The impact of cumulus cell viability and pre-culture with the healthy cell mass on brilliant cresyl blue (BCB) staining assessment and meiotic competence of suboptimal porcine oocytes.

Objectives of the present study were to investigate the characteristics including glucose-6-phosphate dehydrogenase activity, as determined by Brilliant Cresyl Blue (BCB) staining, of suboptimal porcine oocytes and to enhance the meiotic competence of those through pre-culture with cumulus cell masses (CCMs). Percentage of oocyte-cumulus complexes (OCCs) derived from small follicles (SF; <3 mm in diameter) containing the oocytes that were assessed as BCB-negative (BCB-) was significantly higher than those derived from medium follicles (MF; 3-6 mm in diameter). Degrees of dead cumulus cells were significantly higher in OCCs containing BCB- oocytes, regardless of the origin of OCCs (MF vs. SF), than those containing BCB-positive (BCB+) ones. Exposing OCCs containing BCB+ oocytes to the apoptosis inducer, carbonyl cyanide m-chlorophenylhydrazone, for 20 h significantly induced the transition to BCB- and meiotic progression of exposed OCCs were significantly reduced in both SF and MF derived ones. Transit of BCB- oocytes to BCB+ was induced when OCCs were pre-cultured with CCMs of MF derived OCCs containing BCB+ oocytes for 20 h before IVM. This pre-culture also significantly increased the meiotic competence of BCB- oocytes, particularly in SF derived ones. However, reactive oxygen species levels were significantly higher in BCB+ oocytes as compared with BCB- ones, regardless of pre-culture with CCMs, whereas no significant differences were found in the ATP contents among the treatment groups. In conclusion, the BCB result of oocytes could be regulated by the healthy status and content of surrounding cumulus cells and the meiotic competence of suboptimal BCB- porcine oocytes is improved by pre-culture with healthy CCMs.

Rapid thawing of frozen bull spermatozoa by transient exposure to 70 °C improves the viability, motility and mitochondrial health.

Up to now, the definitive conclusion of the positive effects of rapid transient thawing at higher temperatures for shorter durations has not been obtained yet and is still under discussion due to some contradictory findings and limited assessment of post-thawed parameters. The purpose of the current study was to evaluate the effectiveness of rapid thawing in water at 70 °C by using various post-thawed parameters of frozen bull spermatozoa. Experiment 1, monitoring the change of temperature inside frozen bull straw thawed in water at different temperatures. Experiment 2, evaluation of various post-thawed characteristics of frozen bull spermatozoa thawed in water at different temperatures by using a computer-assisted sperm analysis, flow cytometry and immunocytochemistry. The time it took for the temperature inside the straw to warm up to 15 °C was nearly twice as faster when the straw was thawed in 70 °C water compared with 39 °C. Although there were differences among bulls, viability, motility, and mitochondrial membrane potential of spermatozoa thawed at 70 °C for 8 seconds and stabilized at 39 °C for 52 seconds were significantly higher than those of controls (thawed at 39 °C for 60 seconds) at 0 and 3 h after thawing. Just after thawing, however, there were no differences in acrosome integrity and distribution of phospholipase C zeta1, whereas mitochondrial reactive oxygen species production was significantly lower in spermatozoa thawed at 70 °C. From these results, we conclude that rapid thawing at 70 °C and then stabilization at 39 °C significantly improves viability, motility and mitochondrial health of bull spermatozoa rather than conventional thawing at 39 °C. The beneficial effect of rapid transient thawing could be due to shorter exposure to temperatures outside the physiological range, consequently maintaining mitochondrial health.

Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo.

Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.

Negative correlations of mitochondrial DNA copy number in commercial frozen bull spermatozoa with the motility parameters after thawing.

The purpose of the current study was to investigate the relationship between mitochondrial content of commercial frozen-thawed bull spermatozoa and motility. Firstly, mitochondrial DNA copy number per spermatozoon (MDCN), mitochondrial content (MC), the percentage of spermatozoa with high mitochondrial membrane potential (HMMP), intracellular reactive oxygen species (ROS) and motility parameters of frozen-thawed spermatozoa derived from five bulls were determined by using qPCR, flow cytometry and CASA, respectively, and analyzed the relationships. Results showed that all parameters examined, including MDCN, MC, HMMP, ROS and motility indicators, significantly differed among frozen spermatozoa from different bulls. Both MDCN and MC were negatively correlated with HMMP and motility indicators, but positively with ROS, of course, whereas there was a highly positive relationship between MDCN and MC. Secondly, when MDCN and MC were examined in frozen spermatozoa prepared at different points in the lives of four bulls, those did not correlate overall throughout their lives (1.3-14.3 years old), but did correlate significantly in two sires. From these results, we conclude that MDCN and MC of frozen spermatozoa differ among sires, and are negatively correlated with HMMP and sperm motility parameters, probably due to mitochondrial oxidative stress resulted in the presence of ROS, demonstrating that these appear to be useful markers to assess sires' spermatozoa. It should be noted that the MDCN and MC of bull spermatozoa may not vary overall with the age of the sire, whereas those changes with age in some individuals and may affect sperm motility.

Oocyte-cumulus cells crosstalk: New comparative insights.

Mammalian follicles are constituted of a complex structure composed of several layers of granulosa cells surrounding the oocyte and of theca cells that reside beneath its basement membrane. During folliculogenesis, granulosa cells separate into two anatomically and functionally distinct sub-types; the mural cells lining the follicle wall and the oocyte-surrounding cumulus cells, i.e. those in intimate metabolic contact with the oocyte. The cumulus cells connecting with the oocyte have trans-zonal cytoplasmic projections which, penetrating the zona pellucida, form the cumulus-oocyte complex. The connections through gap junctions allow the transfer of small molecules between oocyte and cumulus cells, such as ions, metabolites, and amino acids necessary for oocyte growth, as well as small regulatory molecules that control oocyte development. The bi-directional communication between the oocyte and cumulus cells is crucial for the development and functions of both cell types. Our current knowledge of the relationship between the oocyte and its surrounding cumulus cells continues to change as we gain a greater understanding of factors regulating oocyte development and folliculogenesis. This review will mainly focus on the reciprocal interaction between oocytes and cumulus cells during the latter stages of follicle development i.e. through antral development to periovulatory events including oocyte maturation, expansion, and degradation of the cumulus matrix.

Anti-Müllerian hormone independently affect mtDNA copy number in human granulosa cells.

BACKGROUND: Recently, as a delayed childbearing trend is emerging in modern women's adulthood, diminished reproductive potential due to age-related changes is more prevalent. Reduction in the abundance of mitochondrial DNA (mtDNA) copies and circulating anti-Müllerian hormone (AMH) have been separately reported with aging, contributing to the decrease in successful reproduction. However, there are limited reports on the impact of age on mtDNA and AMH in the same individual and whether mtDNA copy numbers are influenced by age and AMH. METHODS: In the present study, we utilized a real-time quantitative PCR (RT-qPCR) to quantify the mtDNA copy number of granulosa cells obtained from 43 women undergoing an in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) program. RESULTS: According to our analysis, a significant correlation was observed between age and mtDNA copy number (r = -0.54, P < 0.001) and between age and AMH level (r = -0.48, P < 0.001) of the same individual. There was also a positive correlation between mtDNA copy number and AMH (r = 0.88, P < 0.001) with AMH level falling as mtDNA decreases. In our regression, age and AMH were shown to have low collinearity (VIF = 1.297) but only AMH was correlated with mtDNA quantity (P < 0.001). CONCLUSION: Our study suggests that both mtDNA and AMH abundance are influenced by age and that AMH levels independently affect mtDNA copy number regardless of age. Further research is required to understand the role of AMH on mitochondria bioenergetics.

COVID-19 and Attendance Demand for Professional Sport in Japan: A Multilevel Analysis of Repeated Cross-Sectional National Data during the Pandemic.

In the current investigation, we assess the effect of COVID-19 on intention-based spectator demand for professional sports in Japan captured by eight, monthly repeated cross-sectional national surveys from May to December 2020 (n = 20,121). We regress spectator demand on individual (e.g., gender), prefecture-wave (e.g., COVID-19 infection status), and prefecture-level factors (i.e., with or without quality professional teams). The results of multilevel logistic regression demonstrate that individual (i.e., male, younger, full-time employment, and with children status) and prefecture-level team factors (i.e., with teams) were associated with intention-based spectator demand. Nevertheless, COVID-19-related factors were found to be unrelated to spectator demand. The findings imply that sports fans are likely to return to the stadium once behavioral restrictions are lifted. The current research provided further evidence that individual factors and team quality serve as influential antecedents of spectator demand in the context of the COVID-19 epidemic.

Large-scale preparation of sialyl-Tn antigen-containing peptides from mucin-like glycoproteins in boar seminal gel.

Sialyl-Tn antigen, a tumor antigen, is a valuable ligand for the purification of proteins that specifically bind to it. Here, we developed a new method for the preparation of large amounts of sialyl-Tn antigen-containing peptides from an unused resource, boar seminal gel. The glycopeptides were prepared from the actinase E digests by a combination of gel filtration and hydrophilic partitioning.

The Thickness and Density of the Ovarian Tunica Albuginea Increases with Age in Transgender Patients.

It is known that the extracellular matrix structure and composition changes with aging in many organs. Despite this, knowledge on how does the extracellular part of the ovary change with increasing age in women and how those changes might be related to women's loss of fertility is still lacking. For this, we propose that recurrent injury and repair events on the outermost layers of the ovary due to ovulation are partly responsible for those changes women experience with aging. The histological analysis of the ovaries from 18 female-to-male transgender patients revealed that the ovarian tunica albuginea (TA) increases its thickness and density correlatively with increasing age of the patient (r = 0.52 and r = 0.55, P < 0.05 respectively). The increase in thickness is independent of the total androgen dose received and occurs because of the appearance of defined fibrotic areas underneath the TA layer which increase the total distance of dense connective tissue from the ovarian surface. In conclusion, the ovarian TA increases in its thickness and density with aging because of the appearance of fibrotic areas underneath the layer in transgender patients. This fact might contribute to reduce oocyte quality and cause ovulation difficulties in older women.

Relative transcript abundance in porcine cumulus cells collected from different sized follicles.

Crosstalk between the oocyte and surrounding cumulus cells (CCs) is essential for the production of competent oocytes. Previous studies have analysed the relative transcript abundance in oocytes derived from small (SF: <3 mm diameter)- and medium-sized (MF: 3-6 mm diameter) follicles to determine the potential use of SF-derived oocytes in assisted reproductive technologies (ART). The aim of this study was to examine the relative transcript abundance of CCs obtained from cumulus-oocyte complexes (COCs) derived from SF and MF. Nine genes were selected according to their importance for developmental competence: AT-rich interaction domain 1B (ARID1B), bone morphogenic protein receptor 2 (BMPR2), CD44, follicle-stimulating hormone receptor (FSHR), follistatin (FST), inhibin beta-A (INHBA), luteinizing hormone receptor (LHR), nuclear receptor subfamily 2 group F member 6 (NR2F6) and vascular endothelial growth factor A (VEGFA). The expression of these genes was analysed by RT-qPCR. The results pointed to significant differences in five genes, and the relative transcript abundance of SF-derived CCs was lower in the case of INHBA, but higher in FSHR, FST, LHR and NR2F6 compared with MF-derived CCs. We provide information of gene activity in the porcine CCs from different sized follicles, thus improving our understanding of oocyte biology and providing new markers that identify viable and competent oocytes.

The autophagic inducer and inhibitor display different activities on the meiotic and developmental competencies of porcine oocytes derived from small and medium follicles.

This study aimed to examine the effect of rapamycin (autophagy inducer) and 3-methyladenine (3-MA, autophagy inhibitor) on the meiotic and developmental competencies of porcine oocytes derived from medium follicles (MF, 3-6 mm in diameter) and small follicles (SF, 1-2 mm in diameter) during in vitro maturation (IVM) process. The presence of 1 nM but not 10 nM rapamycin significantly increased the maturation rate of MF-derived oocytes (P < 0.05). However, the maturation rate of SF-derived oocytes was not affected by rapamycin at both concentrations (1 nM and 10 nM). The maturation rate of MF-derived oocytes decreased significantly (P < 0.05) in the presence of 0.2 mM but not 2 mM 3-MA than non-supplemented control. In contrast, in SF-derived oocytes, 3-MA at both 0.2 and 2 mM concentrations did not affect the maturation rates. The presence of 1 nM rapamycin significantly increased the blastocyst formation rate of MF-derived mature oocytes following parthenogenetic activation (P < 0.05). However, the blastocyst formation rate of SF-derived mature oocytes was not affected by the presence of rapamycin. The presence of 3-MA significantly reduced the blastocyst formation rate of MF-derived mature oocytes but did not change that of SF-derived oocytes. In conclusion, our study results show differences in activity of the autophagy inducer and inhibitor on the meiotic and developmental competencies of MF- and SF-derived porcine oocytes.

Removal of cumulus cells around 20 h after the start of in vitro maturation improves the meiotic competence of porcine oocytes via reduction in cAMP and cGMP levels.

We examined the effect of the timing of removing cumulus cells surrounding porcine oocytes from small follicles (SFs, < 3 mm in diameter) and medium follicles (MFs; 3-6 mm in diameter) on the meiotic and developmental competence of the oocytes. Cumulus-oocyte complexes (COCs) were collected from SFs and MFs, and the oocytes were denuded at 0, 20, and 44 h after the start of in vitro maturation (IVM), and the meiotic progression of the oocytes was assessed at the end of the IVM period. The incidence of mature oocytes was significantly affected by both the origin of the COCs and the time when the oocytes were denuded. Although the percentage of mature oocytes was always higher when the COCs were collected from MFs than that when the COCs were collected from SFs, the maturation rate was significantly higher when the oocytes were denuded at 20 h than when they were denuded at 44 h after the start of IVM. When the mature oocytes were activated electrically, the developmental competence of the oocytes denuded at 20 and 44 h to reach the blastocyst stage did not differ, whereas the competence of the MF-derived oocytes was significantly higher than that of SF-derived oocytes. When the intracellular cAMP and cGMP levels in SF-derived oocytes were examined at 24 h of IVM, the levels of both were significantly decreased only in the oocytes denuded at 20 h. In conclusion, denuding oocytes at 20 h of IVM caused a significant reduction in ooplasmic cAMP and cGMP levels and increased the meiotic competence of the oocytes without any reduction in blastocyst formation, even in the case of SF-derived oocytes.

Levels of cyclic-AMP and cyclic-GMP in porcine oocyte-cumulus complexes and cumulus-free oocytes derived from small and middle follicles during the first 24-hour period of in vitro maturation.

The objective of this study was to compare the cAMP and cGMP levels in cumulus-oocyte complexes (COCs) derived from the middle follicles (MFs, 3-6 mm in diameter) and small follicles (SFs, 1-3 mm in diameter) of pre-pubertal gilts during the first 24-h period of maturation in vitro (IVM). Both cAMP and cGMP levels in MF- and SF-derived oocytes did not change during this period. Although the cAMP levels increased in the COCs at 10 and 20 h after the start of IVM, the levels of cAMP were significantly higher in MF-derived COCs than in SF-derived COCs at 20 h after the start of IVM. On the other hand, the cGMP levels in COCs decreased to basal levels between 10 and 20 h after the start of the IVM, whereas cGMP levels were lower in SF-derived COCs than in MF-derived COCs during the first 10 h. The number of cumulus cells was larger in the MF-derived COCs than in the SF-derived COCs during the first 20-h period of IVM. The estimated cAMP level per cumulus cell at 10 h after the start of the IVM was higher in SF-derived COCs than in MF-derived COCs, whereas the estimated cGMP level per cumulus cell was no different between MF- and SF-derived COCs. From these results, we conclude that cAMP and cGMP levels in COCs, but not in oocytes, drastically change during the first 20-h period of IVM, and that both cAMP and cGMP levels significantly differ between MF- and SF-derived COCs.

Presence of vascular endothelial growth factor during the first half of IVM improves the meiotic and developmental competence of porcine oocytes from small follicles.

The aim of the present study was to investigate the effect of vascular endothelial growth factor (VEGF) on the meiotic and developmental competence of porcine oocytes from small follicles (SF; 0.5-3mm diameter). When cumulus-oocyte complexes (COCs) from medium-sized follicles (MF; 3-6mm diameter) and SF were cultured for IVM, the maturation rates were significantly higher for oocytes from MF than SF. Concentrations of VEGF in the medium were significantly higher for COCs cultured from MF than SF. When COCs from SF were exposed to 200ngmL-1 VEGF during the first 20h of IVM, the maturation rate improved significantly and was similar to that of oocytes derived from MF. The fertilisability of oocytes was also significantly higher than that of VEGF-free SF controls. Following parthenogenetic activation, the blastocyst formation rate improved significantly when SF COC culture was supplemented with 200ngmL-1 VEGF, with the rate similar to that of oocytes from MF. The results of the present study indicate that VEGF markedly improves the meiotic and developmental competence of oocytes derived from SF, especially at a concentration of 200ngmL-1 during the first 20h of IVM.

Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived from small and medium follicles during IVM on the apoptotic status and meiotic progression of the oocytes.

The present study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small follicle (SF; 0.5-2 mm in diameter) and medium follicle (MF; 3-6 mm in diameter) when the oocytes were denuded before, during, and after IVM. Cumulus-oocyte complexes (COCs) were collected from SF or MF of prepubertal gilt ovaries. Before or 20 hours after the start of IVM culture, some oocytes were denuded and cultured for IVM. At the end of IVM, apoptotic status and meiotic progression of the oocytes were compared with oocytes matured in the presence of cumulus cells (CCs) by Annexin-V/PI assay and 4',6-Diamidino-2-phenylindole staining. Apoptotic status of the oocytes was only affected by time when the oocytes were denuded. In both oocytes from SF and MF, although the incidence of early and late apoptotic oocytes was significantly higher when the CCs were removed before IVM, the rate was significantly lower when CCs were removed 20 and 44 hours after the start of IVM. The incidence of mature oocytes was significantly affected by both the origin of COCs and time when oocytes were denuded from the COCs. Although the percentage of mature oocytes was higher in MF than SF, maturation rates were significantly higher when oocytes were denuded 20 hours, as compared with 0 and 44 hours after the start of IVM. However, the percentage of mature oocytes with a morphologically normal spindle was significantly higher when oocytes were denuded 44 hours, rather than 22 hours of IVM. In conclusion, removing CCs 20 hours after the start of IVM seems to promote meiotic progression of the oocytes to the metaphase-II stage even when the COCs were collected from SF, although factor(s) from or communication with CCs during IVM may need to obtain a morphologically normal spindle in mature oocytes.

Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos.

Viable sperm from sex-sorted semen without centrifugal treatment was separated by a microfluidic sperm sorter (MFSS) for IVF to improve in vitro embryo production of dairy cattle. The MFSS was originally developed to isolate motile human sperm by two laminar flows in the micro-channel (there are four chambers in an MFSS. Chamber A is the inlet for semen, chamber B is the inlet for the medium, chamber C is the exit chamber for motile sperm, and chamber D is the outlet for nonmotile sperm). Sex-sorted sperm were adjusted to 1 × 10(7) spermatozoa/mL (2 million cells/dose, sperm motility was 30% above after thawing). In a first experiment, diluted sex-sorted semen was mixed with modified Medium199(mM199) containing 5-mM caffeine for 5 minutes, resulting in variations in sperm concentration and quality parameters at chambers A, C, and D. In a second experiment, medium containing sperm from three MFSS chambers was collected and mitochondrial activity of the sperm was determined by flow cytometry, the relative activity of sperm mitochondria in chamber C (1.56 ± 0.03) was the highest in three observation areas (P < 0.05). Thus, sperm motility and mitochondrial activity of sperm was high in chamber C. In a third experiment, different concentrations of sperm were added to chamber A and dairy cattle IVM oocytes were placed in chamber C, where motile spermatozoa will accumulate, with mM199 containing 5-mM caffeine for 5 minutes, and then cultured in caffeine-free mM199 for 8 hours. The results showed that sperm penetration rate, the monospermic penetration rate, and blastocyst rate of the 10 × 10(6) group (10 × 10(6) sperm/mL) were higher than in the 1 × 10(6) and 5 × 10(6) groups (P < 0.05). In the last experiment, we compared sperm penetration in the MFSS-IVF system with a modified standard IVF method (cocultured in droplets for 8 hours). The normal fertilization index (the ratio of monospermic oocytes to the number of oocytes examined) 8 hours after insemination was higher in the MFSS-IVF system than the modified standard IVF system (P < 0.05). Developmental competence of fertilized oocytes to the blastocyst stage was also higher in the MFSS-IVF system (40.12% ± 2.61%) than the modified standard IVF technique (24.55% ± 4.54%). These results demonstrate that a short coculture of dairy cattle oocytes with isolated motile sex-sorted spermatozoa gradually accumulated in the MFSS device improves the efficiencies of normally produced fertilized embryos and blastocyst formation.

A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium.

The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 µM), higher sildenafil concentrations (25 and 250 µM) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 µM sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type-5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro.

In vitro fertilization in pigs: New molecules and protocols to consider in the forthcoming years.

Assisted reproduction technology (ART) protocols are used in livestock for the improvement and preservation of their genetics and to enhance reproductive efficiency. In the case of pigs, the potential use of embryos for biomedicine is being followed with great interest by the scientific community. Owing to the physiological similarities with humans, embryos produced in vitro and many of those produced in vivo are used in research laboratories for the procurement of stem cells or the production of transgenic animals, sometimes with the purpose of using their organs for xenotransplantation. Several techniques are required for the production of an in vitro-derived embryo. These include in vitro oocyte maturation, sperm preparation, IVF, and further culture of the putative zygotes. Without doubt, among these technologies, IVF is still a critical limiting factor because of the well-known, but still unsolved, question of polyspermy. Despite the improvements made in the past decade, current IVF systems hardly reach 50% to 60% efficiency and any progression in porcine ARTs requires an unavoidable improvement in the monospermy rate. It is time, then, to learn from what happens under in vivo physiological conditions and to transfer this knowledge into ART. This review describes the latest advances in porcine IVF, from sperm preparation procedures to culture media supplements with special attention paid to molecules with a known or potential role in in vivo fertilization. Oviductal fluid is the natural medium in which fertilization takes place, and, in the near future, could become the definitive supplement for culture media, where it would help to solve many of the problems inherent in ARTs in swine and improve the quality of in vitro-derived porcine embryos.

Rapid thawing and stabilizing procedure improve postthaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a glycerol-free trehalose-based extender.

Thawing process is important in semen cryopreservation as it brings back the sperm cell to physiologic temperature reactivating the metabolism. Aims of the present study were to evaluate survival rate and in vitro penetrability of boar frozen spermatozoa after rapid and transient thawing at a high temperature followed by a warming procedure at 39 °C. Ejaculated semen samples were diluted in an egg yolk-based glycerol-free extender containing 100-mM trehalose and then cryopreserved in 0.5-mL straws according to a common protocol. In experiment 1, when temperature inside the straws was monitored after thawing at 40 °C, 60 °C, 70 °C, and 80 °C, the calculated average warming rate in the straws from -196 °C to 15 °C was much faster when thawed at 70 °C and 80 °C than at 40 °C (P < 0.01). The warming temperature rate inside the straw was 7 to 12 folds faster during the first 2 seconds than the second 2 seconds after immersing in high temperatures. In experiment 2, when frozen straws were thawed at 80 °C for 9 seconds, the viability, motility, and acrosomal integrity were significantly improved (P < 0.05), as compared with controls (at 39 °C). In experiment 3, frozen straws were thawed at 39 °C, 60 °C, 70 °C, and 80 °C for 60, 10, 8, and 6 seconds, respectively, and then maintained at 39 °C for 0, 50, 52, and 54 seconds. Higher viability, motility, mitochondria membrane potential, and acrosome integrity were observed (P < 0.05) when frozen straws were thawed at 70 °C for 8 seconds and then maintained at 39 °C for 52 seconds as compared with the control (39 °C for 60 seconds). In experiment 4, in vitro penetrability of frozen spermatozoa thawed at 70 °C for 8 seconds and maintained at 39 °C for 52 seconds was higher than that of controls. In conclusion, the rapid transient thawing at 70 °C for 8 seconds followed by stabilizing procedure at 39 °C for 52 seconds maintained the viability, motility, mitochondria membrane potential, acrosome integrity, and in vitro penetrability of spermatozoa frozen in a glycerol-free trehalose extender and recommended as an optimum thawing conditions.

Trehalose in glycerol-free freezing extender enhances post-thaw survival of boar spermatozoa.

Cryopreservation of boar semen is still considered suboptimal due to lower fertility as compared with fresh samples when glycerol, a permeating cryoprotectant, is used. Trehalose is a non-permeable cryoprotectant and nonreducing disaccharide known to stabilize proteins and biologic membranes. The aim of this study was to evaluate the cryosurvival and in vitro penetrability of boar spermatozoa when glycerol was replaced with trehalose in a freezing extender. Ejaculated Berkshire semen samples were diluted in egg yolk-based freezing extender containing glycerol (100 mM) or trehalose (0, 50, 100, 150, 200 and 250 mM) and cryopreserved using a straw freezing procedure. Thawed samples were analyzed for motility, viability, mitochondrial membrane potential (MMP), and acrosome integrity. In experiment 2, penetrability of spermatozoa cryopreserved with 100 mM glycerol or trehalose was examined. Replacement of cryoprotectant glycerol (100 mM) with trehalose had no effect on sperm viability, but replacing it with 100 mM trehalose improved motility, MMP and acrosome integrity significantly. Sperm motility and MMP were considerably higher in 100 mM trehalose, whereas the acrosome integrity was substantially higher in 100-250 mM trehalose. The in vitro penetration rate was also significantly higher in spermatozoa cryopreserved with trehalose (61.3%) than in those cryopreserved with glycerol (43.6%). In conclusion, 100 mM non-permeable trehalose can be used to replace glycerol, a permeating cryoprotectant, for maintenance of better post-thaw quality of boar spermatozoa.