Characteristics of donkey spermatozoa along the length of the epididymis

In mammals, the epididymis has numerous interrelated functions including absorptive and secretory activity that affect luminal environment and cell membrane, and the maturation and storage of sperm. Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymis and the motility of epididymal spermatozoa should be a balance between the maturation of flagellum and the inhibition of the flagellar machinery. In this study maturational change in sperm characteristics were evaluated in the epididymis of donkey. Spermatozoa collected from four portions of the epididymis (head, cranial corpus, caudal corpus, tail) were compared before and after ejaculation for viability, mitochondrial activity, kinetic parameters, and morphology. A significant increase in the mitochondrial activity along the epididymis was reported, suggesting a possible involvement in the motion mechanism. This should be corroborated by the significant correlation between mitochondrial activity and the total and progressive motility and the increase in velocities of spermatozoa recorded by computer-assisted sperm analysis. The percentage of most of the abnormal spermatozoa were similar in all tracts, with a great variability between jackasses. Only the bent midpiece percentage decreased significantly along epididymis. A significant increase in the percentage of distal cytoplasmic droplets (DCD), and a simultaneous decrease in the proximal cytoplasmic droplets (PCD), was found. The DCD fell down after ejaculation suggesting the late loss of the cytoplasmic residual (DCD) in the donkey, as hypothesized in the stallion. Because the prevalence of PCD were similar in both tail epididymal and ejaculated spermatozoa, a defect of the maturative process in the PCD sperm should be speculated.     

The degree of resistance to freezing-thawing is related to specific changes in the structures of motile sperm subpopulations and mitochondrial activity in boar spermatozoa

The main aim of this work was to analyze the possible relationship between the structures of motile-sperm subpopulations and boar (Sus scrofa domesticus) sperm resistance to freezability. For this purpose, 45 boar ejaculates were subjected to a standard freezing-thawing protocol, and afterwards they were classified into three groups, in accordance with their resistance to freezing-thawing. Our analysis yielded four separate motile-sperm subpopulations in all of the studied ejaculates, both in fresh samples and after freezing-thawing. Furthermore, whereas curvilinear velocity (VCL), mean velocity (VAP), and dance (DNC) of sperm from Subpopulation 1 underwent significant increases after freezing-thawing in samples with a good response to freezing-thawing, the same parameters of Subpopulation 1 either did not undergo significant variations (VCL and DNC) or even showed a decrease (VAP) (from 20.4 ± 0.4 μm/sec in fresh samples to 15.2 ± 2.2 μm/sec after freezing-thawing) in samples with the poorest response. Similarly, the behavior of other motility parameters in each subpopulation was also very different in the worst samples when comparing them with those with a good or average response to cryopreservation. Additionally, the DNC of all four subpopulations was in all cases lower in samples with the poorest characteristics of freezability. This was not the only difference, and significant changes in parameters such as the VCL of Subpopulations 2 and 4, linearity coefficient (LIN) of Subpopulations 1, 2, and 3, and wobble coefficient (WOB) of Subpopulations 2 and 3 were also observed in samples with different response to freezing-thawing. Meanwhile, the determination of mitochondrial activity and mitochondrial-linked reactive oxygen species formation indicated that the samples with the poorest freezability characteristics were also those with the lowest mitochondrial activity. We conclude that boar ejaculate resistance to cryopreservation seems to be related to the specific, initial motile-sperm subpopulation structure. In turn, this structure would be closely related to the specific, overall mitochondrial activity, which would be a very important indicator of sperm function. Furthermore, and as a practical conclusion, an in-depth analysis of motile sperm subpopulation structure together with functional tests could improve the design of predictive strategies for the freezability of boar sperm.     

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4 h at 5 °C, and at 0 and 2 h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations: Subpopulation 1 showed medium velocity (VCL: 99.4 ± 17.8 μm/sec) and high progressiveness (LIN: 65.1 ± 14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL: 148.7 ± 25.6 μm/sec) but a nonprogressive trajectory (LIN: 33.1 ± 10.5%); Subpopulation 3 represented slowly motile (VCL: 58.3 ± 24.3 μm/sec) and nonprogressive sperm (LIN: 39.6 ± 18.3%); and Subpopulation 4 included very rapid (VCL: 152.8 ± 25.7 μm/sec) and highly progressive sperm (LIN: 70.9 ± 13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P < 0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.     

Factors affecting sperm recovery rates and survival after centrifugation of equine semen

Conventional centrifugation protocols result in important sperm losses during removal of the supernatant. In this study, the effect of centrifugation force (400 or 900 × g), duration (5 or 10 min), and column height (20 or 40 mL; Experiment 1); sperm concentration (25, 50, and 100 × 10⁶/mL; Experiment 2), and centrifugation medium (EZ-Mixin CST [Animal Reproduction Systems, Chino, CA, USA], INRA96 [IMV Technologies, Maple Grove, MN, USA], or VMDZ [Partnar Animal Health, Port Huron, MI, USA]; Experiment 3) on sperm recovery and survival after centrifugation and cooling and storage were evaluated. Overall, sperm survival was not affected by the combination of centrifugation protocol and cooling. Total sperm yield was highest after centrifugation for 10 min at 400 × g in 20-mL columns (95.6 ± 5%, mean ± SD) or 900 × g in 20-mL (99.2 ± 0.8%) or 40-mL (91.4 ± 4.5%) columns, and at 900 × g for 5 min in 20-mL columns (93.8 ± 8.9%; P < 0.0001). Total (TMY) and progressively motile sperm yield followed a similar pattern (P < 0.0001). Sperm yields were not significantly different among samples centrifuged at various sperm concentrations. However, centrifugation at 100 × 10⁶/mL resulted in significantly lower total sperm yield (83.8 ± 10.7%) and TMY (81.7 ± 6.8%) compared with noncentrifuged semen. Centrifugation in VMDZ resulted in significantly lower TMY (69.3 ± 22.6%), progressively motile sperm yield (63.5 ± 18.2%), viable yield (60.9 ± 36.5%), and survival of progressively motile sperm after cooling (21 ± 10.8%) compared with noncentrifuged semen. In conclusion, centrifuging volumes of ≤ 20 mL minimized sperm losses with conventional protocols. With 40-mL columns, it may be recommended to increase the centrifugal force to 900 × g for 10 min and dilute the semen to a sperm concentration of 25 to 50 × 10⁶/mL in a milk- or fractionated milk-based medium. The semen extender VMDZ did not seem well suited for centrifugation of equine semen.     

Seasonal dynamics of sperm morphometric subpopulations and its association with sperm quality parameters in ram ejaculates

Sperm morphologic assessment is considered an irreplaceable part of standard laboratory routine analyses in the diagnosis of male fertility. Thus, in an attempt to quantify the effects of season on sperm morphology and its functional significance in relation to sperm quality parameters, sperm head morphometric traits were analyzed by using an objective computerized analysis combined with principal components analysis (PCA) cluster analysis to establish the relationship between the distribution of the subpopulations found and sperm quality in each season. There were slight variations on sperm motility and sperm membrane integrity indexes (P > 0.05). However, the mean values for sperm concentration substantially changed among seasons in all individuals studied (P < 0.01). There were significant differences in sperm morphometric parameters (P < 0.01) as well as in the distribution of morphometric subpopulations between seasons (P < 0.001). In conclusion, this study confirmed that there was an important seasonal effect on sperm morphometric traits. In addition, the distribution of these subpopulations seems to be related to the season studied and the ejaculate quality which would be a very important indicator of sperm function. The substantial information derived from these morphometric subpopulations has provided new knowledge which can be used in future studies using sperm morphometry as a seasonal indicator in ram ejaculates.     

Donkey jack (Equus asinus) semen cryopreservation: Studies of seminal parameters, post breeding inflammatory response, and fertility in donkey jennies

The aims of this study were (1) to evaluate motility parameters of donkey jack (jack; Equus asinus) semen cryopreserved in INRA-96 (INRA; IMV Technologies, France, 2% egg-yolk enriched) using either glycerol (GLY) or ethylene glycol (EG) as a cryoprotector; (2) to compare in vitro the postthaw re-extension with homologous seminal plasma (SPL) or INRA; (3) to compare fertility in donkey jennies (jennies; Equus asinus) timed artificially inseminated with jack semen cryopreserved using GLY or EG, re-extended with INRA; (4) to compare fertility in jennies timed artificially inseminated with jack semen cryopreserved using GLY re-extended with SPL, INRA, or not re-extended (NN); and (5) to describe some preliminary results of the inflammatory uterine response postbreeding. Semen from two jacks was collected and frozen in an INRA-2% egg yolk extender added of either 2.2% GLY or 1.4% EG. Postthaw motility was evaluated by a computer-assisted motility analyzer. Uterine inflammatory response and fertility were evaluated after artificial insemination (AI) of 13 jennies with frozen-thawed semen, either further extended with INRA (Group GLY-INRA, 13 cycles, and EG-INRA, 8 cycles), or with SPL (Group GLY-SPL, 13 cycles), or not re-extended (GLY-NN, 5 cycles). In each cycle, jennies were bred twice with 500 × 10⁶ sperm cells (250 × 10⁶ from each jack), at fixed times after induction of ovulation, and uterus was flushed at 6 and 10 h after first and second breeding, respectively. Cells in the recovered fluid were counted and distinguished as polymorphonuclear neutrophils (PMN) or other cell types. Total and progressive motility did not differ between cryoprotectants, but were higher when semen samples were re-extended in INRA, compared with SPL (P < 0.05). Pregnancy was diagnosed by transrectal palpation and ultrasonography examinations at 14 and 16 days postovulation. In 7/13 (53.8%) jennies and 12/39 (30.4%) cycles postbreeding intrauterine fluid accumulation was observed, with no differences between treatments (P < 0.05). Polymorphonuclear neutrophil numbers and concentrations were higher in the first flushing compared with the second, and PMN concentration was higher in GLY-SPL than in GLY-INRA (P < 0.05). Pregnancy rates in GLY-SPL, GLY-INRA, EG-INRA, and GLY-NN were 8/13, 3/13, 2/8, and 1/5, respectively. There was no significant difference either between the two cryoprotectants re-extended in INRA, or between re-extension groups. There was however a trend for GLY-SPL to improve pregnancy rates compared with GLY-INRA (P = 0.055). These results indicate that it is possible to obtain similar postthaw sperm motility and pregnancy rates using GLY or EG as a cryoprotectant for donkey semen, and that in the conditions of this study the re-extension in SPL of thawed semen before AI showed a trend toward the improvement of fertility and increased PMN concentration in uterine flushings.     

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.     

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout—Salmo trutta, perch—Perca fluviatilis, burbot—Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml⁻¹) and Ig concentrations (T-Ig: 1.11-1.61 μg ml⁻¹, IgG [measured only in brown trout]: 1.49 μg ml⁻¹) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 μg/g protein, IgG: 0.198 μg ml⁻¹) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.     

Agreement between measures of total motility and membrane integrity in stallion sperm

Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and absolute difference) between TMOT and SMI was high (-0.7 and 5.6%, respectively), but decreased after 24 (10.8 and 15.1%, respectively) and 48 h (23.0 and 23.8%, respectively) of cooled storage as motility declined more rapidly than SMI. We concluded that TMOT and SMI measured separate aspects of sperm quality.     

Effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage

An experiment was conducted to determine whether cooled semen quality could be maintained for a longer interval by conducting daily centrifugation of extended semen, with resuspension of the sperm pellet in fresh extender. Semen treatments included SP10NC and SP50NC which contained 10 and 50% seminal plasma, respectively, were not centrifuged (NC), and were stored at 4 to 7 °C for 96 h. Treatments SP10C and SP50C contained 10 and 50% seminal plasma, respectively, but were centrifuged (C) after 24, 48, and 72 h of cooled storage, with daily resuspension in fresh extender containing 10% seminal plasma. Percent total sperm motility (TMOT) and progressively motile (PMOT) was reduced (P < 0.05) in the SP50NC treatment after 24, 48, 72, and 96 h of storage, and TMOT did not differ (P > 0.05) in the SP10C, SP50C, SP10NC groups after the same storage periods. The % COMP-_αt did not differ (P > 0.05) among treatments at any time period. Percent membrane intact sperm (SMI) was reduced in SP50NC, as compared to SP10C at 48, 72, and 96 h (P < 0.05). Daily centrifugation and resuspension of sperm exposed to 50% seminal plasma for the first 24 h (SP50C) yielded similar TMOT, PMOT, VCL, SMI, % COMP-_αt (P > 0.05) to Groups SP10NC and SP10C after 96 h of storage. Daily centrifugation and resuspension of cool-stored equine semen in fresh extender may be a method to increase sperm longevity.     

Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, P1) have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2), probably in relation to different features of the surrounding seminal plasma (SP). In the present study, we investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing. Moreover, sperm plasma membrane intactness (investigated using SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of P1 spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP, sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions.     

Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L

Damage to spermatozoa during cryopreservation is regarded as a major obstacle to the expansion of sperm storage technology. The authors used two-dimensional polyacrylamide gel electrophoresis and matrix-associated laser desorption/ionization time-of-flight mass spectrometry to explore whether the protein profile of common carp (Cyprinus carpio) spermatozoa is affected by cryopreservation. Fourteen protein spots were significantly altered following cryopreservation. Eleven of these were identified: three as specific membrane proteins (N-ethylmaleimide-sensitive fusion protein attachment protein alpha, cofilin 2, and annexin A4) involved in membrane trafficking, organization, and cell movement; six as cytoplasmic enzymes (S-Adenosylhomocysteine hydrolase, Si:dkey-180p18.9 protein, lactate dehydrogenase B, phosphoglycerate kinase 1, transaldolase 1, and esterase D/formylglutathione hydrolase) involved in cell metabolism, oxidoreductase activity, and signal transduction; and two as transferrin variant C and F. Based on these findings, the authors hypothesize that transferrin in cryopreserved sperm may protect spermatozoa against oxidative damage during the freeze-thaw process. Cryopreservation caused changes in spermatozoa protein profiles that may lead to decreased spermatozoa velocity, motility, and fertilization success, and to subsequent ova hatching rate.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation

The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma membrane disorders (mean ± SEM 9.48 ± 0.65 and 1.62 ± 0.23% in raw and frozen-thawed sperm, respectively). However, both membrane peroxidation and protein phosphorylation were increased (P < 0.05) compared to raw semen (1.74 and 5.20-fold, respectively). There was a correlation (r = 0.73; P < 0.05) between the increase in lipid peroxidation and tyrosine phosphorylation. Seminal plasma, regardless of origin, reduced (P > 0.05) tyrosine phosphorylation present on the surface of cryopreserved sperm; however, lipid peroxidation was not significantly reduced. In conclusion, we inferred that emergence of phosphorylated proteins on the surface of cryopreserved sperm was due to increased lipid peroxidation that occurred during the freezing/thawing process; however, reduced tyrosine phosphorylation that occurred after addition of seminal plasma was triggered by other mechanisms, apparently independent from the reduction in membrane peroxidation.     

Cell biology and functional dynamics of the mammalian sperm surface

Theriogenology has now a 40-year rich history on covering sperm biological aspects with a special emphasis on farm and husbandry animals. The major and most influential of these contributions will be placed into an evolutionary perspective of ongoing and intriguing progresses made in this field. Although many molecular details have been published, it is more the aim of this contribution to provide a guide through the main established aspects and concepts of sperm surface biology and refer only to major molecular players and mechanisms involved in sperm physiology. Those interested in more molecular details and in-depth knowledge can easily access the most relevant literature which is included here for reference purposes. With this approach, a logical and easy to follow buildup can be made of the general picture of sperm surface dynamics and of the ergonomics of sperm physiology and their function in mammalian fertilization. Understanding the ins and outs of sperm surface biology and the dynamics thereof, might challenge future researchers to design novel generation of better sperm-handling procedures. This could be beneficial for assisted reproductive technology and animal breeding industries.     

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen-thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.     

The influence of cryopreservation and seminal plasma on the chromatin structure of dog spermatozoa

It was the aim of the current study to investigate effects of seminal plasma on the chromatin structure of frozen-thawed canine (Canis lupus familiaris) spermatozoa. A total of 20 ejaculates were collected. Ejaculates were divided, and one half was centrifuged for removal of seminal plasma (c) while the other was left uncentrifuged (nc) before cryopreservation. This was performed according to the Uppsala system in a computerized freezing machine. Before freezing (bf) and after thawing (at), samples were investigated for motility (M), viability (CASA), and chromatin status (sperm chromatin structure assay; SCSA). Before freezing, the average DFI% and the SD-DFI from 20 nc ejaculates were 1.7 ± 4.0% and 18.6 ± 1.2, respectively. After thawing, all motility parameters decreased and were significantly lower in centrifuged than in noncentrifuged samples, whereas the percentage of morphologically abnormal spermatozoa (Morph) was significantly higher (nc: M bf, 84.1 ± 20.6%; M at, 51.9 ± 15%; c: M bf, 84.1 ± 20.6%; M at, 43.3 ± 22.2%; Morph nc: 28.3 ± 7.8% vs. c: 31.0 ± 9.8%). Furthermore, only in c samples did the DFI increase within 6 h after thawing (DFI c: bf, 41.8 ± 1.5%; 6 h at, 45.4 ± 6.6%; P < 0.01). The SD-DFI as well as the DFI% increased within 3 h of storage in both groups (SD-DFI nc: bf, 18.6 ± 1.2%; 3 h at, 25.8 ± 5.4%; DFI% nc: bf, 1.1 ± 4.0%; 3 h at, 6.1 ± 12.9%; P < 0.05). For both parameters, there was no significant difference between c and nc samples at any time investigated. In conclusion, centrifugation of semen samples before freezing decreased postthaw motility and increased the percentage of morphologically abnormal spermatozoa as well as the degree of sperm chromatin denaturation over time. Centrifugation of canine ejaculates before cryopreservation can therefore no longer be recommended.     

Cryogenic changes in proteases and antiprotease activities of buffalo (Bubalus bubalis) and cattle (Bos taurus) semen

The postthaw motility and fertility of buffalo and cattle semen is reduced when they are cryopreserved for artificial insemination. In the present study, an attempt was made to characterize the cryogenic changes in proteases and antiprotease activities (APA) of buffalo and cattle semen because these proteolysis regulators have been reported to be associated with sperm motility and fertility. Buffalo sperm demonstrated at least two major proteases of 45 and 42 kDa and three minor proteases of 95, 52, and 33 kDa. Similarly, cattle sperm demonstrated three major proteases of 62, 45, and 42 kDa and two minor proteases of 85 and 78 kDa. Buffalo seminal plasma demonstrated at least three major proteases of 78, 68, and 62 kDa and one minor protease of 98 kDa and cattle seminal plasma demonstrated one major protease of 68 kDa and two minor proteases of 78 and 75 kDa. Except for the 45 kDa protease, most of the previously mentioned proteases were found to be metalloproteinases. Compared with fresh sperm, cryopreserved buffalo and cattle sperm demonstrated a major protease band of 52/49 kDa and the activity of this protease reduced progressively with the duration of cryopreservation. On the contrary, compared with the fresh seminal plasma, cryopreserved buffalo and cattle semen extenders displayed the presence of a new protease band of 45 kDa and demonstrated that this protease activity was leaked from buffalo and cattle cryopreserved spermatozoa. Buffalo and cattle seminal plasmas displayed at least two major APA of 86 and 26 kDa. Compared with buffalo, cattle seminal plasma demonstrated significantly greater APA. Thus, the present study demonstrated the presence of an array of proteases and APA in buffalo and cattle semen and the activities of which changed during cryopreservation. The leakage of the specific protease activity and changes in the proteases and APA might be attributed to reduced motility and fertility of cryopreserved semen in these species.