Thawed Boar Sperm Research Articles

Reducing the Glucose Level in Pre-treatment Solution Improves Post-thaw Boar Sperm Quality.

Frozen–thawed boar sperm was not widely used in pig artificial insemination as the sperm quality was damaged by biochemical and physical modifications during the cryopreservation process. The aim of this study was to investigate whether reduction of the glucose level in diluted medium could protect the post-thaw boar sperm or not. Boar sperm was diluted with the pre-treatment medium with different doses of glucose (153, 122.4, 91.8, 61.2, 30.6, and 0 mM) during the cooling process. The sperm motility patterns and glycolysis were evaluated during the cooling process. Meanwhile, the post-thaw sperm quality, ATP level, mitochondrial function as well as apoptosis were also measured. It was observed that 153 mM glucose treatment showed the highest glycolysis in boar sperm as the activities of hexokinase, fructose-bisphosphate aldolase A, and lactate dehydrogenase are the highest as well as the lactate level. Reduction of the glucose level from 153 to 30.6 mM suppressed sperm glycolysis. In addition, treatment with 153 mM glucose made the sperm demonstrate a circle-like movement along with a high value of curvilinear velocity and amplitude of the lateral head, while decreasing the glucose level reduced those patterns in the cooling process. Moreover, reduction of the glucose level also significantly increased the post-thaw sperm's total motility, progressive motility, straight-linear velocity, membrane integrity, and acrosome integrity. The treatment with 30.6 mM glucose showed the highest value among the treatments. Furthermore, the post-thaw sperm's succinate dehydrogenase activity, malate dehydrogenase activity, mitochondrial membrane potential as well as ATP level were increased by reducing the glucose level from 153 to 30.6 mM. Interestingly, the treatment with 30.6 mM glucose showed the lowest apoptosis of post-thaw sperm among the treatments. Those observations suggest that reduction of the glucose level in diluted medium increased the post-thaw boar sperm quality via decreasing the glycolytic metabolism. These findings provide novel insights that reduction of boar sperm activity via decreasing sperm glycolysis during the cooling process helps to improve the post-thaw sperm quality during cryopreservation.

Comparison of single layer centrifugation and magnetic activated cell sorting for selecting viable boar spermatozoa after thawing

Sperm selection techniques, such as magnetic activated cell sorting (MACS) and colloid centrifugation, are reported to select good quality spermatozoa from semen samples of various species. Although the sperm quality of fresh boar semen is usually good, cryopreservation has a negative effect on parameters such as plasma membrane integrity and mitochondrial activity. Therefore, the objective of the present study was to determine whether MACS or centrifugation through a single layer of colloid (Single Layer Centrifugation, SLC) would be beneficial in enriching thawed boar sperm samples for viable spermatozoa with active mitochondria and good chromatin integrity. Frozen samples from three boars, three ejaculates per boar, were thawed and split. One part was selected by MACS, one was prepared by SLC, and the remainder served as the control. Controls and the selected sperm samples were evaluated for sperm quality (plasma membrane integrity, chromatin integrity, mitochondrial membrane potential and production of reactive oxygen species). Although several aspects of sperm quality were improved in the SLC-selected sperm samples compared to control, the flow-through MACS samples were only improved in having a lower proportion of spermatozoa with immature chromatin (Hi green fluorescence) compared to the labeled control. Sperm quality in the SLC samples was better than in the flow-through samples from MACS. Therefore, despite promising reports of the use of MACS for selecting good quality spermatozoa from semen in other species, the method was not useful for improving sperm quality in the thawed boar sperm samples in this experiment.

134 Trichostatin A as an aging agent during invitro fertilization in pig oocytes

An aged oocyte is one that was not fertilized during the optimal time window after ovulation and potentially has diminished fertilization and embryonic development success. Oocytes (n=2562) were matured with or without trichostatin A (TSA; 100ng mg−1), a known meiotic inhibitor, for 24 or 48h (OMI), then for an additional 16h (OMII) without TSA or hormones. Oocytes were measured for meiosis before maturation (n=95), after OMI (n=365), and after OMII (n=230) as well as cumulus cell expansion (CCE). Oocytes (n=800) were fertilized with frozen–thawed boar sperm and potential embryos were evaluated for IVF characteristics and cleavage and blastocyst formation 48 and 144h after IVF, respectively. Apoptosis was determined in the maturing oocytes by levels of reactive oxygen species (ROS; n=476), mitochondrial electrochemical potential gradient dissipation (n=405), caspase 3 (n=405), phosphatidylserine (n=453), and DNA breakdown (n=192). Data were analysed by ANOVA and Tukey’s test. Oocytes matured without TSA for 48h had a significantly higher (P<0.05) percentage of oocytes at the MI stage of meiosis compared to all other treatment groups (16.00±6.80) and oocytes matured without TSA for 24h had a significantly higher (P<0.05) percentage of oocytes at the MII stage of meiosis compared to all other treatment groups (14.7±11.30). Oocytes matured with or without TSA for 48h had significantly less (P<0.05) CCE compared to oocytes matured without TSA for 24h. Supplementation of TSA to OMI significantly decreased (P<0.05) fertilization penetration rates compared with not supplementing TSA for 24h. Percent of embryos cleaved by 48h and those reaching blastocyst by 144h after IVF were significantly higher (P<0.05) in oocytes matured for 24h compared with those matured for 48h. Oocytes matured without TSA for 24h generated significantly different (P<0.05) levels of ROS compared with oocytes matured with TSA for 48h. Oocytes matured without TSA for 48h had significantly higher (P<0.05) mitochondrial membrane potential compared with all other treatments. Levels of caspase 3 activation and phosphatidylserine (which translocates in response to apoptosis) differed (P<0.05) between treatments. Results from the TUNEL assay indicate oocytes matured through OMII with a short OMI and TSA supplemented (23.8±2.99%) and through OMI with a long OMI and TSA supplemented (24.3±1.11%) had significantly greater (P<0.05) percent of oocytes with fragmented DNA than the other treatments, except for oocytes matured through OMII with a long OMI and TSA supplemented (35.0±3.55%), which was significantly greater (P<0.05) than all other treatments. Results indicate that use of TSA to stimulate aging in pig oocytes is a valid and a reliable option.

Effect of Astragalus polysaccharide addition to thawed boar sperm on in vitro fertilization and embryo development

It is important to utilize an antioxidant to ameliorate oxidative damage during boar sperm cryopreservation and thawing. Some studies have shown that Astragalus polysaccharide (APS) has antioxidant capabilities in sperm storage at low temperatures. However, the effects of APS on thawed sperm are unclear. In this study, the effect of supplementation of thawing boar semen extender with APS (0.5, 1, 5, 10 mg/mL) on sperm quality parameters (viability, motility, acrosome integrity and mitochondrial activity) was evaluated. Next, we investigated the effect of APS (0.5 mg/mL) supplementation on antioxidant parameters. Semen from two straws was thawed and diluted with three volumes of Beltsville Thawing Solution (BTS) and immediately divided into a control group without addition of antioxidants (CTR) and the APS group. Sperm and antioxidant parameters were evaluated in the CTR and APS groups after 1 h of incubation at 37 °C. Finally, we studied the effect of APS (0.5 mg/mL) supplementation on in vitro fertilization (IVF) and embryo development. The addition of different doses of APS to thawed sperm did not induce any significant effects on the sperm viability or motility compared to the sperm without APS treatment. However, the addition of 0.5 mg/mL APS to thawed sperm showed improved mitochondrial activity, higher penetration rate and increased total IVF efficiency compared with those of the control group. Moreover, our results indicate that the supplementation of APS in thawed sperm decreased the concentration of reactive oxygen species (ROS) and improved the activity of superoxide dismutase (SOD) and catalase (CAT). Finally, the addition of APS significantly increased the cleavage rate and blastocyst rate compared to those of the control group. In conclusion, the addition of APS to thawed boar sperm can enhance the antioxidant ability of sperm and improve in vitro fertilization (IVF) parameters and the outcomes of embryonic development. These results imply that APS has practical potential to enhance boar sperm reproductive performance.

Effect of Single Layer Centrifugation Porcicoll (70%, 80% and 90%) or supplementation with reduced glutathione, seminal plasma and bovine serum albumin on frozen-thawed boar sperm

Selecting the optimal sperm population is essential for success with reproductive techniques. Porcicoll (formerly Androcoll-P) is a colloid formulation for selection of high-quality boar spermatozoa by single layer centrifugation (SLC). To date, most studies have been carried out with fresh semen and large volumes. We carried out 2 experiments to test the use of Porcicoll for thawed boar semen in small volumes. In Experiment 1, cryopreserved semen doses were thawed, split in 200-μL aliquots and layered on 1 mL of Porcicoll 70%, 80% or 90%, or buffer without colloid. We assessed sperm recovery (the proportion of the loading dose that appeared in the pellet, %), and the physiology of the selected spermatozoa (flow cytometry: Viability, apoptotic changes, capacitation, mitochondrial activity, intracellular reactive oxygen species). The most suitable proportion was Porcicoll 80%, allowing acceptable sperm recovery (16.9 ± 4.2%, compared to 70% (35.4% ± 3.0, p < 0.001) and 90% (8.2% ± 3.0, P = 0.001), and improved quality (mitochondrial activity: Porcicoll 80%: 77.7 ± 1% vs Control: 60.3 ± 0.7%, P < 0.05). In Experiment 2, we compared 3 supplements to Porcicoll 80%: 500 mM reduced glutathione (GSH), 20% seminal plasma (SP) and 0.5% bovine serum albumin (BSA). Supplementation with GSH or BSA did not cause relevant changes relative to Control. In contrast, SP induced membrane and acrosomal changes resembling capacitation, which might preclude its use in some applications, and decreased recovery (5.5% ± 1.9 vs. 24.3% ± 1.2 Control; P < 0.001). However, it could be useful prior to other applications such as in vitro fertilisation. Overall, Porcicoll is an effective colloid for isolating a high-quality population from thawed boar sperm, 80% being a balanced option for good recovery and high quality. Supplements could be useful depending on the proposed use of the spermatozoa.

Resveratrol and Epigallocatechin-3-gallate addition to thawed boar sperm improves in vitro fertilization

Thawing is one of the most delicate process after semen cryopreservation as spermatozoa pass from a dormant metabolic stage to a sudden awakening in cellular metabolism. The rapid oxygen utilization leads to an overproduction of reactive oxygen species that can damage sperm cells, thus causing a significant decrease of fertilizing potential of frozen-thawed spermatozoa. Resveratrol (Res) is a natural grape-derived phytoalexin and Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to possess high levels of antioxidant activity. The objective of the present study was to assess the effect of different concentrations of Res (0.5, 1 or 2 mM; Experiment 1) or EGCG (25, 50 or 100 μM; Experiment 2) supplementation to thawing boar semen extender on sperm quality parameters (viability and acrosome integrity) and in vitro fertilization (IVF). Semen after thawing and dilution with three volumes of Beltsville Thawing Solution (BTS), was immediately divided in control group without antioxidants addition (CTR) and either Res or EGCG groups. Sperm viability and acrosome integrity were evaluated in CTR, Res or EGCG groups after 1 h of incubation at 37 °C. The addition of different doses of Res or EGCG to thawing extender for 1 h did not induce any effect on boar sperm viability and acrosome integrity. However, both Res and EGCG treated samples exhibited a significantly higher penetration rate compared with CTR when used for IVF. In particular the treatment with all the EGCG concentrations increased the penetration rate (P < 0.01) while only Res 2 mM induced a significant increase of this parameter (P < 0.01). In addition, EGCG 25 and 50 μM supplementation significantly increased total fertilization efficiency as compared to control (EGCG 25 μM: 40.3 ± 8.2 vs 26.8 ± 9.5, P < 0.05; EGCG 50 μM: 40.4 ± 7.8 vs 26.8 ± 9.5, P < 0.01). The same effect was observed with Res 2 mM (51.0 ± 7.6 vs 29.6 ± 11.3, P < 0.01). In conclusion, our results indicate that the addition of different doses of the two antioxidants to thawed spermatozoa for one hour, even if does not exert any effect on sperm viability and acrosome integrity, efficiently improves in vitro penetration rate. Moreover, both molecules (EGCG 25 and 50 μM and Res 2 mM) significantly increases the total efficiency of fertilization.

Protective effect of hyaluronic acid on cryopreserved boar sperm

This study aimed to evaluate the effects of supplementing freezing and thawing media with hyaluronic acid (HA) on the quality parameters of frozen–thawed boar spermatozoa. Boar semen samples were collected from seven mature Yorkshire boars once a week using the gloved hand technique; these samples were frozen–thawed in the extender with added HA. Boar sperm was cryopreserved in the extender with HA added at concentrations of 0 (used as control), 4, 6, 8, 8 and 12mg/L, and their effects on the quality of frozen–thawed boar sperm were evaluated. HA addition to the extender significantly improved sperm motility, sperm membrane integrity, mitochondrial activity, acrosomal integrity, superoxide dismutase and catalase activity, but decreased sperm malondialdehyde level (p<0.05). Therefore, HA could be a promising cryoprotectant for boar sperm.

Protective effect of Rhodiola rosea polysaccharides on cryopreserved boar sperm

Cryopreservation brings sublethal damage to sperm, resulting in reduced fertile life of sperm. Rhodiola rosea polysaccharides (RPs) have antiviral, antioxidant and antitumor activities. In the present study, the cryoprotective effect of RPs on boar sperm quality parameters after frozen–thawed process was investigated. Boar sperm was cryopreserved in the extender with RPs added at concentrations of 0 (used as control), 2, 4, 6, 8 and 10mg/L and their effects on the quality of frozen–thawed boar sperm were assessed. Addition of RPs significantly improved sperm motility, mitochondrial activity, acrosomal integrity, plasma membrane integrity, superoxide dismutase and glutathione peroxidase activity and decreased sperm malonaldehyde level (p<0.05). The results indicated that the addition of RPs to the freezing extender decreased the cryodamage to the boar sperm.

Effect of Salvia miltiorrhiza polysaccharides on boar spermatozoa during freezing–thawing

Salvia miltiorrhiza polysaccharides (SMPs) were extracted from S. miltiorrhiza in this study. The aim of the present study was to evaluate the effect of SMP on the motility of boar sperm, including the antioxidant effect of SMP on boar sperm and the effect of SMP on the in vivo fertilizing ability of frozen–thawed boar sperm. Fifty ejaculates from 5 Swagger boars were collected and diluted with an extender, which contained 3% glycerol (v/v) with five concentrations of SMP (0.2, 0.4, 0.6, 0.8, and 1.0mg/mL). The semen was frozen in 0.25mL straws at 1.0×109 cells/mL. Sixty gilts were inseminated using fresh semen, frozen semen with 0.4mg/mL of SMP and frozen semen without SMP. The results indicate that the addition of SMP to the extender results in a higher percentage of motile sperm post-thaw (P<0.05). The activities of superoxide dismutase, lactate dehydrogenase, glutamic–oxalacetic transaminease and catalase were all determined to be significantly higher than the control group after adding SMP to the extender (P<0.05). The artificial insemination (AI) results demonstrated that the litter size was significantly higher in the 0.4mg/mL of SMP group than in the control group (P<0.05). In conclusion, during the process of freezing, SMP can protect boar sperm from peroxidative damage and increase sperm motility and litter size during the process of freezing–thawing. The optimal concentration of SMP for the frozen extenders in this study was determined to be 0.4mg/mL.

The effect of extender, method of thawing, and duration of storage on in vitro fertility measures of frozen–thawed boar sperm

Frozen–thawed boar sperm (FTS) has reduced in vitro and in vivo life span compared to liquid semen. Experiments tested whether extenders, thawing procedures, and storage temperatures could extend the fertile life span of FTS. Experiment 1 tested the effect of six extenders on postthaw motility (MOT) and viability (VIA). Straws from boars (n = 6) were thawed, diluted into each extender, and evaluated at 20, 60, and 120 minutes. There was a trend (P = 0.08) for an extender-by-time interaction for MOT and effect of extender and time for MOT (P < 0.0001) and extender (P = 0.10) and time (P < 0.0001) for VIA. Experiment 2 evaluated the effect of temperature and time of thawing on in vitro fertility at intervals after thawing. Straws (0.5 mL) from different boar ejaculates (n = 15) were thawed at 50 °C for 10, 20, or 30 seconds or at 70 °C for 5, 10, or 20 seconds and evaluated at 5, 30, and 60 minutes. There was an effect of thawing treatment on MOT, VIA, and ACR (viable sperm with intact acrosomes, P < 0.0001) and an effect of time of evaluation (P < 0.0001) on MOT and ACR. Thawing at 70 °C for 20 seconds reduced (P < 0.05) MOT, VIA, and ACR compared to other treatments. Experiment 3 tested the effects of storage temperature and time after thawing using 20 ejaculates. Samples were thawed, diluted, and allotted to storage at 17 °C, 26 °C, or 37 °C with evaluation at 2, 6, 12, and 24 hours. There was a storage temperature and time effect and an interaction for MOT and VIA (P < 0.0001). Storage at 17 °C and 26 °C increased (P < 0.05) MOT over all times (38.5%) compared to 37 °C (26%), whereas MOT was reduced at intervals. Viability was also greatest with 17 °C and 26 °C compared to 37 °C and was also affected by time and decreased with time. These results indicate that FTS can be held at 17 °C or 26 °C for up to 2 hours before use and would allow for preparation of multiple doses. These data suggest in vitro fertility of FTS is affected by extenders, thawing, and storage.

Centrifugation on Percoll density gradient enhances motility, membrane integrity andin vitrofertilizing ability of frozen–thawed boar sperm

The effects of Percoll density gradient centrifugation on sperm quality, in vitro fertilizability and developmental capacity of frozen-thawed boar sperm were evaluated. Two-step density gradient centrifugation by Percoll enhanced significantly the motility parameters of sperm compared with a simple centrifugation procedure. Percentages of motile sperm and sperm with intact plasma and acrosome membranes after Percoll separation were significantly greater than those after simple centrifugation. The rates of penetration, cleavage and blastocyst formation after in vitro fertilization were significantly improved by Percoll separation compared with simple centrifugation and were influenced positively by the intactness of sperm head membranes, but not any sperm motility parameters. However, insemination with increased concentrations of sperm prepared by Percoll gradient centrifugation did not improve the success of fertilization and embryo development in vitro. Our results indicate that the integrity of sperm head membranes after Percoll separation is important for successful embryo development in vitro, more so than sperm motility.

The effect of Laminaria japonic polysaccharide on sperm characteristics and biochemical parameters in cryopreserved boar sperm

The aim of the present study was to evaluate the cryoprotective effect of Laminaria japonic polysaccharide (LJP) on boar sperm. Semen samples were collected from seven mature Yorkshire boars once a week by the gloved hand technique and frozen–thawed in the extender with LJP added. Extender with LJP added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0mg/mL to the extender and its effects on the quality of frozen–thawed boar sperm were assessed. Results showed: (i) sperm motility and plasma membrane integrity were greater in the extender containing 0.5 and 1.0mg/mL LJP, as compared to other groups (P<0.05); (ii) extender added 1.0mg/mL LJP showed the greatest plasma membrane and acrosomal integrity percentages in comparison with other groups (P<0.05); (iii) mitochondrial activity was significantly higher at the concentration of 0.5 and 1.0mg/mL LJP than those of other groups (P<0.05); (iv) in terms of biochemical assessments, 0.5 and 1.0mg/mL LJP improved SOD (superoxide dismutase) and CAT (catalase) concentrations, compared to other groups (P<0.05). However, no significant difference was found in GSH-Px (glutathione peroxidase) concentration when supplemented with LJP. Interestingly, LJP exhibited a dose-related response and the lesser concentration represented greater protective effects. It is also important to note that 1.0mg/mL LJP provides for an enhanced cryoprotective effect in boar semen.

Effects of low-density lipoproteins extracted from different avian yolks on boar spermatozoa quality following freezing–thawing

Low-density lipoproteins (LDL) is known to protect boar sperm during freezing-thawing, but little information is known about the effects of LDL extracted from different avian egg yolks on post-thaw boar semen quality. The purpose of this study was to compare and analyze the effects of LDL at various concentrations and different species on boar sperm quality after freezing-thawing. LDL extracted from the yolk of hen egg, duck egg, quail egg, pigeon egg or ostrich egg was added to the extender at the concentrations of 0.06, 0.07, 0.08, 0.09 and 0.1 g/ml, respectively, and their effects on frozen-thawed boar sperm quality were assessed. According to all measured parameters, the results showed that sperm motility, acrosome integrity and plasma membrane integrity were 43.20%, 52.57% and 48.13%, respectively, after being frozen-thawed with 0.09 g/ml LDL extracted from pigeon egg yolk. All these quality parameters were higher than that of other groups (P < 0.05). In conclusion, our results confirmed that LDL extracted from pigeon egg yolk had the best cryoprotective effects on frozen-thawed boar sperm among all of the groups supplemented with LDL from five kinds of avian egg in extender. The optimum concentration of LDL extracted from pigeon egg in boar semen freezing extender was 0.09 g/ml.

The addition of calcium ion chelator, EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation-like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal-derived materials. In this study, we focused on the increase of intracellular Ca(2+) ([Ca(2+)](i)) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca(2+)](i) indicator, Fluo-3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca(2+) chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post-thawed sperm motility. When the frozen-thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca(2+)](i) and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen-thawed boar sperm.

Osmolarity of Coconut Water (Cocos nucifera) Based Diluents and their Effect Over Viability of Frozen Boar Semen

Problem statement: Boar sperm cells are sensitive to the freezing process, which compromises viability of frozen-thawed sperm. In a constant search for minimizing or suppressing sperm cell damage caused by the temperature and osmolarity changes during the freezing process, crioprotective and antioxidant substances have been added to the freezing media, such as coconut water, in order to increase the viability of frozen-thawed swine semen. The addition of any substance to the freezing diluent, directly affects osmolarity of the media, which can have positive or negative effects over the sperm cell. Approach: There are no published studies currently that indicate the effect of adding coconut water over the osmolarity of freezing media and their effect over viability of sperm cells, therefore, the objective of the present study was to evaluate the effect of coconut water based diluents osmolarity over the Motility (Mot), Acrosome Integrity (AI), Membrane Integrity (MI) and Mitochondrial Activity (MA) of thawed boar sperm cells. The treatments used were control T1 (LEY with bidistilled water + LEYGO) with an osmolarity range of 296-368 mOsmol Kg-1, T2 (LEY and deionized coconut water + LEYGO) between 381 and 480 mOsmol Kg-1 and T3 (LEY and in natura coconut water + LEYGO) between 519 and 1041 mOsmol Kg-1. The Westendorf modified method was the freezing method used. The obtained data were statistically analyzed by GLM, using the SAS software (SAS, 2000). Results: A significant difference was observed on T2 compared to T1 regarding Mot 41.9 Vs 36.9% and MI 58.0 Vs 50.2. T3 had a detrimental effect on all studied variables. Conclusion: Under our study conditions, the osmolarity range of T2, due to the non ionic solutes content, contributed to improve the viability of frozen-thawed sperm cells.

Assessment of viability of fresh, liquid and frozen–thawed boar sperm

Assessment of viability of fresh, liquid and frozen–thawed boar sperm

Practical application of seminal plasma

Natural mating deposits a large number of sperm in a high volume of seminal plasma. Semen processed for AI has the seminal plasma greatly diluted, and processing of boar sperm for cryopreservation removes all seminal plasma. Boar sperm were evaluated for the impact of seminal plasma on the functional status of boar sperm. Seminal plasma prevented or reversed capacitation in frozen–thawed boar sperm. However, supplementing frozen–thawed semen doses with 10% seminal plasma did not significantly affect the number of sperm found in the utero-tubal sperm reservoir, nor did it affect farrowing rates.

Ubiquitination and its influence in boar sperm physiology and cryopreservation

Recent reports document the potential use of the ubiquitin protein as an indicator of mammalian sperm quality or fertility, based on poor morphology, sperm count, and other cellular qualities. However, its influence on cellular physiologic mechanisms and boar sperm cryopreservation are unknown. The objective of this research was to determine the influence of boar sperm ubiquitination ( n = 12 boars) on motility (using CASA), and flow cytometry and fluorescent probes (in parentheses) to evaluate mitochondrial activity (JC-1), plasma and acrosomal membrane integrity (PI and FITC-PNA), membrane fluidity (M540), and chromatin stability (TUNEL) for fresh and frozen–thawed samples. The effects of ubiquitination (determined flow cytometrically) on the ability of frozen–thawed boar sperm to capacitate (FLUO-3AM) and acrosome react (FITC-PNA) were also investigated using flow cytometry. Cryopreservation induced a decrease in the percentage of sperm that were ubiquitinated from 29 to 20% ( P < 0.0001), but no significant effects of ubiquitin on sperm quality (motility, membrane integrities and organization) were detected. The ability of sperm to capacitate and acrosome react was influenced by ubiquitination. Samples with more ubiquitinated boar sperm were able to maintain plasma membrane integrity (PMI) better and have fewer live acrosome-reacted cells over 120 min of induced capacitation ( P < 0.05). In conclusion, frozen–thawed ubiquitinated boar sperm were better able to survive the physical stresses of induced capacitation, yet were still capable of capacitating and acrosome reacting, which may enable use of this assay for in the vitro evaluation of the quality of boar sperm.

44 EFFECT OF CYTOPLAST VOLUME ON FERTILIZATION AND EMBRYO DEVELOPMENT IN PORCINE M-II OOCYTES RECONSTRUCTED WITH KARYOPLASTS AND CYTOPLASTS OBTAINED BY THE 'CENTRI-FUSION' METHOD

Metaphase-II chromosome transfer (M-II transfer) of oocytes is considered to be one of the advanced procedures to improve fertilization and developmental abilities of oocytes with poor cytoplasmic maturation. The aim of this study was to investigate the developmental capacity after IVF and IVC of porcine oocytes reconstructed from karyoplasts and cytoplasts produced by centri-fusion (Fahrudin et al. 2007 Cloning Stem Cells 9, 216–228). In brief, IVM oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) with a visible first polar body were centrifuged at 13 000g for 9 min to stratify the cytoplasm. Then the zonae pellucidae were removed with pronase treatment. Zona-free oocytes were layered on a 300-µL discontinuous gradient of Percoll in TCM-HEPES with 5 µg mL–1 of cytochalasin B. After centrifugation at 6000g for 4 s, fragmented cytoplasms with approximately equal volumes were obtained, stained with Hoechst-33342, and classified into cytoplasm with (K; karyoplast) or without (C; cytoplast) chromosomes. One karyoplast was fused with 0, 1, 2, 3, and 4 cytoplasts (K, K + 1C, K + 2C, K + 3C, and K + 4C, respectively) by an electric stimulation with a single DC pulse (1.5 kV cm–1 for 20 µs) and cultured for 1 h. Zona-free oocytes without any reconstruction served as control oocytes. The diameters of the reconstructed and control oocytes were measured. All specimens were fertilized in vitro with frozen–thawed boar sperm, and cultured using the well of the well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Their fertilization status and developmental competence were examined. Data were analyzed by ANOVA followed by Duncan's multiple range tests. The diameter differed significantly among K to K + 4C oocytes (75.0–127.1 µm; P &lt; 0.05), whereas the diameter of K + 2C oocytes was similar to that of the control oocytes (110.5 µm). Regardless of the cytoplast volume, sperm penetration rates (73.1–93.8%) for K to K + 4C oocytes were not significantly different compared to control oocytes (78.0%). Male pronuclear formation rates of K to K + 4C oocytes (92.3–97.1%) were also not different significantly different compared to control oocytes (96.6%). However, monospermy rates of K oocytes was significantly higher (61.6%; P &lt; 0.05) than those of the reconstructed (K + 1C to K + 4C; 18.2–34.9%) and control oocytes (32.9%). The blastocyst formation rates in K, K + 1C, K + 2C, and K + 3C groups (0.0–9.8%; P &lt; 0.05) were significantly lower than those in the control and K + 4C groups (17.8% and 15.3%, respectively; P &lt; 0.05). The total cell numbers per blastocyst in K + 1C and K + 2C groups (7.5 and 8.3 cells, respectively) were significantly lower than in the control, K + 3C, and K + 4C groups (15.3–26.2 cells; P &lt; 0.05). These results suggest that the cytoplast volume of porcine M-II transferred oocytes, produced by reconstruction from a karyoplast and cytoplast(s) and centri-fusion, is important for their ability to develop to the blastocyst stage and influences cell number.

Non-surgical deep intra-uterine transfer of in vitro produced porcine embryos derived from sex-sorted frozen–thawed boar sperm

Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen–thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen–thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen–thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients ( n = 7) non-surgically ( n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen–thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex ( n = 16). Delayed return to oestrus (>23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen–thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.