of motile and viable boar sperm after vitrification with different methods (pearls and mini straws) using sucrose as a cryoprotectant

Vitrification of sperm by direct contact with liquid nitrogen is increasing in popularity as an alternative to conventional (slow) freezing. Although slow freezing is very challenging in boar sperm cryopreservation, this is currently the standard method used. We compared vitrification in “pearls” and in “mini straws” using the in vitro fertilization media Porcine Gamete Media with 0.3 M sucrose with the standard (slow) method used to preserve boar sperm. Both vitrification methods reduced the viability of the sperm sample more than slow freezing (42.2 ± 4.3% total motility and 71.4 ± 2.3% alive), however, both protocols allowed for the successful recovery of the sperm samples. By comparing two different methods of vitrification and two different methods of post-thaw preparation we were able to determine the optimal vitrification-thaw protocol for boar sperm. When comparing pearls and mini-straws, the smaller liquid volume associated with pearls had a positive effect on the survivability of the samples, reducing sperm DNA damage (1.2 ± 0.2% vs. 5.1 ± 0.1.7%) and preserving motility (26.15 ± 2.8% vs 9.39 ± 0.9%) after thawing. In conclusion, the pearl method was the most suitable of the vitrification techniques for use with boar sperm.

Phosphocreatine addition to extender enhances the quality and antioxidant capacity of cryopreserved boar sperm.

Boar sperm are less resistant to drastic changes in the external environment during cryopreservation, mainly because their plasma membranes are rich in unsaturated fatty acids but lack cholesterol and are thus susceptible to lipid peroxidation caused by the attack of reactive oxygen species. This study evaluated the effect of adding phosphocreatine to cryopreservation extenders on boar sperm quality and antioxidant capacity. Different concentrations (0, 5.0, 7.5, 10.0 and 12.5 mmol/L) of phosphocreatine were added to the cryopreservation extender. After thawing, sperm were analysed for morphological parameters, kinetic parameters, acrosome integrity, membrane integrity, mitochondrial activity, DNA integrity and antioxidant enzyme activity. The results showed that 10.0 mmol/L phosphocreatine samples enhanced the boar sperm motility, viability, average path velocity, straight-line velocity, curvilinear velocity and beat cross frequency after cryopreservation and reduced the malformation rate compared to the control group (p < .05). The acrosome integrity, membrane integrity, mitochondrial activity and DNA integrity of boar sperm were higher than those of the control group after adding 10.0 mmol/L phosphocreatine to the cryopreservation extender (p < .05). Extenders containing 10.0 mmol/L phosphocreatine maintained high total antioxidant capacity; elevated the activities of catalase, glutathione peroxidase and superoxide dismutase; reduced malondialdehyde and H2 O2 content (p < .05). Therefore, adding phosphocreatine to the extender is potentially beneficial for boar sperm cryopreservation at an optimal 10.0 mmol/L concentration.

Exogenous spermidine effectively improves the quality of cryopreserved boar sperm.

Boar sperm are less resistant to the dramatic environmental changes that occur during in vitro preservation. Spermidine has various physiological functions including the anti-oxidative effect. The main objective of this study was to clarify whether spermidine could protect boar sperm from the attack of reactive oxygen species under cryopreservation treatment. We set the concentrations of spermidine at 0, 2, 4, 6, and 8mmol/L and evaluated the effects of spermidine on sperm motility, viability, malformation rates, kinetic parameters, membrane integrity, mitochondrial activity, DNA integrity, H2 O2 content, malondialdehyde content, total antioxidant capacity, and antioxidant enzyme activity. Finally, the effects of spermidine on the sperm fertility were assessed by artificial insemination. The results showed that spermidine improved various physiological parameters of sperm in a dose-dependent manner. The quality and antioxidant capacity of sperm cryopreserved with 6mmol/L spermidine were significantly less reduced (P < 0.05), and the contents of malformation rate, H2 O2 , and malondialdehyde content were significantly decreased (P < 0.05). The significant increase in the number of litters indicated the possibility that spermidine had important practical value in pig reproduction (P < 0.05). Therefore, the addition of appropriate concentrations of spermidine to cryopreservation extenders may effectively improve the quality of boar sperm for in vitro preservation.

A novel experimental design for boar sperm cryopreservation.

Cryopreservation of sperm is a routine technology in many livestock species, but not in swine. Frozen sperm must result in acceptable conception rates and produce 11 to 12 piglets/litter to be competitive with traditional cooled semen. The development of an extender that results in high post-thaw sperm quality and acceptable litter size requires the identification of factors that markedly affect post-thaw semen quality. The present study aims to first identify factors in boar sperm cryopreservation that significantly affect post-thaw sperm quality using an efficient, cost-effective, and relatively rapid approach. The Plackett-Burman experimental design is ideal for the screening of factors at their extreme, greatly reducing the amount of time and resources needed for a follow-up, full factorial design. Using commercial semen, a 9-factor, 12-run Plackett-Burman design was used on 10 boars split between 12 treatments. Through this method, glycerol concentration, cooling rate, antioxidant supplementation with GameteGuard (Membrane Protective Technologies, Inc. Fort Collins, CO), and straw size were identified as highly influential factors that affect post-thaw sperm quality. Extender type, starting osmolality, sodium dodecyl sulfate addition, and stepwise addition of glycerol were also influential for some but not all post-thaw sperm parameters (P < 0.05). Equilibration time in the straws before freezing was determined to have no impact on post-thaw sperm quality parameters. Using the Plackett-Burman design, it can be concluded that four of the nine factors warrant detailed investigation in full factorial experiments in the development of boar sperm cryopreservation extenders.

Використання полівінілалкоголю та полівінілпіролідону для підготовки деконсервованих еякульованих сперматозоїдів кнурів до запліднення методом ICSI

The aim of the study was to explore the effect of PVP (polyvinylpyrrolidone) and PVA (polyvinyl alcohol) media on deconserved ejaculated boar sperm and their preparation for artificial insemination to optimize biotechnological approaches. The studies used ejaculated cryopreserved sperm of a boar of the Myrhorod breed Dnipro 641. Genetic material was stored in the Bank of Genetic Resources of Animals IABG nnamed after M.V. Zubets NAAS for eight years. The sperm suspension was thawed in a water bath at +37 °C for 5 min until completely thawed. Separation of sperm from cryopreservative agent and diluent was performed using the swim up method in Sp-TALP medium. After the presence of sperm in the 10.0% solution of PVP for 10 min, motility decreased by 68.2% (P &lt; 0.05) and amounted to 3.4%, and after the next 10 min of incubation decreased to 1.4% (P &lt;0.01), which is 10 times lower than the initial mobility. In 10.0% of PVA mobility after 10 min of incubation decreased by 37.4% (P &lt;0.05) and amounted to 6.7%, and after 10 min decreased to 5.7% (P &lt; 0.01), which is 1.8 times lower than the initial mobility. It was found that in the case of 10.0% of PVP solution ejaculated deconserved boar sperm lose motility by 86.9% (P &lt;0.01) from the initial motility, which makes it impossible to select a suitable sperm for fertilization by ICSI (intracytoplasmic sperm injection). It is shown that 10.0% PVA solution can be used for immobilization of boar sperm, as it reduces motility by 46.7% (P &lt;0.01) of the initial sperm motility. It is proved that the mobility in the case of incubation of deconserved ejaculated boar sperm in 5.0% PVA solution decreases only by 28.0% (P &lt;0.05) from the initial, which is optimal when using cryopreserved boar sperm, material which are limited and convenient for the operator and safe for oocytes.

A comparative study of two methods to determine acrosome integrity of frozen-thawed boar sperm

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P&amp;gt;0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P&amp;gt;0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.

PSV-9 Effects of Natural Honey Inclusion in Dilution and Freezing Extenders on Frozen-thawed Semen Quality in Boars

Abstract Improvements in the post-thaw quality of frozen semen could allow increased utilization of this technology in the swine industry. The objective of this study was to investigate the effects of natural honey inclusion in semen extender and freezing media on motility, mobility, and morphology of cryopreserved boar sperm. Ejaculates from 6 terminal cross-bred boars were collected using the gloved-hand technique for 3 weeks and used in a 2 x 3 factorial study design. Following collection, semen samples were incubated overnight in dilution extender with and without natural honey (D0: Androhep Plus; D1: Androhep Plus + 0.25% honey). The following day, the semen samples were cooled to 4 C in LEY cooling extender then frozen in freezing media containing 93% cooling extender + 6% glycerol + 1% Equex-STM Paste (F1), or freezing media with natural honey replacing 50% of the glycerol (F2) or 50% of the Equex-STM paste (F3). Semen samples were frozen using a controlled-rate freezer and stored in liquid nitrogen. Two straws per treatment for each boar were thawed and semen quality assessed. The inclusion of natural honey in dilution extender had no effect on post-thaw motility (P=0.733), progressive motility (P=0.562), or other mobility parameters (0.995≤P≥0.081). However, D1 had a higher percentage of normal acrosomes (P=0.001) and morphologically normal cells (P&amp;lt; 0.001) resulting from lower tail abnormalities compared to D0 (P=0.006). Post-thaw motility (P&amp;lt; 0.001) and progressive motility (P&amp;lt; 0.001) were increased in F3 compared to both F2 and F1. F1 had reduced normal acrosomes (P=0.009) and morphologically normal cells (P&amp;lt; 0.001) resulting from higher tail abnormalities (P&amp;lt; 0.001). In conclusion, the inclusion of natural honey, at 0.25%, in dilution extender helps maintain normal sperm and acrosome morphology, and replacing 50% Equex-STM Paste with honey in freezing extender improves post-thaw sperm motility and progressive motility of frozen-thawed boar semen.

10.4314/sajas.v40i5.65338

The study evaluated the effect of different cryopro tectants on post-thaw survival and motility of Kolbroek sperm. Semen from Kolbroek boars was collected with the gloved hand technique. Ejaculates wer e diluted with Beltsville thawing solution (BTS) at a ratio of 1 : 1 prior to freezing. Semen was dilute d with egg yolk tris; thereafter, one of the three cryopro tectants (14% glycerol, 14% DMSO or 7% glycerol + 7% DMSO) were added. Diluted samples were then loaded into 0.5 mL straws and cooled with a programmable freezer. Thereafter the semen straws were plunged d irectly into liquid nitrogen (-196 °C) and stored f or 48 h. Frozen straws were thawed at 39 °C for a minute and evaluated for sperm motility and survival at 0, 30 , 60 and 90 min post-thaw. The post-thaw sperm survival frozen using glycerol as a cryoprotectant was significantly higher immediately after thawing, com pared to DMSO, however, similar to the combination of glycerol and DMSO. There was no significant difference on motility rate immediately (0 min) post-thaw between the three cryoprotectants. Sperm cryopreser ved with glycerol exhibited a significantly higher percentage motility at 30, 60 and 90 min post-thaw than in the other cryoprotectants. Based on sperm motility, glycerol was a better cryoprotectant for cryopreservation of Kolbroek boar sperm.

Cryopreservation of Semen from Domestic Livestock: Bovine, Equine, and Porcine Sperm.

In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5°C, packaging of insemination doses in straws, freezing at a defined cooling rate in liquid nitrogen vapor, cryogenic storage, and thawing. Two-step dilution approaches, with commonly used diluents, are presented, namely, TRIS-egg yolk (TEY) extender for bull sperm, skim milk (INRA-82) extender for stallion sperm, and lactose-egg yolk (LEY) extender for boar sperm. Furthermore, simple methods are presented for cooling and freezing of sperm at defined cooling rates.

Effect of astaxanthin in extenders on sperm quality and functional variables of frozen-thawed boar semen

The aim of the study was to determine whether the presence of astaxanthin (ASX) protects boar spermatozoa against damage related to cryopreservation. Pooled ejaculates extended in Beltsville Thawing Solution (BTS) were used. Three experiments were conducted: 1) sperm samples were pre-incubated overnight (17 °C) with ASX (0, 0.5, 5, 15 μM) prior to freezing and then frozen using cooling and thawing extenders supplemented with ASX (0, 0.5, 5, 15 μM); 2) sperm samples were treated with ASX (0, 0.5, 5, 15 μM) only during overnight pre-incubation (17 °C) prior to cryopreservation; and 3) a thawing extender was supplemented with ASX (0, 0.5, 5, 15 μM). The groups were as follows: control (C; no treatment), ASX 1 (0.5 μM), ASX 2 (5 μM) and ASX 3 (15 μM). Total (TM) and progressive (PM) motility was analyzed using CASA, while sperm viability, reactive oxygen species generation, lipid peroxidation and apoptoticlike changes were analyzed using flow cytometry. Sperm variables were evaluated prior to freezing as well as 30 and 150 min after thawing. In Experiment 1, the values of TM and sperm viability post-thaw were less in the ASX 3 than C group. In Experiment 2, there was no effect of ASX on any of the sperm variables evaluated, while in Experiment 3, apoptotic-like changes were less in the ASX 1 than C group. In conclusion, there was a subtle beneficial effect on cryopreserved boar spermatozoa after addition of ASX to thawing media.

Safety assessment of poly(N-vinylcaprolactam) as a potential drug carrier in extenders for boar sperm cryopreservation

Polymers may be used to deliver compounds in freezing extenders to minimize injuries in spermatozoa during cryopreservation, although their activity and toxicity for boar sperm are unknown. This study investigated the effects of the polymer (N-vinylcaprolactam) (PNVCL), when included in extenders for boar sperm cryopreservation. In Experiment 1, sperm was exposed to PNVCL at: 0 (control); 39.1; 78.1; 156.3; and 312.5 μg/mL. Spermatozoa structure, kinetics and biochemical functions were unaltered in contact with PNVCL at 38 °C (P > .05) but declined with prolonged exposure (10, 60 and 120 min) in all treatments (P > .05). In Experiment 2, after inclusion of PNVCL in the freezing extender at the same concentrations, post-thawing sperm quality did not differ compared to the control (P > .05). Lipid peroxidation and the production of reactive oxygen species were the only parameters of sperm quality that were unaffected in both experiments, even after contact with PNVCL for 120 min (P > .05). As no negative effects were observed in post-thawing boar sperm quality, PNVCL did not incur in cytotoxicity and may be a potential carrier for antioxidants in freezing extenders.

Strategies to improve the fertility of frozen-thawed boar semen for artificial insemination

Although cryopreservation of boar semen for artificial insemination (Al) was developed 35 years ago, cryopreservation conditions and Al strategies are still considered sub-optimal. Al with excessive numbers of frozenthawed sperm (5-6 x 109 cells), still does not achieve fertility levels similar to Al using liquid semen because of reduced sperm survival. Frozenthawed (FT) spermatozoa have therefore not been the preferred option for commercial breeding programmes. However, substantial progress has been made regarding boar sperm cryopreservation. Adjustment of cooling and re-warming rates to biophysical properties of boar spermatozoa, new sperm package systems and the achievement of accurately consistent freezing of large numbers of samples using programmable freezers have contributed to post-thaw survival rates above 50%, a threshold similar to that used for bull AI-semen. Moreover, these post-thaw sperm survival rates are consistent within a large population of boars selected for sperm freezability potential, as occurs with Al-bull sires. When such post-thaw boar semen is deposited intra-utero, acceptable fertility (in terms of farrowing rates and litter size) is obtained. Currently, the most effective application of FT-semen for Al is achieved using deep uterine-Al (DUI) which allows placement of a minimal semen dose (in volume 0.5 to 10 mL and sperm number 0.5 to 1 x109 total spermatozoa) into the anterior 1/3 of one uterine horn, with levels of fertility close to Al with liquid semen. However, owing to their shorter life span, FT-boar spermatozoa require an AI-to-ovulation interval not longer than 4-6 h, making peri-ovulatory Al a pre-requisite to obtain the highest possible fertility. Spontaneous ovulation most often occurs when two-thirds of oestrus has passed. Estimation of the duration of oestrus, taking into account the weaning-to-oestrus interval, is helpful when establishing appropriate AI-schedules. However, as the length of oestrus varies within and between farms, different Al strategies should be established a priori. The development of bio-sensors for spontaneous ovulation will widen the use of Al with frozen-thawed frozen semen.

Effect of Alpha-Linolenic Acid with Bovine Serum Albumin or Methyl-Beta-Cyclodextrin on Membrane Integrity and Oxidative Stress of Frozen-Thawed Boar Sperm.

The study was conducted to investigate the effects of alpha-linolenic acid (ALA) combined with bovine serum albumin (BSA) or methyl-beta-cyclodextrin (MBCD) on plasma and acrosomal membrane damages, mitochondrial activity, morphological abnormality, motility, and oxidative stress in frozen-thawed boar sperm. In previous our study, 3 ng/mL ALA had been shown protective effect during freezing process of boar sperm. Therefore, we used 3 ng/mL ALA in present study and ALA was combined with same molar ratio of BSA or MBCD (ALA+BSA and ALA+MBCD, respectively). To confirm the effect of two carrier proteins, same volume of BSA and MBCD without ALA were added during cryopreservation. Membrane damage, mitochondrial activity, reactive oxygen species (ROS) and lipid peroxidation (LPO) levels were measured using flow cytometry, and movement of sperm tail as motility parameter and morphological abnormality were observed under light microscope. In results, all of sperm parameters were enhanced by ALA combined with BSA or MBCD compared to control groups (p<0.05). Mitochondrial activity, morphological abnormality, ROS and LPO levels in ALA+BSA or MBCD groups were no significant difference compared with ALA, BSA and MBCD treatment groups. On the other hand, plasma and acrosomal membrane intact, and sperm motility in ALA+MBCD group were higher than single treatment groups (p<0.05), whereas ALA+BSA did not differ. Our findings indicate that carrier proteins such as BSA and MBCD could improve the effect of ALA during cryopreservation of boar sperm, and treatment of ALA with carrier proteins enhance membrane integrity, mitochondrial activity through reduction of ROS-induced LPO.

The effect of melatonin as antioxidant for the cryopreservation of boar spermatozoa

The effect of melatonin as antioxidant for the cryopreservation of boar spermatozoa

Effects of ice-binding protein from Leucosporidium on the cryopreservation of boar sperm*

Effects of ice-binding protein from Leucosporidium on the cryopreservation of boar sperm*