An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm.

BACKGROUND: In vitro incubation of epididymal and vas deferens sperm with Mn2+ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn2+ and Mg2+) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry. RESULTS: Incubation with Mn2+/Ca2+ activated SCF in a dose-dependent (P < 0.05) albeit not time-dependent manner (P > 0.05); in contrast, Mg2+/Ca2+ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn2+/Ca2+ or Mg2+/Ca2+, SCF generated DNA fragments of 33-194 Kb, compatible with the size of one or multiple toroids. Besides, Mn2+/Ca2+ affected sperm motility in a dose-dependent manner (P < 0.05), whereas Mg2+/Ca2+ only impaired this variable at high concentrations (P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn2+/Ca2+ or Mg2+/Ca2+ treatments. CONCLUSION: Mn2+/Ca2+ and Mn2+/Ca2+ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death.

Clustering of self-thermophilic asymmetric dimers: the relevance of hydrodynamics.

Self-thermophilic dimers are characterized by a net phoretic attraction which, in combination with hydrodynamic interactions, results in the formation of crystalline-like aggregates. To distinguish the effect of the different contributions is frequently an important challenge. We present a simulation investigation done in parallel in the presence and the absence of hydrodynamic interactions for the case of asymmetric self-thermophoretic dimers. In the absence of hydrodynamics, the clusters have the standard heads-in configurations. In contrast, in the presence of hydrodynamics, clusters with heads-in conformation are being formed, in which dimers with their propulsion velocity pointing out of the cluster are assembled and stabilized by strong hydrodynamic osmotic flows. Significant variation in the material properties is to be expected from such differences in the collective behavior, whose understanding and control is of great relevance for the development of new synthetic active materials.

Self-phoretic Brownian dynamics simulations.

A realistic and effective model to simulate phoretic Brownian dynamics swimmers based on the general form of the thermophoretic force is here presented. The collective behavior of self-phoretic dimers is investigated with this model and compared with two simpler versions, allowing the understanding of the subtle interplay of steric interactions, propulsion, and phoretic effects. The phoretic Brownian dynamics method has control parameters which can be tuned to closely map the properties of experiments or simulations with explicit solvent, in particular those performed with multiparticle collision dynamics. The combination of the phoretic Brownian method and multiparticle collision dynamics is a powerful tool to precisely identify the importance of hydrodynamic interactions in systems of self-phoretic swimmers.

A Review on the Role of Bicarbonate and Proton Transporters during Sperm Capacitation in Mammals.

Alkalinization of sperm cytosol is essential for plasma membrane hyperpolarization, hyperactivation of motility, and acrosomal exocytosis during sperm capacitation in mammals. The plasma membrane of sperm cells contains different ion channels implicated in the increase of internal pH (pHi) by favoring either bicarbonate entrance or proton efflux. Bicarbonate transporters belong to the solute carrier families 4 (SLC4) and 26 (SLC26) and are currently grouped into Na+/HCO3- transporters and Cl-/HCO3- exchangers. Na+/HCO3- transporters are reported to be essential for the initial and fast entrance of HCO3- that triggers sperm capacitation, whereas Cl-/HCO3- exchangers are responsible for the sustained HCO3- entrance which orchestrates the sequence of changes associated with sperm capacitation. Proton efflux is required for the fast alkalinization of capacitated sperm cells and the activation of pH-dependent proteins; according to the species, this transport can be mediated by Na+/H+ exchangers (NHE) belonging to the SLC9 family and/or voltage-gated proton channels (HVCN1). Herein, we discuss the involvement of each of these channels in sperm capacitation and the acrosome reaction.

Collective behavior of thermophoretic dimeric active colloids in three-dimensional bulk.

Colloids driven by phoresis constitute one of the main avenues for the design of synthetic microswimmers. For these swimmers, the specific form of the phoretic and hydrodynamic interactions dramatically influences their dynamics. Explicit solvent simulations allow the investigation of the different behaviors of dimeric Janus active colloids. The phoretic character is modified from thermophilic to thermophobic, and this, together with the relative size of the beads, strongly influences the resulting solvent velocity fields. Hydrodynamic flows can change from puller-type to pusher-type, although the actual flows significantly differ from these standard flows. Such hydrodynamic interactions combined with phoretic interactions between dimers result in several interesting phenomena in three-dimensional bulk conditions. Thermophilic dimeric swimmers are attracted to each other and form large and stable aggregates. Repulsive phoretic interactions among thermophobic dimeric swimmers hinder such clustering and lead, together with long- and short-ranged attractive hydrodynamic interactions, to short-lived, aligned swarming structures.

The triple role of glutathione S-transferases in mammalian male fertility.

Male idiopathic infertility accounts for 15-25% of reproductive failure. One of the factors that has been linked to this condition is oxidative stress (OS), defined as the imbalance between antioxidants and reactive oxygen species. Amongst the different factors that protect the cell against OS, the members of the glutathione S-transferase (GST) superfamily play an important role. Interestingly, reduction or lack of some GSTs has been associated to infertility in men. Therefore, and to clarify the relationship between GSTs and male fertility, the aim of this work is to describe the role that GSTs play in the male reproductive tract and in sperm physiology. To that end, the present review provides a novel perspective on the triple role of GSTs (detoxification, regulation of cell signalling and fertilisation), and reports their localisation in sperm, seminal plasma and the male reproductive tract. Furthermore, we also tackle the existing correlation between some GST classes and male fertility. Due to the considerable impact of GSTs in human pathology and their tight relationship with fertility, future research should address the specific role of these proteins in male fertility, which could result in new approaches for the diagnosis and/or treatment of male infertility.

Blocking NHE Channels Reduces the Ability of In Vitro Capacitated Mammalian Sperm to Respond to Progesterone Stimulus.

Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus.

Inhibition of Potassium Channels Affects the Ability of Pig Spermatozoa to Elicit Capacitation and Trigger the Acrosome Exocytosis Induced by Progesterone.

During capacitation, sperm undergo a myriad of changes, including remodeling of plasma membrane, modification of sperm motility and kinematic parameters, membrane hyperpolarization, increase in intracellular calcium levels, and tyrosine phosphorylation of certain sperm proteins. While potassium channels have been reported to be crucial for capacitation of mouse and human sperm, their role in pigs has not been investigated. With this purpose, sperm samples from 15 boars were incubated in capacitation medium for 300 min with quinine, a general blocker of potassium channels (including voltage-gated potassium channels, calcium-activated potassium channels, and tandem pore domain potassium channels), and paxilline (PAX), a specific inhibitor of calcium-activated potassium channels. In all samples, acrosome exocytosis was induced after 240 min of incubation with progesterone. Plasma membrane and acrosome integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and total and progressive sperm motility were evaluated after 0, 120, and 240 min of incubation, and after 5, 30, and 60 min of progesterone addition. Although blocking potassium channels with quinine and PAX prevented sperm to elicit in vitro capacitation by impairing motility and mitochondrial function, as well as reducing intracellular calcium levels, the extent of that inhibition was larger with quinine than with PAX. Therefore, while our data support that calcium-activated potassium channels are essential for sperm capacitation in pigs, they also suggest that other potassium channels, such as the voltage-gated, tandem pore domain, and mitochondrial ATP-regulated ones, are involved in that process. Thus, further research is needed to elucidate the specific functions of these channels and the mechanisms underlying its regulation during sperm capacitation.

HVCN1 but Not Potassium Channels Are Related to Mammalian Sperm Cryotolerance.

Little data exist about the physiological role of ion channels during the freeze-thaw process in mammalian sperm. Herein, we determined the relevance of potassium channels, including SLO1, and of voltage-gated proton channels (HVCN1) during mammalian sperm cryopreservation, using the pig as a model and through the addition of specific blockers (TEA: tetraethyl ammonium chloride, PAX: paxilline or 2-GBI: 2-guanidino benzimidazole) to the cryoprotective media at either 15 °C or 5 °C. Sperm quality of the control and blocked samples was performed at 30- and 240-min post-thaw, by assessing sperm motility and kinematics, plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and intracellular O2-⁻ and H2O2 levels. General blockade of K+ channels by TEA and specific blockade of SLO1 channels by PAX did not result in alterations in sperm quality after thawing as compared to control samples. In contrast, HVCN1-blocking with 2-GBI led to a significant decrease in post-thaw sperm quality as compared to the control, despite intracellular O2-⁻ and H2O2 levels in 2-GBI blocked samples being lower than in the control and in TEA- and PAX-blocked samples. We can thus conclude that HVCN1 channels are related to mammalian sperm cryotolerance and have an essential role during cryopreservation. In contrast, potassium channels do not seem to play such an instrumental role.

HVCN1 Channels Are Relevant for the Maintenance of Sperm Motility During In Vitro Capacitation of Pig Spermatozoa.

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca2+ entrance to the sperm head.

The Presence of Seminal Plasma during Liquid Storage of Pig Spermatozoa at 17 °C Modulates Their Ability to Elicit In Vitro Capacitation and Trigger Acrosomal Exocytosis.

Although seminal plasma is essential to maintain sperm integrity and function, it is diluted/removed prior to liquid storage and cryopreservation in most mammalian species. This study sought to evaluate, using the pig as a model, whether storing semen in the presence of seminal plasma affects the sperm ability to elicit in vitro capacitation and acrosomal exocytosis. Upon collection, seminal plasma was separated from sperm samples, which were diluted in a commercial extender, added with seminal plasma (15% or 30%), and stored at 17 °C for 48 or 72 h. Sperm cells were subsequently exposed to capacitating medium for 4 h, and then added with progesterone to induce acrosomal exocytosis. Sperm motility, acrosome integrity, membrane lipid disorder, intracellular Ca2+ levels, mitochondrial activity, and tyrosine phosphorylation levels of glycogen synthase kinase-3 (GSK3)α/ß were determined after 0, 2, and 4 h of incubation, and after 5, 30, and 60 min of progesterone addition. Results showed that storing sperm at 17 °C with 15% or 30% seminal plasma led to reduced percentages of viable spermatozoa exhibiting an exocytosed acrosome, mitochondrial membrane potential, intracellular Ca2+ levels stained by Fluo3, and tyrosine phosphorylation levels of GSK3α/ß after in vitro capacitation and progesterone-induced acrosomal exocytosis. Therefore, the direct contact between spermatozoa and seminal plasma during liquid storage at 17 °C modulated their ability to elicit in vitro capacitation and undergo acrosomal exocytosis, via signal transduction pathways involving Ca2+ and Tyr phosphorylation of GSK3α/ß. Further research is required to address whether such a modulating effect has any impact upon sperm fertilizing ability.

Red-light stimulation of boar semen prior to artificial insemination improves field fertility in farms: A worldwide survey.

A survey of in vivo fertility data from 31 pig farms distributed worldwide was conducted to determine whether stimulating boar semen with LED-based red light increases its reproductive performance following artificial insemination (AI). Red-light stimulation with MaXipig® was found to increase farrowing rates (mean ± SEM, control: 87.2% ± 0.4% vs. light stimulation 90.3% ± 0.5%) and the number of both total and live newborn piglets. Red-light stimulation increased farrowing rates in 27 farms, with an increase ranging from 0.2% to 9.1%. Similar results were observed in litter sizes. Suboptimal management after AI was suggested in those farms with no response to red-light stimulation. Our results indicate that a routine use of red-light stimulation of boar semen can have a positive effect on the reproductive performance. However, the effectiveness of this system appears to highly rely upon proper management of pig farms.

Effect of AQP Inhibition on Boar Sperm Cryotolerance Depends on the Intrinsic Freezability of the Ejaculate.

Aquaporins (AQPs) are transmembrane channels with permeability to water and small solutes that can be classified according to their structure and permeability into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs. In boar spermatozoa, AQPs are related to osmoregulation and play a critical role in maturation and motility activation. In addition, their levels differ between ejaculates with good and poor cryotolerance (GFE and PFE, respectively). The aim of this work was to elucidate whether the involvement of AQPs in the sperm response to cryopreservation relies on the intrinsic freezability of the ejaculate. With this purpose, two different molecules: phloretin (PHL) and 1,3-propanediol (PDO), were used to inhibit sperm AQPs in GFE and PFE. Boar sperm samples were treated with three different concentrations of each inhibitor prior to cryopreservation, and sperm quality and functionality parameters were evaluated in fresh samples and after 30 and 240 min of thawing. Ejaculates were classified as GFE or PFE, according to their post-thaw sperm viability and motility. While the presence of PHL caused a decrease in sperm quality and function compared to the control, samples treated with PDO exhibited better quality and function parameters than the control. In addition, the effects of both inhibitors were more apparent in GFE than in PFE. In conclusion, AQP inhibition has more notable consequences in GFE than in PFE, which can be related to the difference in relative levels of AQPs between these two groups of samples.

Elucidating the Role of K+ Channels during In Vitro Capacitation of Boar Spermatozoa: Do SLO1 Channels Play a Crucial Role?

This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during in vitro sperm capacitation. Sperm samples from 14 boars were incubated in a capacitation medium for 300 min in the presence of paxilline (PAX), a specific SLO1-channel blocker, added either at 0 min or after 240 min of incubation. Negative controls were incubated in capacitation medium, and positive controls in capacitation medium plus tetraethyl ammonium (TEA), a general K+-channel blocker, also added at 0 min or after 240 min of incubation. In all samples, acrosome exocytosis was triggered with progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium levels and acrosin activity were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. In boar spermatozoa, SLO1 channels were found to have 80 kDa and be localized in the anterior postacrosomal region and the mid and principal piece of the tail; their specific blockage through PAX resulted in altered calcium levels and acrosome exocytosis. As expected, TEA blocker impaired in vitro sperm capacitation, by altering sperm motility and kinematics and calcium levels. In conclusion, SLO1 channels are crucial for the acrosome exocytosis induced by progesterone in in vitro capacitated boar spermatozoa.

Evaluation of sperm motility with CASA-Mot: which factors may influence our measurements?

Computer-aided sperm analysis (CASA) is now routinely used in IVF clinics, animal breeding centres and research laboratories. Although CASA provides a more objective way to evaluate sperm parameters, a significant number of factors can affect these measurements. This paper classifies these factors into four categories: (1) sample and slide (e.g. preincubation time, type of specimen and type of chamber slide); (2) microscope (e.g. light source and microscope stage); (3) hardware and software, including the settings of each system; and (4) user-related factors. We review the effects of the different factors in each category on the measurements made and emphasise the need to take measures to standardise evaluations. The take-home message of the present article is that there are several commercial and useful CASA systems, and all are appropriate for routine analysis. Non-commercial systems may also be good choices when the user needs to adapt the device to specific experimental conditions. In both cases (commercial and non-commercial), it is important that standard protocols are put in place for evaluation, as well as methods to validate the system.

Aquaporin 11 is related to cryotolerance and fertilising ability of frozen-thawed bull spermatozoa.

Aquaporins (AQPs) are channel proteins involved in the transport of water and solutes across biological membranes. In the present study we identified and localised aquaporin 11 (AQP11) in bull spermatozoa and investigated the relationship between the relative AQP11 content, sperm cryotolerance and the fertilising ability of frozen-thawed semen. Bull ejaculates were classified into two groups of good and poor freezability and assessed through immunofluorescence and immunoblotting analyses before and after cryopreservation. AQP11 was localised throughout the entire tail and along the sperm head. These findings were confirmed through immunoblotting, which showed a specific band of approximately 50 kDa corresponding to AQP11. The relative amount of AQP11 was significantly (P<0.05) higher in both fresh and frozen-thawed spermatozoa from bull ejaculates with good freezability compared with those with poorer freezability. In addition, in vitro oocyte penetration rates and non-return rates 56 days after AI were correlated with the relative AQP11 content in fresh spermatozoa. In conclusion, AQP11 is present in the head and tail of bull spermatozoa and its relative amount in fresh and frozen-thawed spermatozoa is related to the resilience of the spermatozoa to withstand cryopreservation and the fertilising ability of frozen-thawed spermatozoa. Further research is needed to elucidate the actual role of sperm AQP11 in bovine fertility.

Artificial insemination with frozen-thawed boar sperm.

Artificial insemination with frozen-thawed semen in pigs is not a routine technique; its use is restricted to specific cases, such as preservation of valuable genetic material (germplasm banks), safety strategies in case of natural disasters, long-distance transport of sperm, and in combination with sex-sorting. Cryoinjuries resulting from freeze-thawing protocols are a major concern with regard to the fertilization capacity of the treated sperm, which is lower than that of liquid-stored semen. Here, we provide an overview of artificial insemination using cryopreserved sperm, and summarize the factors that influence cryopreservation success before, during, and after freeze-thaw (i.e., sperm selection before starting the cryopreservation process, holding time, use of cryoprotectants, and rates of freezing and thawing) and that are driving the identification of biomarkers to predict sensitivity to cryodamage. Three different artificial insemination techniques (conventional or intracervical; intrauterine; and deep intrauterine) are also discussed with regards to their relevance when using frozen-thawed semen. Finally, we review the use of additives to freezing and thawing media, given reports that they may maintain and improve the quality and fertilizing capacity of frozen-thawed sperm. In sum, artificial insemination with frozen-thawed boar sperm can provide reasonable fertility outcomes, if freezable ejaculates, specific additives, and appropriate insemination techniques are used.

Aquaglyceroporins 3 and 7 in bull spermatozoa: identification, localisation and their relationship with sperm cryotolerance.

The present study aimed to determine the localisation of aquaglyceroporins 3 (AQP3) and 7 (AQP7) in bull spermatozoa and their relationship with the sperm cell's resilience to withstand cryopreservation (i.e. cryotolerance). A total of 18 bull ejaculates were cryopreserved and their sperm quality analysed before and after freeze-thawing. The presence and localisation of AQP3 and AQP7 was determined through immunoblotting and immunocytochemistry. AQP3 was found in the mid-piece and AQP7 in the mid-piece and post-acrosomal region of bull spermatozoa. Immunoblotting showed specific signal bands at 30 and 60kDa for AQP3 and at 25kDa for AQP7. Neither the relative abundance of AQP3 and AQP7 nor their localisation patterns was altered by cryopreservation but individual differences between bull ejaculates were found in immunoblots. In order to determine whether these individual differences were related to sperm cryotolerance, bull ejaculates were classified as having good (GFE) or poor freezability (PFE) on the basis of their sperm quality after thawing. While the relative abundance of AQP3 before cryopreservation did not differ between ejaculates with GFE and PFE, the abundance of AQP7 was higher in GFE than in PFE ejaculates. This finding was further confirmed through principal component and linear regression analyses. In conclusion, the relative abundance of AQP7 in fresh semen may be used as a marker to predict bull sperm cryotolerance.

Aquaporins in boar spermatozoa. Part II: detection and localisation of aquaglyceroporin 3.

The proteins belonging to the aquaporin family play a fundamental role in water and solute transport across biological membranes. While the presence of these proteins has been extensively studied in somatic cells, their function in mammalian spermatozoa has been studied less. The present study was designed to identify and localise aquaglyceroporin 3 (AQP3) in boar spermatozoa. With this purpose, 29 fresh ejaculates from post-pubertal Piétrain boars were classified into two groups based upon their sperm quality and subsequently evaluated through western blot and immunofluorescence assessments. Western blotting showed the specific signal band of AQP3 at 25 kDa, whereas immunofluorescence assessments allowed us to identify two different AQP3 localisation patterns: (1) spermatozoa presenting a clear labelling located only in the mid-piece and (2) spermatozoa exhibiting a distribution pattern in the head and along the entire tail. The first staining pattern was predominant in all studied ejaculates. Despite individual differences in AQP3 content and localisation between boar ejaculates, these differences were not correlated with sperm quality. In conclusion, although AQP3 is present in boar spermatozoa in two different localisation patterns, neither the AQP3 content nor its localisation have been found to be associated with conventional sperm parameters.

Cryotolerance of porcine in vitro-produced blastocysts relies on blastocyst stage and length of in vitro culture prior to vitrification.

The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24h. Re-expansion rates were recorded at 3 and 24h and total cell number and apoptotic cells were determined at 24h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification-warming procedures and length of in vitro culture, as expanding and hatching-hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential.