The ubiquitous transmembrane protein Na+⁄K+-ATPase affects sperm fertility and capacitation through ion transport and cell signaling, but its effect on in vivo boar fertility, and any controlling mechanism(s) are unknown. The isoforms of the α and β subunits of Na+⁄K+-ATPase (α1, α2, α3, β1, β2, β3) were assessed in one ejaculate from each of six high and six low fertility boars (HF, LF respectively) which differed in their in vivo direct boar effects (DBE) for both farrowing rate (FR) and litter size (p < 0.05). Sperm function assessed by CASA was similar in HF and LF ejaculated sperm. Immunocytochemistry of the ejaculate sperm found more intact HF than LF sperm had α3 equatorially (p < 0.05), and fewer HF than LF methanol-permeabilized sperm had detectable α2 (p < 0.05). Image Quant analysis of Western blots rigorously quantified the amount of each isoform in the isolated head plasma membrane from each ejaculate, determining that the total amount of the isoform α3, and the amounts of 7 individual α3 bands, differed significantly (HF > LF, p < 0.05), and linear regression confirmed a highly significant relationship of total amount of α3 and α1 with FR (r2 = 0.90; p < 0.0001 and r2 = 0.36; p < 0.05, respectively). The β1, β2 and β3 isoforms in LF lacked several molecular weight bands common to HF, and the total volume of β1 tended to correlate with DBE for FR (r2 = 0.2652; p = 0.09). These findings suggest that specific isoforms of Na+/K+-ATPase in the sperm head are correlated to boar in vivo fertility, probably through Na+⁄K+-ATPase's role in capacitation.

Sodium-potassium-ATPase (Na+K+-ATPase), a target to treat congestive heart failure, is the only known receptor for cardiac glycosides implicated in intracellular signaling and additionally functions enzymatically in ion transport. Spermatozoa need transmembrane ion transport and signaling to fertilize, and Na+K+-ATPase is identified here for the first time in boar spermatozoa. Head plasma membrane (HPM) isolated from boar spermatozoa was confirmed pure by marker enzymes acid and alkaline phosphatase (218 ± 23% and 245 ± 38% enrichment, respectively, versus whole spermatozoa). Western immunoblotting detected α and β subunits (isoforms α1, α3, β1, β2, and β3) in different concentrations in whole spermatozoa and HPM. Immunofluorescence of intact sperm only detected α3 on the post-equatorial exterior membrane; methanol-permeabilized sperm also had α3 post-equatorially and other isoforms on the acrosomal ridge and cap. Mass spectrometry confirmed the presence of all isoforms in HPM. Incubating boar sperm in capacitating media to induce the physiological changes preceding fertilization significantly increased the percentage of capacitated sperm compared to 0 h control (33.0 ± 2.6% vs. 19.2 ± 2.6% capacitated sperm, respectively; p = 0.014) and altered the β2 immunofluorescence pattern. These results demonstrate the presence of Na+K+-ATPase in boar sperm HPM and that it changes during capacitation.

Capacitation begins in the sperm head plasma membrane (HPM). Membrane rafts could house signaling molecules, but although these specialized microdomains have been microscopically visualized in sperm heads, rafts have been isolated for study only from homogenized whole sperm or tails, never purified HPM. Sodium/potassium ATPase (Na+ K+ -ATPase) is a membrane-bound signaling protein that induces capacitation in bull sperm in response to the steroid hormone ouabain, and its subunit isoforms α1, α3, β1, β2, and β3 are known in HPM. This study hypothesized that rafts exist in the HPM of bull sperm, with Na+ K+ -ATPase subunit isoforms preferentially localized there. Western immunoblotting (WB) of HPM from fresh, uncapacitated bull sperm (n = 7 ejaculates), and detergent-resistant membranes isolated by density gradient centrifugation from this HPM, contained the raft-marker protein Flotillin-1; the non-raft fraction did not. HPM, raft, and non-raft contained all known Na+ K+ -ATPase isoforms including, for the first time, the previously unknown α2 isoform. Quantification (ImageQuant Software) found α3 and β1 were relatively dominant isoforms in the HPM raft. WB profiles of raft isoforms differed significantly from HPM and non-raft profiles, with unique banding patterns and amounts, hinting that the capacitation signaling in the now-identified HPM rafts may depend on unique sequences within the isoform structure.

The endogenous steroid hormone ouabain induces capacitation of bull sperm acting through its receptor Na+/K+-ATPase on the sperm plasma membrane. Progesterone (P4) is believed to act through the sperm membrane P4 receptor (mPR) to induce non-genomic signalling leading to capacitation and/or acrosome reaction (AR) in the sperm of some species, but the exact nature of this receptor molecule on bull sperm is not known. In amphibian oocytes, P4 acts through the low-affinity ouabain binding site on Na+/K+-ATPase to induce signalling highly reminiscent of ouabain's signalling that initiates capacitation. This study hypothesized that ouabain and P4 interact agonistically or antagonistically to induce bull sperm capacitation. Sperm were incubated with 0, 12.5, 25, 50 and 100 μM ouabain, P4 or ouabain + P4 (12.5, 25 and 50 μM each) under capacitating conditions, and capacitation was assessed microscopically looking at the acrosome status of sperm and as the amount of protein tyrosine phosphorylation (Tyr-P). Both steroids stimulated Tyr-P of certain sperm proteins, but ouabain caused tyrosine phosphorylation of more proteins than P4 and stimulated significantly more overall Tyr-P (P < 0.05). Ouabain also was the only steroid to stimulate significant microscopically-evident AR. When sperm were co-incubated with the two steroids, P4 partially inhibited ouabain-induced Tyr-P and AR. These results suggest that P4 and ouabain may both interact with Na+/K+-ATPase, but ouabain is the more effective hormone. Ouabain may, therefore, be the primary physiological inducer of bovine capacitation.

Although sperm and seminal plasma differ in their origin, biophysical and biochemical properties of seminal plasma influence the sperm function. Seminal plasma is a fluid medium containing substances from testes, epididymides and accessory glands. Composition of seminal plasma varies among animal species and in boars, prostate and bulbourethral glands are major contributors to the volume and contents. While the origin of some components of seminal plasma are known, the source of recently discovered seminal plasma microRNAs remains unknown, in part due to the difficulty of recovering and characterizing RNA from porcine sperm and seminal plasma. To test the hypothesis that seminal plasma miRNAs interact with sperm, the first objective was to validate protocols for recovering RNAs from porcine seminal plasma and sperm, whereas the second objective was to characterize expression patterns of 84 prioritized microRNAs employing real time PCR methodology. The study identified a relationship between sperm and seminal plasma microRNAs, based on the normalized threshold cycle of amplifying cDNA in sperm and seminal plasma from the same semen of Landrace boars. Therefore, it was concluded that seminal plasma miRNAs may originate from sperm or these miRNAs may shuttle between sperm and seminal plasma in order to facilitate cell-to-cell communication.

ContentsLiposomes are artificial membrane vesicles that can be used to test and model the functions and interactions of various biological membranes, or as a carrier system to deliver biologically active substances into the cells, or to incorporate lipids into the plasma membrane of target cells to modify membrane structure–function relationships. Sperm plasma membrane undergoes lipid modification during maturation in epididymis and during capacitation in the female reproductive tract to facilitate fertilization. Natural variation in the amounts and composition of lipids in the sperm plasma membrane may also contribute to the species‐specific sperm sensitivities to handling and storage conditions. Boar sperm are notoriously susceptible to membrane damage and are resistant to compositional alteration by artificial liposomes. This study used flow cytometry to demonstrate stable incorporation of nanoliposomes prepared from a complex mixture of various phospholipids (phosphatidylcholine : phosphatidylethanolamine : sphingomyelin : phosphatidylserine : phosphatidylinositol) with high fusion efficiency. Over 90% of sperm rapidly took up fluorescently labelled liposomes and retained the lipids for at least 60 min, in a significant time‐ and concentration‐dependent manner. This unique fusion efficacy could be used to alter sperm plasma membrane composition and hence membrane‐based functional responses.

This study proposes a framework for the collection and management of knowledge related to food security in Canada. This paper has several goals. First, the paper summarizes the current state of food security knowledge in Canada. Second, the paper presents data from an experiment where food security issues were discussed by stakeholders at two fora, the first of which was held in western Canada and the second held in eastern Canada. Finally, based on the notes taken during and feedback received after the fora, this paper suggests a framework for organizing and managing the multiple perspectives and complex types of knowledge about food security and sustainable development from a Canadian context. Two fora were held in Canada where food security issues were presented and discussed by multiple stakeholders. Most provinces in Canada were represented in at least one of the two sessions. Sessions were designed to be informative and interactive; agenda were designed to take advantage of the needs and experiences of multiple stakeholders in both western and eastern regions of Canada. To develop the framework, the sessions were also designed to assess the types of issues and knowledge about food security in Canada. Evidence presented from this experiment supports arguments that food security work must be approached in a collaborative manner, no matter the approach or discipline. Data from fora held in western and eastern regions of Canada indicate a number of specific ways in which those along the food continuum have the desire to share knowledge and enter into partnerships to work toward secure and sustainable food systems. Data also indicate how academia and institutions of higher learning might play a key role in sharing food security-based knowledge. The data from this study suggest that academia could play a leadership role in collecting and sharing information about food security-based knowledge from all disciplinary approaches that could help collaborative in addressing the complexity of food security challenges. The framework developed in this paper could provide the guide for organizing knowledge about food security and sustainable development. The framework could be used as a map to guide understanding about the different ways in which food security can be approached and understood; this could help reduce tensions among partners in projects where a wide variety of experiences are attempting to work collaboratively. The two food security fora brought together stakeholders with specialized knowledge about food security in a Canadian context. The analysis of data arising from the fora permitted unique insights to arise about the nature of knowledge by region. The data in this study also allowed us to build a framework for food security knowledge from these regional knowledge bases. This paper suggests that academia actively take a leadership role and openly share knowledge about food security. Open sharing of knowledge will help collaborates in dealing with complex food security issues understand in-depth other approaches; this sharing may help to make explicit the tensions that arise during collaborative work.

Awda, B. J., Miller, S. P., Montanholi, Y. R., Vander Voort, G., Caldwell, T., Buhr, M. M. and Swanson, K. C. 2013. The relationship between feed efficiency traits and fertility in young beef bulls. Can. J. Anim. Sci. 93: 185–192. Great efforts have been taken in recent years for improving feed efficiency in beef cattle. Despite there being several studies on biological factors associated with this trait, little is known about the potential relationships between fertility and feed efficiency in beef cattle. The objective of this study was to examine the relationship between feed efficiency traits and bull fertility traits [sperm motility, viability and scrotal circumference (SC)]. From a total of 328 crossbred beef bulls that were subjected to a performance test (112 d in each of 6 yr), SC was measured and semen collected from 110 bulls using the electroejaculation method (average age±SE=417±2.5 d). Sperm were extended, cooled, and frozen in liquid nitrogen. Two residual feed intake (RFI) measures were considered with different prediction models for dry matter intake (DMI), RFI Koch included size and growth rate and RFI bkft included the additional adjustment for backfat thickness (BKFT). Sperm viability, motility and progressive motility of the 10 bulls with the greatest RFI Koch (Hi-RFI Koch ) were greater than those of the 10 bulls with the lowest RFI Koch (Lo-RFI Koch ; P<0.05, 0.01, and 0.05, respectively). Sperm motility (P<0.01), progressive motility and SC (P<0.05) of the 10 bulls with the greatest RFI bkft (Hi-RFI bkft ) were greater than those of the 10 bulls with the lowest RFI bkft (Lo-RFI bkft ). In summary, these data indicate that young beef bulls with greater feed efficiency have decreased sperm motility, sperm viability and SC, which is an undesirable effect of selection for improved feed efficiency that needs to be addressed through multiple trait selection.

Existing as a ubiquitous transmembrane protein, Na(+)K(+)-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na(+)K(+)-ATPase is a dimer of α and β subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na(+)K(+)-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against α1 and 3, and all β isoforms. Relative quantity of Na(+)K(+)-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that α3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of β1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of β1 revealed three distinct spots. Based on the unique quantity, location and structure Na(+)K(+)-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm.

The African elephant population in North American zoos is not self-sustaining, in part due to the prevalence of ovarian acyclicity. While little is known about the cause of this condition, earlier research has shown that females without cyclic corpus luteum (CL) function rank higher in the dominance hierarchy than females with cyclic CL function. The goal of this study was to measure longitudinal serum testosterone concentrations in captive female African elephants to determine if there is a relationship among serum testosterone concentrations, social dominance rank and ovarian cyclicity status. Weekly blood samples from 49 female African elephants (24 having and 25 not having cyclic CL function at 22 facilities) were collected over a 12-month period and analyzed for serum testosterone using an enzymeimmunoassay. A progesterone radioimmunoassay was used to quantify serum progestagen concentrations and categorize ovarian cyclicity status. The dominance hierarchy of individual elephants within each herd was assessed by a written temperament survey, which identified 19 dominant, 15 middle and 15 subordinate females. No clear patterns of serum testosterone secretion were observed in females with and without cyclic CL function. Furthermore, no significant relationships were found among serum testosterone concentrations, dominance rank, and ovarian cyclicity status. These data suggest that increased circulating testosterone concentrations are not associated with greater rates of ovarian acyclicity or dominance status in captive female African elephants.

This paper reviews stresses boar sperm undergo during processing and presents preliminary results of dietary modification that minimize this damage. Processing for artificial insemination (AI) stresses boar sperm by osmotic effects; altering cell size, shape and membranes; intracellular ice formation; and production of reactive oxygen species (ROS). Sperm response to ROS is concentration-dependent, with low levels activating the ERK pathway to stimulate tyrosine phosphorylation (Tyr-P) and capacitation, but high concentrations or inappropriately timed onset of ROS pathways can harm sperm. Fresh boar sperm exposed to ROS increased intracellular hydrogen peroxide (H(2) O(2) ) phospholipase and lipid peroxidation, maintained viability but lost motility and underwent acrosome reactions (AR). Direct incorporation of lipids ± the antioxidant Vitamin E improves the survival of liquid- and frozen-stored semen. Boars fed dietary flaxseed for 8 weeks to increase n-3 fatty acids displayed improved sperm morphology (p < 0.05), increased membrane fluidity (p < 0.05) and better retention of motility and viability during 5-7 day storage (p < 0.05). Processes reducing oxidative damage to stored sperm should be evaluated.

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na(+) K(+)-ATPase or α-amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the β subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na(+) K(+)-ATPase within the membranes.

The objective of this study was to determine the effect of in utero and lactational exposure to nicotine on the male reproductive tract. Dams were randomly assigned to receive saline or nicotine bitartrate (1mg/kg-d s.c.) daily for two weeks prior to mating until weaning (postnatal day 21). Male offspring were sacrificed at 7 (peri-pubertal) and 26 (adult) weeks of age. Nicotine-exposure resulted in retention of spermatids after stage VIII, tubular vacuolation, degeneration of pachytene and round spermatids at stage VII in the testes; and lymphocyte infiltration, germ cell exfoliation, and hypospermia in epididymides, at 7 weeks of age. Nicotine-exposure had no effect on testis or epididymal morphology, daily sperm production, epididymal sperm reserve, sperm viability at 26 weeks of age, and circulating testosterone levels at either age examined. We conclude that maternal nicotine-exposure during pregnancy and lactation can induce transient structural changes in the testis and epididymis of male offspring.

The extracellular signal-regulated kinase (ERK) family of the mitogen-activated protein kinase (MAPK) pathway is identified for the first time in boar sperm and is associated with capacitation and tyrosine phosphorylation (tyr-P). Reactive oxygen species (ROS) modulate this signal transduction. Western immunoblotting detected the ERK pathway components RAF1, MEK1/2, and ERK1/2 in extracts from fresh boar spermatozoa and determined that their phosphoprotein profiles differed in a capacitation-dependent fashion. Capacitation was accompanied by appearance of two new ERKs (158 and 161 kDa) and disappearance of others. Capacitation was verified with increased tyr-P, which was inhibited by a 30-min pre-exposure of fresh boar sperm to a xanthine/xanthine oxidase ROS-generating system prior to the capacitating incubation; ROS pre-exposure also affected the phosphorylation of RAF1, MEK1/2, and ERK1/2. Preincubating sperm with inhibitors of the ERK components with or without the ROS generator affected subsequent capacitation. Inhibiting ERK1/2 inhibited tyr-P of capacitated boar spermatozoa proteins of 172, 97, and 66 kDa (P ≤ 0.04); with ROS, this inhibition increased (P < 0.002) and tyr-P of 111 kDa declined (P < 0.028). Pre-exposure to ROS plus MEK1/2 inhibitor prevented capacitation-induced tyr-P of proteins of 187 (P < 0.01) and 112 kDa (P < 0.04) versus capacitation with or without ROS. Therefore, ERK1/2 components of the MAPK pathway significantly regulate boar sperm capacitation, and RAF1 and MEK1/2 may have some lesser influence through crosstalk with different pathways. ROS affect RAF1, MEK1/2, and ERK1/2 and could influence the sequential events of boar sperm capacitation.

Na+K+-ATPase acts as a signaling molecule when ouabain binds, inducing dose-dependent protein tyrosine phosphorylation (p-tyr) during sperm capacitation. Src is present in head membranes of bull sperm and its expression alters during ouabain-induced p-tyr, so this study investigated Src's role in capacitation. Fresh bull sperm (n=3-6 ejaculates per trial) were pre-incubated with or without Src inhibitors herbimycin A (inhibits kinase) or SU6656 (prevents ATP binding to Src), followed by incubation in the presence or absence of ouabain or heparin. Extracted proteins were assessed by western blotting, band intensity measured, and quantities of each protein band compared by ANOVA. Both protein p-tyr and LPC-induced acrosome reaction increased (p<0.05) upon exposure of sperm to ouabain (242, 176, 81, 49 kDa) or heparin (176kDa). Inhibition of Src activity by herbimycin or SU6656 antagonized (P<0.05) various ouabain- and heparin-induced p-tyr, and herbimycin inhibited the ouabain-induced acrosome reaction, confirming Src's integral role in capacitation. Herbimycin actually reduced some Src itself (p<0.05) during ouabain- or heparin-induced capacitation incubation. However, since Src was activated by auto-phosphorylation in the presence of ouabain or heparin, it was of greater interest that herbimycin significantly reduced ouabain-enhanced Src autophosphorylation of the 59kDa phospho-Src and the other ouabain- (166-31kDa) and heparin-induced phospho-Src (130, 40kDa) (P<0.05) implying that those are coupling proteins or substrates of Src. Src can also be activated by serine phosphorylation (p-ser) and both ouabain and heparin significantly stimulated p-ser of various sperm proteins (254-18kDa). SU6656 inhibited ouabain-induced p-ser at 40 and 32kDa, but heparin overcame this inhibition, implying that the Na+K+-ATPase pathway requires ATP binding to Src. In contrast, heparin is less ATP sensitive, perhaps primarily stimulating the cAMP/PKA pathway. Ouabain appears to induce capacitation through the ERK pathway, since phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187) during capacitation were ouabain-sensitive. SU6656 antagonized ouabain-enhanced phosphorylation of ERK2, and other ERKs (39-17kDa) at 2h and herbimycin inhibited ERK1 in control sperm incubated for 5h. Heparin overcame the Src inhibitors' effect on ERKs possibly through the heparin-induced cAMP/PKA pathway. In summary, this study demonstrates Src's activation and involvement in tyrosine phosphorylation-dependent signaling through involving and activating ERK1/2 (potentially ERK2) in ouabain-induced bull sperm capacitation or acrosome reaction. The ouabain-sensitive Na+K+-ATPase signaling cascade is unique and distinct from that of heparin, but this novel research does identify for first time that Src cross talks with the heparin signaling pathway to induce capacitation. (poster)