Detrimental effects of non-functional spermatozoa on the freezability of functional spermatozoa from boar ejaculate

In the present study, the impact of non-functional spermatozoa on the cryopreservation success of functional boar spermatozoa was evaluated. Fifteen sperm-rich ejaculate fractions collected from five fertile boars were frozen with different proportions of induced non-functional sperm (0--native semen sample-, 25, 50 and 75% non-functional spermatozoa). After thawing, the recovery of motile and viable spermatozoa was assessed, and the functional of the spermatozoa was evaluated from plasma membrane fluidity and intracellular reactive oxygen species (ROS) generation upon exposure to capacitation conditions. In addition, the lipid peroxidation of the plasma membrane was assessed by the indirect measurement of malondialdehyde (MDA) generation. The normalized (with respect to a native semen sample) sperm motility (assessed by CASA) and viability (cytometrically assessed after staining with Hoechst 33342, propidium iodide and fluorescein-conjugated peanut agglutinin) decreased (p<0.01) as the proportion of functional spermatozoa in the semen samples before freezing decreased, irrespective of the semen donor. However, the magnitude of the effect differed (p<0.01) among boars. Moreover, semen samples with the largest non-functional sperm subpopulation before freezing showed the highest (p<0.01) levels of MDA after thawing. The thawed viable spermatozoa of semen samples with a high proportion of non-functional spermatozoa before freezing were also functionally different from those of samples with a low proportion of non-functional spermatozoa. These differences consisted of higher (p<0.01) levels of intracellular ROS generation (assessed with 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; CM-H(2)DCFDA) and increased (p<0.01) membrane fluidity (assessed with Merocyanine 540). These findings indicate that non-functional spermatozoa in the semen samples before freezing negatively influence the freezability of functional spermatozoa.     

Impact of epididymal maturation, ejaculation and in vitro capacitation on tyrosine phosphorylation patterns exhibited of boar (Sus domesticus) spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation and ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to investigate the impact of epididymal maturation, ejaculation and capacitation on phosphotyrosine content of sperm proteins. Western blot, immunocytochemical and flow cytometry analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues of the sperm head followed by a subtle increase after in vitro capacitation. As cells pass from caput to cauda epididymis, tyrosine phosphorylation becomes confined to a triangular band over the posterior part of midacrosome region, whereas in vitro capacitation causes a spread labeling over the whole head. Different bands with phosphotyrosine residues were detected during epididymal maturation and after in vitro capacitation: 1) 93, 66 and 45 kDa bands with specific phosphotyrosine expression in immature spermatozoa; 2) 76, 23 and 12 kDa bands with specific phosphotyrosine expression in mature spermatozoa, being significantly increased in their expression after in vitro capacitation; 3) 49, 40, 37, 30, 26 and 25 kDa constitutive bands that increased their phosphotyrosine expression after maturation and/or in vitro capacitation; and 4) 28 and 20 kDa bands with a specific phosphotyrosine expression in in vitro capacitated spermatozoa. These results provided integral novel data of expression and location of phosphotyrosine residues during epididymal maturation, ejaculation and in vitro capacitation of boar spermatozoa. Two new constitutive proteins bands of 26 and 25 kDa with phosphotyrosine residues were also identified.     

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.     

The effect of sperm concentration in the ejaculate on morphological traits of bull spermatozoa

Experiments were performed on 75 ejaculates obtained from 19 bulls representing different cattle breeds used at the Masovian Centre for Animal Breeding and Reproduction in ?owicz. Fresh ejaculates were measured in respect to their volume and sperm count in the ejaculates was determined. The ejaculates were classified based on the criterion of sperm concentration and divided into five groups. Sperm morphometric measurements were taken from each bull and assessment of semen morphology was done on the basis of examination under a microscope using preparations made from fresh ejaculates. For each slide, morphometric measurements were taken of 15 randomly selected spermatozoa characterised by normal morphology and well visible under the microscope. Additionally, in each preparation morphometry of 500 spermatozoa was evaluated, numbers of spermatozoa with normal morphology and morphological abnormalities were recorded and these were categorized into spermatozoa with major and minor defects. An insignificant correlation was observed between the sperm concentration in the ejaculate and morphological traits, dimensions and shapes of bull spermatozoa. The less concentrated ejaculates contained spermatozoa with a slightly larger head circumference and a more elongated head shape in comparison with the spermatozoa in the more concentrated ejaculates. The highest frequency of morphologically malformed spermatozoa, both in the case of primary and secondary alterations, was observed in ejaculates with sperm concentration of no more than 1000 x 10(3)/mm3.     

A morphologic study of the ductus of the epididymis of Sus domesticus

The head, body, and tail regions of the epididymal duct (or caput, corpus, and cauda epididymis) in two healthy and sexually mature Sus domesticus males were examined by light microscopy and by scanning or transmission electron microscopy. The epididymal duct is lined with a pseudostratified epithelium with stereocilia and covered by a muscular-connective tissue sheath that is thickest in the tail region. Diameter of the epididymal duct and height of epididymal epithelium are maximal in the head region. Length of the sterocilia and spermatic density are higher in the head and body regions. Somatic cells are abundant in the tail region. The epididymal epithelium is made up of five cell types: basal cells, principal cells, clear cells, narrow cells, and basophilic cells. Abundant secretory units are observed in the supranuclear cytoplasm of columnar principal cells. Each mature secretory unit is constituted by electron-dense secretion granules covered by more than eight layers of cisternae of reticulum between which the mitochondria are intercalated. In the apical cytoplasm the isolated secretion granules become larger and less electron dense. The apical surface is covered by numerous sterocilia. Basal cells are pyramidal and less high than principal cells. The clear cells, arranged between the principal cells, are characterized by the presence of abundant vesicular elements and electron-lucid secretion granules, and by an apocrine secretory process. The narrow cells are characterized by their highly vacuolized cytoplasm. Intermediate cell typologies can be found among basal, principal, clear, and narrow cells, which could be four developmental stages of the same cell type. The basophilic cells are spheroidal and are found at different levels between the epithelial cells and in the connective tissue underlying the epithelium. © 1993 Wiley-Liss, Inc.     

Glucagon and tolbutamide as insulin secretagogues in the pig (Sus domesticus)

1. Glucagon tolbutamide, either alone or in combination, were injected i.v. into pigs and the effect upon plasma glucose and insulin concentrations measured. 2. Glucagon gave similar insulin responses to those seen in humans, but insulin responses to tolbutamide were less than in humans. 3. Combined doses of glucagon and tolbutamide gave similar, though reduced, responses to those seen in humans. At the highest combined doses applied, glucose concentration remained reduced for up to 6 hr. The insulin responses were approximately equal to the sum of the responses to each substance given alone.     

Polypeptides from serum low-density lipoproteins of pigs (Sus domesticus).

Low-density lipoproteins floating between densities 1-006 and 1-063 g cm-3 were isolated by centrifugation of blood serum obtained from 24-h fasted pigs (Sus domesticus). This lipoprotein fraction contained two components with Sf 1-063 values of 3-4 and 2-3 at 20 degrees C when examined by analytical ultracentrifugation. Delipidation of the lipoprotein yielded 15% recovery of soluble protein. Chromatography on Sephadex G100 in 8 M urea of these delipidation products yielded three fractions of different sizes which were present in both native and succinylated apoproteins. These fractions from the succinylated apolipoproteins were further characterized. A polypeptide fraction comprising 70% of the total protein had an apparent molecular weight of 34000 and contained greater amounts of amino acids with hydrophobic side chains than did the second fraction of apparent molecular weight 22000 which contained 15% of the protein. The third fraction of apparent molecular weight 12500 contained 15% of the protein.     

Dominance Rank and Parental Investment in Swine (Sus scrofa domesticus)

Using domestic swine, we tested the general prediction from life history theory that females increase their investment in offspring with increasing age and parity. Because increased investment may have a greater beneficial impact on the lifetime reproduction of sons than daughters, we also tested the prediction that older females would invest more in sons than in daughters compared to younger females. Finally, we examined whether age- or parity-related patterns of change in reproductive effort were associated with differences in the social dominance ranks of females. Female swine from a large number of domestic breeds were assigned to social groups, and their dominance ranks were determined based on the outcome of agonistic encounters. The prediction that older females produce larger litters was supported, but the increase was related only to age, not to parity. Across all ages, high-ranking females produced a greater proportion of sons than low-ranking females. Contrary to our prediction, there was no rank-related change in the proportion of sons born with increasing age or parity. However, the mean body masses offspring born to high-ranking females increased with increasing maternal age and parity, but this was not the case for offspring of low-ranking females. Studies of free-ranging groups of swine are needed to determine whether an increase in body mass at birth would have different effects on the reproduction of sons or daughters.     

Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10 mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca²⁺]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P < 0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca²⁺]i between P1 and SRF-P1 in fresh as well as in frozen–thawed semen. A higher (P < 0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32 kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.     

Ontogenic development of swine (Sus domesticus) adipose tissue lipases.

1. The in vitro activities of lipoprotein lipase (LPL) and hormone sensitive lipase (HSL) were examined in adipose tissue preparations from pigs 0-150 days of age. 2. The activities of both LPL and HSL increased 3- to 4-fold between birth and day 2 postpartum, remained at relatively high levels through weaning, and fell sharply in the oldest animals (150 days). 3. The decline in enzyme activities at older ages could partially be attributed to an increase in adipocyte size.     

Intramural chronotopography of myocardial depolarization in the heart ventricles of the pig (Sus scrofa domesticus)

Multichannel synchronous cardioelectrotopography is used to analyze the sequence of myocardial depolarization in the ventricles of the pig heart. Formation of early depolarization areas has been established in subendocardium of interventricular septum and in the base of papillary muscles of the left ventricle; of multiple foci-in the thickness of myocardial walls; of areas of late depolarization-in subepicardium of the dorsolateral side of the left ventricle. In pig heart ventricles, as compared with other ungulates (reindeer and sheep) there are revealed differences in location of areas of the early and late depolarization areas and the breakthrough of excitation wave onto subepicardium.     

Quality of boar spermatozoa from the sperm-peak portion of the ejaculate after simplified freezing in MiniFlatpacks compared to the remaining spermatozoa of the sperm-rich fraction

Boar sperm viability post-thaw differs depending on the ejaculate fraction used, with spermatozoa present in the first 10 mL of the sperm-rich fraction (SRF) (portion 1, P1, sperm-peak portion) displaying the best cryosurvival in vitro compared with that of spermatozoa from the rest of the ejaculate (portion 2 of the SRF plus the post-spermatic fraction), even when using simplified freezing routines. This viability apparently relates to the specific profile of seminal plasma in P1 (i.e., glycoprotein and bicarbonate concentrations, and pH). However, spermatozoa from P1 have not been compared with spermatozoa from the rest of the SRF (SRF-P1, usually 30-40 mL of the SRF), which is routinely used for freezing. We compared P1 with SRF-P1 in terms of sperm kinematics (using the QualiSperm™ system), while membrane integrity (SYBR-14/PI), acrosome integrity (FITC PNA/PI), and sperm membrane stability (Annexin-V) were explored using flow cytometry. As well, total protein concentration and the proteomics of the seminal plasma (SP) of both portions of the SRF were studied using two-dimensional electrophoresis (2DE), mass fingerprinting (MALDI-TOF), and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) on selected peptides. The SRF portions were collected weekly from four mature boars (4-5 replicates per boar, sperm concentration: P1, 1.86 ± 0.20; SRF-P1, 1.25 ± 0.14 × 10⁹ spz/mL) and processed using a quick freezing method in MiniFlatPacks. Post-thaw sperm motility reached 50%, without differences between SRF portions, but with clear inter-boar variation. Neither plasma membrane nor acrosome integrity differed (ns) between fractions. These results indicate that there are no differences in cryosurvival after quick freezing of boar spermatozoa derived from either of the two SRF portions. While P1 and SRF-P1 clearly differed in relative total protein contents, as expected, they displayed very similar protein profiles as assessed using 2DE and mass spectrometry (tryptic peptide mass fingerprint analysis and CID-MS/MS), indicating a similar emission of epididymal protein content.