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.

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.

Neuronal signaling repertoire in the mammalian sperm functionality.

The common embryonic origin has been a recurrent explanation to understand the presence of "neural receptors" in sperm. However, this designation has conditioned a bias marked by the classical neurotransmission model, dismissing the possibility that neurotransmitters can play specific roles in the sperm function by themselves. For instance, the launching of acrosome reaction, a fundamental sperm function, includes several steps that recall the process of presynaptic secretion. Unlike of postsynaptic neuron, whose activation is mediated by molecular interaction between neurotransmitter and postsynaptic receptors, the oocyte activation is not mediated by receptors, but by cytosolic translocation of sperm phospholipase (PLCζ). Thus, the sperm has a cellular design to access and activate the oocyte and restore the ploidy of the species by an "allogenic pronuclear fusion." At subcellular level, the events controlling sperm function, particularly the capacitation process, are activated by chemical signals that trigger ion fluxes, sterol oxidation, synthesis of cyclic adenosine monophosphate, protein kinase A activation, tyrosine phosphorylations and calcium signaling, which correspond to second messengers similar to those associated with exocytosis and growth cone guidance in neurons. Classically, the sperm function associated with neural signals has been analyzed as a unidimensional approach (single ligand-receptor effect). However, the in vivo sperm are exposed to multidimensional signaling context, for example, the GABAergic, monoaminergic, purinergic, cholinergic, and melatoninergic, to name a few. The aim of this review is to present an overview of sperm functionality associated with "neuronal signaling" and possible cellular and molecular mechanisms involved in their regulation.

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.

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.

Effects of reduced glutathione on acrosin activity in frozen-thawed boar spermatozoa.

In pigs, acrosin activity in extended semen is correlated with reproductive performance and has recently been identified as a freezability marker. Reduced glutathione (GSH) is known to decrease sperm cryodamage and increase the reproductive performance of frozen-thawed boar spermatozoa. However, the effects of GSH on the acrosin activity of good and poor freezability ejaculates (GFE and PFE, respectively) is yet to be examined. The present study investigated how supplementing cryopreservation media with GSH affected acrosin activity in GFE and PFE, as well as the relationship between acrosin activity and reproductive performance in frozen-thawed boar spermatozoa. In addition, we examined whether the increase in fertility rates and litter sizes observed after the addition of 2mM GSH to cryopreservation extenders was related to acrosin activity. Supplementing freezing media with 2mM GSH partially counteracted the cryopreservation-related decrease in acrosin activity in GFE but not PFE. Acrosin activity was found to be significantly correlated with in vivo reproductive performance of frozen-thawed boar semen. In conclusion, the effects of adding GSH to freezing extenders on the acrosin activity of frozen-thawed boar spermatozoa rely on the intrinsic freezability of the ejaculate. Furthermore, the maintenance of proper acrosin activity could contribute to the increase in reproductive performance mediated by GSH.

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.

Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates.

It has been proposed that seminal plasma proteins (SPP) support survival of ram spermatozoa, exerting a dual effect, both capacitating and decapacitating. In this study, changes in motility patterns of ram spermatozoa capacitated in the presence of epidermal growth factor (EGF) were evaluated. Clustering procedures were used to determine the presence of sperm subpopulations with specific motion characteristics. Four sperm subpopulations (SP) were defined after the application of a principal component analysis procedure. Progressive spermatozoa with high straightness (STR) were found in SP1, reflected in the high linearity (LIN) and STR values and low amplitude of lateral head movement (ALH; rapid, non-hyperactivated spermatozoa). SP2 spermatozoa seemed to be starting to acquire hyperactivated motility, while the SP3 group consisted of rapid, hyperactivated spermatozoa. SP4 showed less-vigorous spermatozoa, with non-linear motility. The addition of SPP before in vitro capacitation with EGF induced a decrease in SP1 and an increase in SP3. However, a reduction in the chlortetracycline-capacitated sperm rate and protein tyrosine phosphorylation was found, which corroborates with the hypothesis that the SPP protective effect on spermatozoa is related to their decapacitating role. These findings allow us to deduce that ram spermatozoa are able to undergo capacitation with no hyperactivation and that SPP are able to induce hyperactivation in spermatozoa but maintain them in a decapacitated state.

The addition of reduced glutathione to cryopreservation media induces changes in the structure of motile subpopulations of frozen-thawed boar sperm.

Adding cryopreservation media with reduced glutathione (GSH) has previously been shown to maintain the motility, membrane integrity and fertilizing ability of frozen-thawed boar sperm, although the effects of GSH on good (GFE) and poor freezability (PFE) ejaculates rely upon the intrinsic ejaculate freezability. The resilience to withstand freeze-thawing procedures has previously been related to the existence of a specific distribution of motile sperm subpopulations, which differs between GFE and PFE. Thus, the main aim of this study was to determine whether the addition of GSH to freezing media has any impact on the distribution of motile sperm subpopulations in GFE and PFE. With this purpose, 18 GFE and 13 PFE were cryopreserved with or without 2 mM GSH. Sperm quality and motile subpopulations were evaluated at 30 min and 4 h post-thawing. Three subpopulations were identified and the percentages of spermatozoa belonging to the fastest and most linear subpopulation, which was referred as 'SP1', decreased over post-thawing time. Good freezability ejaculates that were cryopreserved in the presence of 2 mM exhibited a significantly higher percentage of spermatozoa belonging to SP1 than the other combinations of treatment and freezability both at 30 min (mean ± SEM: GFE-C: 16.6 ± 0.4; GFE-GSH 27.7 ± 0.6) and 4 h post-thawing (GFE-C: 7.8 ± 0.2 vs. GFE-GSH: 16.7 ± 0.4). In conclusion, the positive effect of GSH on the motility of frozen-thawed sperm is related to a specific sperm subpopulation (SP1), which could coincide with the fertile sperm one.

Aquaporins 7 and 11 in boar spermatozoa: detection, localisation and relationship with sperm quality.

Aquaporins (AQPs) are integral membrane water channels that allow transport of water and small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Two aquaporins, AQP7 and AQP11, in boar spermatozoa were identified by western blotting and localised through immunocytochemistry analyses. Western blot results showed that boar spermatozoa expressed AQP7 (25kDa) and AQP11 (50kDa). Immunocytochemistry analyses demonstrated that AQP7 was localised in the connecting piece of boar spermatozoa, while AQP11 was found in the head and mid-piece and diffuse labelling was also seen along the tail. Despite differences in AQP7 and AQP11 content between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11 content showed a significant correlation (P<0.05) with sperm membrane integrity and fluidity and sperm motility. In conclusion, boar spermatozoa express AQP7 and AQP11, and the amounts of AQP11 but not those of AQP7 are correlated with sperm motility and membrane integrity.

AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa.

Spermatozoa undergo energy- and metabolism-dependent processes to successfully fertilize the oocyte. AMP-activated protein kinase, AMPK, is a sensor of cell energy. We recently showed that AMPK controls spermatozoa motility. Our aims are i) to investigate the intracellular localization of AMPK in boar spermatozoa by immunofluorescence, ii) to study whether AMPK plays a role in other relevant processes of spermatozoa: mitochondrial membrane potential (∆Ψm), plasma membrane lipid disorganization, outward phosphatidylserine (PS) exposure, acrosome integrity and induced-acrosome reaction by flow cytometry and iii) to investigate intracellular AMPK pathways by western blot. Spermatozoa were incubated under different conditions in the presence or absence of compound C (CC, 30µM), an AMPK inhibitor and/or cAMP analog 8Br-cAMP. AMPKα protein is expressed at the entire acrosome and at the midpiece of spermatozoa flagellum, whereas phospho-Thr(172)-AMPK is specifically localized at the apical part of acrosome and at flagellum midpiece. CC treatment rapidly confers head-to-head aggregation-promoting property to spermatozoa. Long term AMPK inhibition in spermatozoa incubated in TCM significantly reduces high ∆Ψm. Moreover, AMPK inhibition significantly induces plasma membrane lipid disorganization and simultaneously reduces outward PS translocation at plasma membrane in a time-dependent manner. Acrosomal integrity in TCM is significantly enhanced when AMPK is inhibited. However, neither acrosome reaction nor membrane lipid disorganization induced by ionophore A23187 are affected by CC. AMPK phosphorylation is potently stimulated upon PKA activation in spermatozoa. This work suggests that AMPK, lying downstream of PKA, regulates at different levels mammalian spermatozoa membrane function.

Viable and morphologically normal boar spermatozoa alter the expression of heat-shock protein genes in oviductal epithelial cells during co-culture in vitro.

The principal aim of this study was to determine if boar spermatozoa influence the expression of four selected chaperone and heat-shock protein (HSP) genes-namely clusterin (CLU), HSP90AA1, HSPA5, and HSPA8-in oviductal epithelial cells (OECs) during in vitro co-culture. All corresponding proteins of these genes were previously identified in a sperm-interacting, 70-kDa soluble fraction derived from apical plasma membranes of OECs. The present study also sought to determine whether or not: (i) spermatozoa must directly bind to OEC for an effect on gene expression to be elicited and (ii) reproductive and nonreproductive epithelial cell types (LLC-PK1, pig kidney) respond equivalently, in terms of alterations in chaperone and HSP gene expression, during co-culture with sperm. Spermatozoa induced a significant upregulation (P < 0.05) in HSP90AA1 and HSPA5 in OECs after 3 hr, and in HSPA8 after 6 hr of co-culture when they were in direct contact with epithelial cells. Conversely, no upregulation of HSP transcription was observed when spermatozoa did not directly bind to OECs. Spermatozoa also induced a significant upregulation (P < 0.05) of the same three genes when in direct contact with LLC-PK1 cells, but the timing occurred later than with OECs. Interestingly, the extent of HSP gene upregulation induced by direct contact of spermatozoa with epithelial cells was dependent on sperm-binding index and on the viability and morphological quality of the bound sperm population. In conclusion, the upregulation of HSP genes caused by direct contact between spermatozoa and OECs, rather than nonreproductive epithelial cells, suggests HSPs could play an integral role in the modulation of sperm function in the oviductal reservoir.

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.

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.

Heat shock protein 70 kDa (HSP70) is involved in the maintenance of pig sperm function throughout liquid storage at 17 °C.

At present, liquid storage is the most efficient method for pig semen preservation. This approach relies upon reducing sperm metabolism, allowing for the maintenance of cell lifespan. In this context, the study of proteins that could protect sperm during liquid storage is of high relevance. The 70 kDa Heat Shock Protein (HSP70) is an anti-apoptotic protein that has been reported to be relevant to sperm survival. Thus, we explored the role of HSP70 during prolonged storage of pig semen at 17 °C. Six semen pools were incubated with YM-1 (0, 0.05, 0.1 and 0.2 µM), an HSP70 inhibitor, and stored at 17 °C for 21 days. On days 0, 4, 10, 14 and 21, sperm quality and function were evaluated through flow cytometry and Computer-Assisted Sperm Analysis (CASA), and HSP70 activity and chromatin condensation were also determined. While inhibition of HSP70 increased progressive motility, Ca2+ and Reactive Oxygen Species (ROS) levels, and mitochondrial activity during the first 10 days of storage, it had a detrimental effect on sperm motility after 14 and 21 days. In spite of this, sperm viability was not altered. We can conclude that HSP70 contributes to the liquid storage of pig semen because it keeps mitochondrial activity low, which is needed for the maintenance of sperm function.

Voltage-dependent anion channels are involved in the maintenance of pig sperm quality during liquid preservation.

Pigs are usually bred through artificial insemination with liquid semen preserved at 15-20 °C. While this method of preservation brings many benefits, including a greater reproductive performance compared to frozen-thawed sperm, the period of storage is a limiting factor. As the mitochondrion regulates many facets of sperm physiology, modulating its activity could have an impact on their lifespan. Aligned with this hypothesis, the present study sought to investigate whether inhibition of voltage-dependent anion channels (VDACs), which reside in the outer mitochondrial membrane and regulate the flux of ions between mitochondria and the cytosol in somatic cells, influences the resilience of pig sperm to liquid preservation at 17 °C. For this purpose, semen samples (N = 7) were treated with two different concentrations of TRO19622 (5 µM and 50 µM), an inhibitor of VDACs, and stored at 17 °C for 10 days. At days 0, 4 and 10, sperm quality and functionality parameters were evaluated by flow cytometry and computer-assisted sperm analysis (CASA). The effects of inhibiting VDACs depended on the concentration of the inhibitor. On the one hand, the greatest concentration of TRO19622 (50 µM) led to a decrease in sperm motility, viability and mitochondrial membrane potential, which could be related to the observed intracellular Ca2+ increase. In contrast, total sperm motility was higher in samples treated with 5 µM TRO19622 than in the control, suggesting that when VDACs channels are inhibited by the lowest concentration of the blocking agent the resilience of pig sperm to liquid storage increases. In conclusion, the current research indicates that mitochondrial function, as regulated by ion channels in the outer mitochondrial membrane like VDACs, is related to the sperm resilience to liquid preservation and may influence cell lifespan.

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.

The effects of red LED light on pig sperm function rely upon mitochondrial electron chain activity rather than on a PKC-mediated mechanism.

While irradiation with red LED light has been reported to modulate sperm function in different mammalian species, the mechanisms underlying their response are poorly understood. This work sought to provide new insights into whether this effect relies on a direct action upon mitochondrial electron chain and/or on PKC-linked mechanisms such as those related to opsins. For this purpose, pig semen was light-stimulated for 1, 5 or 10 min in the presence/absence of antimycin A, an inhibitor of the mitochondrial electron chain, or PKC 20-28® (PKCi), a PKC inhibitor. Antimycin A completely blocked the effects of light at all the performed irradiation patterns. This effect was linked to a complete immobility of sperm, which was accompanied with a significant (p < 0.05) drop in several markers of mitochondrial activity, such as JC-1 staining and O2 consumption rate. Antimycin A, however, did not affect intracellular ATP levels, intramitochondrial calcium, total ROS, superoxides or cytochrome C oxidase (CCO) activity. In the case of PKCi, it did also counteract the effects of light on motility, O2 consumption rate and CCO activity, but not to the same extent than that observed for antimycin A. Finally, the effects observed when sperm were co-incubated with antimycin A and PKCi were similar to those observed with antimycin A alone. In conclusion, red LED light acts on sperm function via a direct effect on mitochondrial electron chain. Additionally, light-activated PKC pathways have a supplementary effect to that observed in the electron chain, thereby modulating sperm parameters such as motility and CCO activity.

Advances in sperm cryopreservation in farm animals: Cattle, horse, pig and sheep.

Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. In some farm animals, the use of cryopreserved sperm has so many benefits for which relevance has become more evident in recent decades. Values for post-thaw sperm quality, however, are variable among species and within individuals of the same species. There is no standardized methodology for each of the stages of the cryopreservation procedure (andrological examination, semen collection, dilution, centrifugation, resuspension of the pellet with the freezing medium, packaging, freezing and post-thaw sperm evaluation), which also contributes to differences among studies. Cryotolerance markers of sperm and seminal plasma (SP) have been evaluated for prediction of ejaculate freezability. In addition, in previous research, there has been a focus on supplementing cryopreservation media with different substances, such as enzymatic and non-enzymatic antioxidants. In most studies, inclusion of these substances have led to improved post-thaw sperm quality and fertilizing capacity as a result of minimizing the adverse effects on sperm structure and function. Another approach is the use of different cryoprotectants. The aim with this review article is to provide an update on sperm cryopreservation in farm animals. The main detrimental effects of cryopreservation are described, including the negative repercussion on reproductive performance. Furthermore, the potential use of molecular biomarkers to predict sperm cryotolerance is discussed, as well as the addition of substances that can mitigate the harmful impact of freezing and thawing on sperm.