T. Rigau

Regression analyses and motile sperm subpopulation structure study as improving tools in boar semen quality analysis

A precise estimation of the fertilizing ability of a boar ejaculate would be very useful to improve pig assisted reproduction results. For this purpose, we tested the mathematical combination of several parameters of the boar semen quality analysis, including the computer-assisted semen motility analysis (CASA), as a predictive fertility tool. The utilized mathematical relations among parameters were logistic and linear regressions. Two mathematical models obtained by logistic regression involving Osmotic Resistance Test (ORT Test), Hyperosmotic Resistance Test (HRT Test) and viability of fresh samples, showed a significant (P<0.05) correlation between semen characteristics and conception rate. However, none of the obtained models produced a significant correlation model between semen characteristics and prolificacy. The CASA analyses show that three separate subpopulations of spermatozoa with different motility characteristics coexist in boar ejaculates. There were significant (P<0.001) differences in the distribution of these subpopulations among boars, but no clear relationship between motile subpopulation structure and fertility was obtained. Our results support the belief that the predictive use of the results obtained in a standard boar semen quality analysis can reasonably be achieved by applying logistic correlation analyses among several function parameters of boar semen quality analysis and in vivo conception rates obtained after artificial insemination (AI).

Effects of glucose and fructose on motility patterns of dog spermatozoa from fresh ejaculates.

This study was performed to gain insight about how fructose and glucose modulate dog spermatozoa motility in the absence of other motility-modulating factors. Incubation of dog spermatozoa from fresh ejaculates in a basal medium without sugars for 60 min at 37 degrees C induced a progressive decrease in the percentage of motile spermatozoa and in some mean motility parameters, such as mean velocity (VAP), linear coefficient (LIN) and dance (DNC), and an increase in the mean frequency of head displacement (BCF). This indicates a progressive loss of linearity and an increase in oscillatory movement. Addition of 10 mM fructose prevented these effects. Incubation in a basal medium with 10 mM glucose for 60 min at 37 degrees C provoked a fast and intense decrease of LIN and a slight increase of DNC, inducing a less linear and more oscillatory mean movement. Neither fructose nor glucose modified the percentage of motile spermatozoa. The response to both sugars was dose-dependent, with differences appearing at concentrations as low as 1 mM. An analysis of the spermatozoa subpopulation placed above the 95th percentile of the whole population and a factorial analysis of the data indicated that the changes in the mean values of the motility parameters were mainly due to a specific motile subpopulation that had a strong reaction to the two sugars. Our results indicate that fructose, at concentrations from 1 to 10 mM, induced a more linear and less oscillatory motility pattern than glucose. Moreover, from our results we suggest the presence of motile dog sperm subpopulations with an increased sensitivity to fructose and glucose.

Effects of hypoosmotic incubation on acrosome and tail structure on canine spermatozoa

Hypoosmotic tests are widely used as valuable tests for determining sperm quality in species as varied as the human and the porcine. However, there is little information about the use of these tests in canine spermatozoa. This work evaluates the response of canine spermatozoa in hypoosmotic media in order to introduce the use of the hypoosmotic tests in the canine standard semen analysis. In this way, the incubation of canine spermatozoa in hypoosmotic media containing citrate (ORT medium, osmotic pressure = 100 mOsm) or citrate plus fructose (HOS medium, osmotic pressure = 150 mOsm) resulted in the swelling of the sperm tail. These reactions were time-dependent, reaching maximum percentages after 45 to 60 min. Optimal percentage of tail swelling with minimal effect on the viability of spermatozoa was observed at 100 to 150 mOsm. Response on sperm viability, tail swelling and acrosome detachment to hypoosmotic tests of both undiluted fresh, and 24 h-stored samples were similar. The percentage of swollen tails after both tests showed a good correlation to viability and to gross and progressive motility but not to concentration. However, acrosome detachment after both hypoosmotic tests did not correlate to any of the studied parameters. Our results indicate that the swelling observed after hypoosmotic shock could be used as a useful test in improving the standard semen analysis in the dog.

Evidence for a functional glycogen metabolism in mature mammalian spermatozoa.

The glycogen content in fresh raw dog spermatozoa was 0.22 ± 0.03 μmol/mg protein. This matched with the presence of a glycogen‐like staining in the head and midpiece. Glycogen levels lowered to 0.05 μmol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 μmol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35 ± 0.07, measured in the absence and the presence of glucose 6‐P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25 ± 0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10–20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo. Mol. Reprod. Dev. 56:207–219, 2000.

Effects of freezing/thawing on motile sperm subpopulations of boar and donkey ejaculates

The main aim of this study is to assess the influence of freeze/thawing on motile sperm subpopulations in ejaculates from two phylogenetically different mammalian species, boar and donkey. Our results indicate that, whereas boar and donkey sperm respond very differently in their mean motion characteristics to freezing/thawing, this process did not change the existence of a 4-subpopulations structure in the ejaculates in either species when these subpopulations were defined by taking values of curvilinear velocity (VCL) as reference. Moreover, the freezing/thawing-linked changes in mean sperm-motion characteristics in both boar and donkey semen were especially due to changes in the proportion among each concrete subpopulation. In this way, the freezing/thawing-induced mean increase in motion characteristics observed in boar sperm was a result of the decrease in the percentage of sperm in Subpopulation 1 (from 53.9%+/-4.7% to 31.2%+/-3.9% after thawing) and a concomitant increase of sperm from Subpopulations 3 (from 13.3%+/-2.5% to 32.6%+/-3.9% after thawing) and 4 (from 3.4%+/-0.9% to 8.0%+/-1.1% after thawing). On the contrary, changes in mean motility of frozen/thawed donkey sperm were linked to an increase in the percentage of sperm in Subpopulation 1 (from 31.5%+/-4.3% to 58.8%+/-4.9% after thawing) and a concomitant decrease of sperm from Subpopulations 3 (from 32.4%+/-3.2% to 6.6%+/-1.8% after thawing) and 4 (from 12.2%+/-2.5% to 7.3%+/-1.9% after thawing). In conclusion, our results seem to indicate that motility changes induced by the freezing/thawing protocol are linked to concomitant changes in both the specific parameters and, more importantly, to the specific percentage of each of the motile sperm subpopulations. These changes did not affect the overall proportion of motile sperm present in both boar and donkey, which is conserved despite the detrimental effect caused by freezing/thawing in both species. Finally, the presence of some kind of motile sperm subpopulations structure has been described in mammalian species with a very great phylogenetic distance, thus suggesting that this structure could play some role in the maintenance of the overall function of mammalian ejaculates.

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.

Gluconeogenesis-Linked Glycogen Metabolism Is Important in the Achievement of In Vitro Capacitation of Dog Spermatozoa in a Medium Without Glucose

Abstract In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [14C]glycogen after the addition to l-CCM with [14C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

Subjecting horse spermatozoa to hypoosmotic incubation: Effects of ouabain

Although hypoosmotic tests are widely used to assess spermatozoal quality in different species, they have not been used extensively in the stallion. Moreover, the role of the Na (+)K (+), ouabain sensitive-ATP-ase in the response of equine sperm to hypoosmotic shock is not well understood. This study tests two hypotheses: 1) that equine spermatozoa will respond to a hypoosmotic medium by swelling of the tail, and 2) that addition of ouabain will increase the percentage of swollen sperm tails. Ejaculates from 3 stallions were collected with an artificial vagina and diluted in Kenneys medium (Time = 0). Aliquots were randomly selected to be incubated in an isoosmotic (297 mOsm) or different hypoosmotic media that were composed of citrate or of citrate wïth fructose. The osmolarity of the hypoosmotic media with citrate ranged from 18 to 96 mOsm, and the medium composed of citrate plus fructose (HOS medium) was of 153 mOsm. Moreover, aliquots of spermatozoa pretreated with ouabain were added to the isoosmotic medium and also to the HOS and the 96 mOsm citrate medium (ORT medium). Incubation of equine sperm in the hypoosmotic media resulted in a time- and osmolarity-dependent swelling of the sperm tail, reaching maximum values after incubation for 20-30 min in both the HOS and ORT media. Ouabain induced a dose-dependent effect on swollen tails and viability in fresh semen and also affected some parameters related to motility. Ouabain also increased the swelling response in a hypoosmotic medium although viability decreased. The percentage of swollen tails after incubation in ORT and HOS media snowed significant correlations to viability, altered acrosomes and total motility, but not to other parameters of horse semen analysis. Our results suggest that hypoosmotic tests could be used to improve standard horse semen analysis. Additionally, Na (+)K (+)-ATP-ase activity could be related to the response against hypoosmotic shock of horse spermatozoa.

Effects of slight agitation on the quality of refrigerated boar sperm

Abstract The main objective of this study was to test the effect of slight agitation upon characteristics of seminal quality in refrigerated boar semen. Storage of refrigerated (15–17°C) boar insemination doses for 48 h with slight agitation increased percentages of viability and total motility compared with similar doses stored without agitation. Agitation also reduced the percentage of altered acrosomes. Incubation in an iso-osmotic medium with fructose (osmotic pressure 300 mOsm) increased the percentage of osmotic resistance (ORT), and L-lactate production. The form of storage did not alter the ability to detach an acrosome in a hypo-osmotic medium (osmotic pressure 100 mOsm), as reflected in the percentage of hypo-osmotic sensitive spermatozoa (HSS). Similar results were observed when doses were stored for 92 h. While these data indicate that storage of refrigerated, diluted boar sperm with agitation may improve quality by increasing the percentage of viable spermatozoa, the HSS results suggest that the quality of the individual viable sperm was unaffected.

Cryopreservation-induced alterations in boar spermatozoa mitochondrial function are related to changes in the expression and location of midpiece mitofusin-2 and actin network

The authors analyzed changes in mitochondrial activity of boar semen during a standard cryopreservation protocol. For this purpose, mitochondrial activity was evaluated simultaneously with the rhythm of mitochondrial formation of reactive oxygen species (mROS) through a double MitoTracker Red/proxylfluorescamine stain. Moreover, we analyzed changes in the expression and location of two key regulatory elements of mitochondrial function, namely mitofusin-2 (Mfn2) and actin, during the freezing-thawing protocol. Our results indicate that mitochondrial activity and mROS formation decreased during cyropreservation, with an initial decrease during the cooling phase of the protocol. This decrease was accompanied by an increase in the amount of solubilized Mfn2, which was concomitant with a progressive extension of Mfn2 location from the apical zone of the midpiece to the whole midpiece. Simultaneously, cryopreservation induced a decrease in solubilized actin, which was concurrent with significant changes in the midpiece actin location. The observed changes in the expression and location of both Mfn2 and actin were already present after the cooling phase of the cryopreservation protocol. Our results suggest that freezing-thawing impaired mitochondrial function. This impairment was concomitant with a decrease in the mitochondrial capacity to synthesize mROS. This impairment is attributed to changes in mitochondrial volume as a result of alterations in the expression and location of both Mfn-2 and the actin network. Finally, the alterations of mitochondrial function induced by the cryopreservation protocol were already apparent at the cooling phase. This observation indicates that the cooling phase is a crucial stage in which mitochondrial alterations occur during cryopreservation.