Efrén Estrada

Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze-thawing but not in ROS levels.

The main aim of the present study was to determine whether differences in the amounts of free cysteine residues in sperm nucleoproteins, which are a direct marker of the integrity of the disulfide bonds between nucleoproteins, existed between good (GFE) and poor boar freezability ejaculates (PFE) during the different steps of the freeze-thawing process. The analyzed steps were: (1) immediately before starting cryopreservation (17 °C), (2) at the end of the cooling step (5 °C), and (3) 30, and (4) 240 minutes after thawing. In addition, the present study also sought to determine whether GFE and PFE differed in the amounts of peroxides and superoxides generated during freeze-thawing as an overall measure of the boar sperm reactive oxygen species (ROS) accumulation rate. According to our results, PFE present lower resistance than GFE to cryopreservation-induced alterations of disulfide bonds between nucleoproteins, because levels of cysteine free residues were higher in PFE than in GFE at 30 and 240 minutes after thawing. On the other hand, no significant differences were observed between GFE and PFE in ROS levels during freeze-thawing. In conclusion, PFE are less resistant than GFE to cryopreservation not only in terms of sperm motility and membrane integrity, but also in the integrity of nucleoprotein structure. However, this difference between PFE and GFE in the resistance of the nucleoprotein structure to freeze-thawing is not linked with concomitant changes in ROS levels.

Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze–thawing by stabilising disulfide bonds

One important change the head of boar spermatozoa during freeze-thawing is the destabilisation of its nucleoprotein structure due to a disruption of disulfide bonds. With the aim of better understanding these changes in frozen-thawed spermatozoa, two agents, namely reduced glutathione (GSH) and procaine hydrochloride (ProHCl), were added at different concentrations to the freezing media at different concentrations and combinations over the range 1-2mM. Then, 30 and 240 min after thawing, cysteine-free residue levels of boar sperm nucleoproteins, DNA fragmentation and other sperm functional parameters were evaluated. Both GSH and ProHCl, at final concentrations of 2mM, induced a significant (P<0.05) increase in the number of non-disrupted sperm head disulfide bonds 30 and 240 min after thawing compared with the frozen-thawed control. This effect was accompanied by a significant (P<0.05) decrease in DNA fragmentation 240 min after thawing. Concomitantly, 1 and 2mM GSH, but not ProHCl at any of the concentrations tested, partially counteracted the detrimental effects caused by freeze-thawing on sperm peroxide levels, motility patterns and plasma membrane integrity. In conclusion, the results show that both GSH and ProHCl have a stabilising effect on the nucleoprotein structure of frozen-thawed spermatozoa, although only GSH exerts an appreciable effect on sperm viability.

Supplementing cryopreservation media with reduced glutathione increases fertility and prolificacy of sows inseminated with frozen-thawed boar semen

The main aim of this work was to evaluate how supplementing freezing media with reduced glutathione (GSH) affected the ‘in vivo’ fertilizing ability of boar semen subjected to cryopreservation procedures. With this purpose, 12 ejaculates coming from 12 boars were cryopreserved in the presence or absence of 2 mm GSH, whereas the same number of extended ejaculates coming from the same boars was used as negative/farm controls. Eight different sperm parameters (levels of free‐cysteine residues in sperm nucleoproteins, DNA fragmentation, sperm viability, acrosome‐membrane integrity, intracellular peroxide and superoxide levels, and total and progressive sperm motility) were evaluated before freezing and after 30 and 240 min of thawing. In addition, a total of 180 multiparous sows were used in the field fertility trials, the females being randomly divided into three groups and inseminated with extended, frozen‐thawed control or frozen‐thawed semen supplemented with 2 mm GSH. The presence of GSH in the freezing media significantly (p < 0.05) increased farrowing rates and the number of total born piglets and alive born piglets, and partially counteracted the cryopreservation‐induced damages inflicted on frozen‐thawed spermatozoa. We can thus conclude that supplementing freezing media with 2 mm GSH greatly improves boar sperm cryopreservation technology, as it significantly improves the fertilizing ability of frozen‐thawed spermatozoa.

Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus

Although cryopreservation of stallion spermatozoa allows long‐term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze‐thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) and poor (poor freezability ejaculates: PFE) freezability stallion ejaculates in other sperm parameters, including peroxide and superoxide levels, potential of mitochondrial membrane and nuclear integrity. With this purpose, a total of 24 stallion ejaculates were cryopreserved and classified into two groups (GFE vs. PFE), depending on their sperm membrane integrity and motility after freeze‐thawing. From the total of 24 ejaculates, 13 were classified as GFE and the other 11 were classified as PFE. Apart from differences in sperm membrane permeability and lipid disorder after freeze‐thawing, GFE presented significantly (p < 0.05) higher percentages of viable spermatozoa with high content of peroxides and of superoxides than PFE. In contrast, and despite cryopreservation of stallion spermatozoa increasing DNA fragmentation and disrupting disulphide bonds in sperm head proteins, no significant differences between GFE and PFE were seen. We can thus conclude that good and poor freezability stallion ejaculates differ in their reactive oxygen species levels after cryopreservation, but not in the damage extent on sperm nucleus.

The improving effect of reduced glutathione on boar sperm cryotolerance is related with the intrinsic ejaculate freezability.

Reduced glutathione (GSH) improves boar sperm cryosurvival and fertilising ability when added to freezing extenders. Poor freezability ejaculates (PFE) are known to present lower resistance than good freezability ejaculates (GFE) to cryopreservation procedures. So far, no study has evaluated whether the ability of GSH to counteract the cryopreservation-induced injuries depends on ejaculate freezability (i.e. GFE vs. PFE). For this reason, thirty boar ejaculates were divided into three equal volume fractions and cryopreserved with or without GSH at a final concentration of either 2 or 5mM in freezing media. Before and after freeze-thawing, sperm quality was evaluated through analysis of viability, motility, integrity of outer acrosome membrane, ROS levels, integrity of nucleoprotein structure, and DNA fragmentation. Ejaculates were classified into two groups (GFE or PFE) according to their post-thaw sperm motility and viability assessments in negative control (GSH 0mM), after running cluster analyses. Values of each sperm parameter were then compared between treatments (GSH 0mM, GSH 2mM, GSH 5mM) and freezability groups (GFE, PFE). In the case of GFE, GSH significantly improved boar sperm cryotolerance, without differences between 2 and 5mM. In contrast, PFE freezability was significantly increased when supplemented with 5mM GSH, but not when supplemented with 2mM GSH. In conclusion, PFE need a higher concentration of GSH than GFE to improve their cryotolerance.

The Increase in Phosphorylation Levels of Serine Residues of Protein HSP70 during Holding Time at 17°C Is Concomitant with a Higher Cryotolerance of Boar Spermatozoa

Boar-sperm cryopreservation is not usually performed immediately after semen collection, but rather a holding time (HT) of 4 h–30 h at 17°C is spent before starting this procedure. Taking this into account, the aim of this study was to go further in-depth into the mechanisms underlying the improving effects of HT at 17°C on boar-sperm cryotolerance by evaluating the effects of two different HTs (3 h and 24 h) on overall boar-sperm function and survival before and after cryopreservation. Given that phospho/dephosphorylation mechanisms are of utmost importance in the overall regulation of sperm function, the phosphorylation levels of serine residues (pSer) in 30 different sperm proteins after a 3 h- or 24 h-HT period were also assessed. We found that a HT of 24 h contributed to a higher sperm resistance to freeze-thawing procedures, whereas mini-array protein analyses showed that a HT of 24 h induced a significant (P<0.05) increase in pSer (from 100.0±1.8 arbitrary units in HT 3 h to 150.2±5.1 arbitrary units in HT 24 h) of HSP70 and, to a lesser extent, in protein kinases GSK3 and total TRK and in the cell-cycle regulatory protein CDC2/CDK1. In the case of HSP70, this increase was confirmed through immunoprecipation analyses. Principal component and multiple regression analyses indicated that a component explaining a percentage of variance higher than 50% in sperm cryotolerance was significantly correlated with pSer levels in HSP70. In addition, from all the parameters evaluated before freeze-thawing, only pSer levels in HSP70 resulted to be able to predict sperm cryotolerance. In conclusion, our results suggest that boar spermatozoa modulate its function during HT, at least partially, by changes in pSer levels of proteins like HSP70, and this is related to a higher cryotolerance.

Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels

Incubation of boar spermatozoa in a capacitation medium with oligomycin A, a specific inhibitor of the F0 component of the mitochondrial ATP synthase, induced an immediate and almost complete immobilisation of cells. Oligomycin A also inhibited the ability of spermatozoa to achieve feasible in vitro capacitation (IVC), as measured through IVC-compatible changes in motility patterns, tyrosine phosphorylation levels of the acrosomal p32 protein, membrane fluidity and the ability of spermatozoa to achieve subsequent, progesterone-induced in vitro acrosome exocytosis (IVAE). Both inhibitory effects were caused without changes in the rhythm of O2 consumption, intracellular ATP levels or mitochondrial membrane potential (MMP). IVAE was accompanied by a fast and intense peak in O2 consumption and ATP levels in control spermatozoa. Oligomycin A also inhibited progesterone-induced IVAE as well as the concomitant peaks of O2 consumption and ATP levels. The effect of oligomycin on IVAE was also accompanied by concomitant alterations in the IVAE-induced changes on intracellular Ca(2+) levels and MMP. Our results suggest that the oligomycin A-sensitive mitochondrial ATP-synthase activity is instrumental in the achievement of an adequate boar sperm motion pattern, IVC and IVAE. However, this effect seems not to be linked to changes in the overall maintenance of adequate energy levels in stages other than IVAE.

Intracellular calcium movements of boar spermatozoa during ‘in vitro’ capacitation and subsequent acrosome exocytosis follow a multiple‐storage place, extracellular calcium‐dependent model

This work analysed intracellular calcium stores of boar spermatozoa subjected to ‘in vitro’ capacitation (IVC) and subsequent progesterone‐induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico‐chemical properties, Fluo‐3 and Rhod‐5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo‐3 was located at both the midpiece and the whole head. Rhod‐5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca2+ signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca2+ labellings concomitantly with the sperms inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca2+ signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo‐3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca2+. The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca2+‐induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

Artificial Insemination in Boar Reproduction

This chapter is focused on the main aspects related to utilizing artificial insemination (AI) in pig production. The importance of AI in pig farming is discussed, as well as the main techniques used in its application. The first section is devoted to a brief description of the historical development of AI and its application in the pig industry, followed by a brief justification of its current importance. Subsequently, the application of AI with frozen-thawed and sex-sorted semen samples is discussed. A last section regarding current legislation in AI pig farming has also been included.

Specific LED-based red light photo-stimulation procedures improve overall sperm function and reproductive performance of boar ejaculates.

The present study evaluated the effects of exposing liquid-stored boar semen to different red light LED regimens on sperm quality and reproductive performance. Of all of the tested photo-stimulation procedures, the best pattern consisted of 10 min light, 10 min rest and 10 min of further light (10-10-10 pattern). This pattern induced an intense and transient increase in the majority of motility parameters, without modifying sperm viability and acrosome integrity. While incubating non-photo-stimulated sperm at 37 °C for 90 min decreased all sperm quality parameters, this reduction was prevented when the previously-described light procedure was applied. This effect was concomitant with an increase in the percentage of sperm with high mitochondrial membrane potential. When sperm were subjected to ‘in vitro’ capacitation, photo-stimulation also increased the percentage of sperm with capacitation-like changes in membrane structure. On the other hand, treating commercial semen doses intended for artificial insemination with the 10-10-10 photo-stimulation pattern significantly increased farrowing rates and the number of both total and live-born piglets for parturition. Therefore, our results indicate that a precise photo-stimulation procedure is able to increase the fertilising ability of boar sperm via a mechanism that could be related to mitochondrial function.