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Biomedical subjects

M M Buhr

Publications and source records attributed to M M Buhr.

At least 19 recordsLinked to original sources

Spontaneous uptake of exogenous DNA by bull spermatozoa.

Sperm-mediated DNA transfer can be used to transfer exogenous DNA into the oocyte for the production of transgenic animals. In spite of controversy in the literature, sperm-mediated DNA transfer is a simple and quick technique that can be used in routine breeding programs (AI, embryo transfer and IVF). The main objective of this study was to determine the factors affecting the spontaneous uptake of exogenous DNA by bull spermatozoa. For this purpose, fresh and frozen spermatozoa (0.25 x 10(6)), from the same ejaculate from each of four bulls were co-incubated with fluorescent-labeled green fluorescent protein (GFP) and chloremphenicol acetyltransferase (CAT) plasmids at 37 degrees C for 30 min. Neither bull nor plasmid significantly affected the uptake of exogenous DNA. However, transfection efficiency was higher in frozen-thawed versus fresh spermatozoa (P<0.001). Regardless of whether transfected spermatozoa were alive or dead, all transfected spermatozoa were immotile. It can be concluded that a population of spermatozoa is present in bull semen which has the ability to uptake exogenous DNA spontaneously. There is tremendous scope to improve transfection efficiency of spermatozoa while maintaining motility; this needs to be achieved in order to more easily use this technique in transgenesis. However, live-transfected bull spermatozoa clearly can incorporate exogenous DNA and should be usable in intracytoplasmic sperm injection protocols.

Animals↗

Impact of genetic selection on management of boar replacement.

Boars in an artificial insemination centre have been selected for their superior genetic potential, with 'superior' being defined as having traits the customer wants transmitted to his herd. The ability to meet the customers' needs depends on the heritability of the trait, the geneticist's success in devising a selection scheme for the trait in balance with other economically important traits, and the boar's ability to produce sperm that can fertilise oocytes. Genetic evaluation research over the past 20 years has greatly increased the number of traits for which a boar can be selected: currently in the Canadian national program, these include age at 100 kg, backfat at 100 kg, feed efficiency, lean yield and litter size. In the near future, traits that are very likely to be added to this selection list include piglet survival, marbling, loin eye area and structure traits. In Canada, sires are ranked on two estimated breeding value (EBV) indices; one, focused on development of terminal sire lines, is based on the growth and yield traits and another, primarily focused on maternal line development, de-emphasises these traits and incorporates litter size. Boars that are in Canadian AI centres because of their excellent growth traits are typically in the top 5-10% of the national population for terminal sire line index, but they may be only average or substandard for litter size. Conversely, boars selected to be in the top 5-10% for conveying such reproductive traits as litter size may only be in the top 33% for growth traits. The more offspring from a superior boar in either of these indices, the faster the population average for the trait improves. The original sire gets knocked out of the elite group, is culled and replaced by a higher ranked young boar from the now improved general population. Although genetic superiority should govern an AI centre's selection and culling of boars, decision-making in real life is seldom that simple. Selection criteria may be contradictory as above, or a boar with truly superior traits may be excluded because a newly-developed molecular genetics test determines he carries an undesirable gene such as PSS, RN or others being developed. Selection for terminal sire or maternal line traits can ignore important practical factors that affect an AI centre--boars with superior genetics may not produce good semen because skeletal or penile problems prevent ejaculation, or because sperm production is poor due to a genetic flaw, disease, or some other cause. Interestingly, selection pressure for one trait may inadvertently select for a trait that is linked but whose linkage is unrecognised, and such unintentionally selected genes could benefit, harm, or have no effect on production traits. An AI centre serving a variety of customers must select boars in anticipation of their customers' needs (including new, foreign and niche markets). A centre should also review its genetic evaluation results and progeny records, both to critique its own selection success and to try to detect unexpected linkages. Finally, an AI centre needs to predict its own future, selecting not just for production traits for the swine producer, but also for factors that enhance the centre's efficiency including boar conformation and temperament, and sperm quantity, quality and hardiness. Can we select for efficiency? Our colleagues in dairy cattle AI evaluate bull performance--should the swine industry consider evaluation of male fertility traits?

Animals↗

Cloning and characterization of chicken stromal cell derived factor-1.

Stromal cell derived factor-1, SDF-1, belongs to the CXC family of chemokines and has been identified in mammals, amphibians, and fish. This chemokine has a diverse array of functions in organogenesis, hematopoeisis, B cell development and recruitment of immune system cells. Here, we report the cloning of the chicken SDF-1 ortholog and examine its temporal and spatial expression. The chicken SDF-1 cDNA contained an open reading frame encoding a predicted protein of 89 amino acids, which shared 40-75% identity to SDF-1 protein in other species. Protein folding simulation predicted a tertiary structure very similar to that obtained for human SDF-1. Recombinant chicken SDF-1 was produced using a prokaryotic expression system and the recombinant protein was shown to be biologically active in a calcium flux assay. The SDF-1 gene was found to be expressed ubiquitously and constitutively in adult tissues and was present as early as the primitive streak stage of chicken embryos.

Amino Acid Sequence↗

Liquid storage of Asian elephant (Elephas maximus) sperm at 4 degrees C.

The Asian elephant (Elephas maximus) population in the wild has been in decline for several decades and breeding in captivity has not been self-sustaining. The use of artificial insemination (AI) can help overcome many of the difficulties associated with breeding elephants in captivity; however, the ability to store semen for extended periods of time is critical to the successful application of AI to elephants. The objective of the present study was to assess the effects of four different semen extenders and the presence of egg yolk on the viability and motility of Asian elephant semen stored at 4 degrees C. High quality ejaculates (n=4) were collected from two Asian elephant bulls by rectal massage. Aliquots of each ejaculate were extended in four different diluents (Beltsville thawing solution (BTS); Tris-citric acid (TCA)/fructose-based; Beltsville F5 (BF5); dextrose-supplemented phosphate-buffered saline (PBS)) with or without egg yolk then cooled and stored at 4 degrees C. The percentages of viable (viability) and motile (motility) sperm were evaluated at 8, 24 and 48 h following collection. The addition of egg yolk significantly reduced the percentage loss in viability from initial collection to 48 h compared to extenders without egg yolk (17.0 +/- 8.2 versus 32.6 +/- 8.9 decline in percent viable sperm in the population, respectively; P<0.05). Extender and egg yolk affected (P<0.005) total motility and percent progressively motile sperm at all evaluation times during incubation. TCA + egg yolk maintained higher (P<0.05) levels of progressive motility compared to other extenders supplemented with egg yolk. These results indicate that Asian elephant semen extended in TCA diluent supplemented with egg yolk can maintain at least 50% viability and motility when stored at 4 degrees C for 48 h.

Animals↗

Optimizing and quantifying fusion of liposomes to mammalian sperm using resonance energy transfer and flow cytometric methods.

BACKGROUND: Liposomes are used to carry pharmaceutical agents and to alter the lipid composition of cell membranes. This study compared resonance energy transfer (RET), fluorescence dequenching, and flow cytometry as monitors and quantifiers of fusion between liposomes and mammalian spermatozoa. METHODS: Preliminary experiments used RET to determine the optimum sperm concentration for fusion of DL-alpha-phosphatidylcholine dipalmitoyl (PC)/DL-alpha-phosphatidylethanolamine dipalmitoyl (PE) liposomes at 35 degrees C +/- 5 mM Ca2+. Microscopy confirmed the fusion of liposomes, not just adhesion (n = 3). Dequenching tested the time-dependent fusion of liposomes of two different lipid compositions to sperm, both, (n = 3) +/- 1 mM Ca2+ and (n = 3) without Ca2+ at two sperm concentrations. Finally, flow cytometry absolutely quantified the percentage of sperm fusing to liposomes at different liposome-to-sperm ratios (n = 4) and with sperm from different donors (n = 3). RESULTS: RET detected fusion of liposomes with sperm and microscopy confirmed the interaction to be true fusion. Dequenching detected more fusion of liposomes with sperm at 100 x 10(6) sperm per milliliter than at lower concentrations (P < 0.05). Fusion dynamics differed with lipid composition but Ca2+ had no effect. Flow cytometry reliably quantified the percentage of sperm fusing with liposomes, which varied from bull to bull (P < 0.05). CONCLUSION: Liposome fusion with mammalian sperm membranes can be quantified cytometrically and varies with lipid composition, sperm-to-liposome ratio, and individual animals.

Animals↗

Isolation and unique composition of purified head plasma membrane from rooster sperm.

The structure, composition, and function of membranes from organelles of mammalian spermatozoa differ from each other and from the sperm's plasma membrane. Avian sperm studies have suffered from the lack of a technique to isolate these various membranes, which the current study now provides. Nitrogen cavitation and differential centrifugation separated head plasma membranes (HPM) of rooster sperm from sperm debris, acrosomal membranes, and mitochondrial membranes and characterized these membranes enzymatically and microscopically. The HPM was enriched in acid phosphatase (marker enzyme for HPM; 1,814.81 +/- 470.43 micromol phosphate released/microg protein vs. 868.53 +/- 75.55 for whole semen; a 202.5 +/- 37.8% enrichment, mean +/- SE, P < 0.001), with less (P < 0.001) mitochondrial and acrosomal enzyme activity. The mitochondrial fraction had 515.1 +/- 167.6% more succinate dehydrogenase activity (marker for mitochondria, P < 0.001) and the acrosomal fraction had 315.4 +/- 61.2% more acetylglucosaminidase activity (marker for acrosome, P < 0.0001) than whole semen. Thin layer and gas chromatography showed that HPM lipids had more (P < 0.05) sphingomyelin and phosphatidylserine, and less phosphatidylcholine and phosphatidylethanolamine than did the sperm body membranes (SBM). Overall, HPM had less polyunsaturated fatty acids than SBM (36.8 +/- 3.4 vs. 44.5 +/- 1.7% of total phospholipids, P < 0.05). HPM had slightly more n3 (3.2 +/- 0.5 vs. 1.3 +/- 0.2%, P < 0.01) but much less n6 (33.6 +/- 3.3 vs. 43.3 +/- 1.9%, P < 0.01), specifically less C22:4n6. Future study of avian sperm will be able to reliably characterize the structure-function relationships of specific sperm membranes.

Acid Phosphatase↗

Incorporating lipids into boar sperm decreases chilling sensitivity but not capacitation potential.

Fresh boar sperm were incubated with small unilamellar liposomes composed of either the total lipids extracted from head plasma membranes (HPM) of fresh boar sperm or selected lipids (SL) of five defined phospholipids with specific acyl chains. To optimize fusion, liposomes with 2 mol% octadecyl rhodamine fluorophore in Beltsville Thawing Solution +/- 1 mM CaCl(2) were incubated at 35 degrees C with 1;ts 10(7) or 10(8) spermatozoa/ml and monitored over 60 min, using flow cytometry and fluorescence microscopy. The HPM fused to both sperm concentrations faster than SL but was equivalent by 30 min (10(8) sperm/ml) or 60 min (10(7) sperm/ml; 57.5 +/- 3% and 67.1 +/- 8% sperm fused to HPM and SL, respectively) +/- Ca(2+). Neither HPM nor SL affected onset of capacitation or spontaneous or ionophore-induced acrosome reactions at 0 or 3 h (chlortetracycline and fluorescein isothiocyanate-Pisum sativum agglutinin; n = 3). During cooling and after cryopreservation (n = 4 ejaculates), SL but not HPM significantly improved sperm motility and viability (Sybr14/propidium iodide staining) +/- 20% egg yolk, but egg yolk alone was more effective than SL alone. Liposomes of complex composition can fuse to boar sperm without harming in vitro capacitation or acrosome reaction and reduce sperm chilling sensitivity.

Acrosome Reaction↗

Assessment of stallion spermatozoa viability by flow cytometry and light microscope analysis.

Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of each aliquot by 1) motility; 2) eosin-nigrosin staining; and 3) dual fluorescent staining. For the latter, aliquots incubated with SYBR-14 and propidium iodide had live and dead spermatozoa quantitated by fluorescent microscope (2 x 100 sperm/sample) and flow cytometry (10,000 sperm/sample). We found a linear relationship between the ratio of live:dead spermatozoa and the percentage of spermatozoa counted as live (P<0.0001). For fresh spermatozoa, correlation coefficients of the known live:dead ratio were high for all methods (eosin-nigrosin, r>0.75; fluorescent microscope, r>0.76; flow cytometry, r>0.75; motility, r>0.76). To determine viability of cryopreserved equine spermatozoa, we froze 17 fresh ejaculates from 6 stallions in a glycine extender. Each sample was thawed, extended 1:1 with EZ Mixin and evaluated as above. Cryopreserved spermatozoa assessed by flow cytometry tended to be less well correlated (r<0.68) with the other methods, and estimates were significantly higher with eosin-nigrosin staining (P<0.001). This study shows that different methods may equally estimate viability of fresh equine spermatozoa. However, evaluation by flow cytometry appears to be less precise with cryopreserved spermatozoa.

Aniline Compounds↗

Relationship between heparin binding to spermatozoa and the fertility of dairy bulls.

The presence of heparin in in vitro media has been implicated in improved fertility parameters of bull spermatozoa. In a previous study, Zhang et al. (25) obtained an estimate of bull nonreturn rates based on spermatozoal concentration, motility and zona pellucida binding (24). The objective of this study was to test for a relationship between fertility parameters previously estimated for the same batch of cryopreserved semen (25) and amount of heparin bound to spermatozoa. 3H-heparin binding to spermatozoa was assessed by radioimmunoassay, and statistical correlations were drawn to previously measured sperm characteristics. Preliminary experiments established optimal binding conditions of 25 degrees C, and 60 min incubation with 3H-heparin at a concentration of 50,000 cpm. 3H-heparin bound to an average of 2.2 x 10(6) receptors/cell with a Kd of 2.0 x 10(-7) M. The total 3H-heparin bound to spermatozoa from different bulls was significantly different (P<0.003). However, the total 3H-heparin bound to spermatozoa was not correlated with any measured sperm parameter, including zona pellucida binding, embryo cleavage and blastocyst formation, and 56-day nonreturn rates (P>0.19). Thus, the total amount of heparin bound to the surface of spermatozoa may not be relevant to fertilizing ability.

Animals↗

Effects of urea and trimethylamine N-oxide on fluidity of liposomes and membranes of an elasmobranch.

The effects on membrane fluidity of two solutes of biological importance in elasmobranch fishes, urea and trimethylamine oxide (TMAO), were determined using elasmobranch red blood cell plasma membranes and artificial liposomes. Fluorescence polarizations of three probes with differing sites of insertion (1, 6-diphenylhexatriene, cis-parinaric acid, and trans-parinaric acid) were used to study the effects of physiological levels of urea (400 mM) and TMAO (200 mM) separately and together in a 2:1 urea:TMAO ratio (400 mM:200 mM). In the elasmobranch erythrocyte membrane, there was a trend toward an increase in the order of the gel-phase domains when treated with urea, although this was not statistically significant. This effect was counteracted by the presence of TMAO. To determine if the organic solutes were acting directly on the membrane lipids or on the integral proteins, phase-transition profiles of protein-free dipalmitoyl phosphatidylcholine liposomes were determined. These profiles showed that urea again increased the order of the gel-phase domains of the bilayer; however, this effect was not counteracted by the presence of TMAO. We suggest that the increased order in the gel-phase domains may be an indirect effect of a decrease in the order of the fluid-phase domains. This increase in fluidity may be due either to a disruptive effect of urea on the hydrophobic core of the membrane or to indirect effects mediated by changes in the integral membrane proteins. This study is the first to demonstrate that urea and TMAO may act as counteracting solutes in the elasmobranch erythrocyte membrane and that the counteraction appears to be at the level of the integral proteins rather than the membrane lipids.

Animals↗

Epididymal maturation affects calcium regulation in equine spermatozoa exposed to heparin and glucose.

Spermatozoal function is affected by the ability to regulate intracellular calcium concentrations ([Ca2+]i), and may be influenced by epididymal maturation as well as environmental components. Regulation of [Ca2+]i in ejaculated and epididymal stallion spermatozoa was monitored over time in various media. Spermatozoa from each of 5 pony stallions (3 ejaculate samples and 1 caput and cauda sample) were labeled with the fluorescent calcium indicator probe Indo-1 in a calcium-free modified Tyrode's buffer. Fluorescent emissions were monitored by a dual wavelength spectrofluorometer over 5 h. Calcium (1 mM) was added at T = 15 min, and heparin (HEP; 10 micrograms/ml) or heparin plus glucose (hGLUC; 5 mM in 10 micrograms/ml heparin) was added at T = 30 min. Spermatozoal Ca2+ content and regulation differed among males (P = 0.0066). Relative initial [Ca2+]i differed significantly among all stages of maturity (0.84 +/- 0.104, 0.76 +/- 0.023, 1.20 +/- 0.036 LSM of relative Ca2+ units for caput, cauda and ejaculate spermatozoa respectively; P = 0.001). Rate of Ca2+ uptake was similar for ejaculate and cauda spermatozoa (0.021 +/- 0.005 and 0.026 +/- 0.002 relative Ca2+ units/sec) but slower for caput spermatozoa (0.012 +/- 0.001; P = 0.0006). There was no immediate effect of HEP or hGLUC in any stage (P > 0.05), and caput spermatozoa did not differ from cauda spermatozoa for any treatment or time period. A significant increase in [Ca2+]i was seen in ejaculate spermatozoa treated with HEP from 2 h on (P < 0.05). This study demonstrates that both the absolute Ca2+ concentration and the rate of Ca2+ internalization in equine spermatozoa is dependent on the stage of maturation. Ejaculate spermatozoa respond to heparin through increased [Ca2+]i, which may play a role in the fertilizing ability of ejaculate spermatozoa.

Animals↗

Environmental, management, and genetic factors affecting semen production in Holstein bulls.

The objective of this study was to evaluate the importance of environment, management, physiological status, and genetics on semen quality (volume of the ejaculate, sperm concentration, sperm motility, number of sperm, and number of motile spermatozoa per ejaculate) of Canadian Holstein bulls. For this purpose, semen production data from 198 bulls were analyzed using mixed linear models. Young bulls (up to 30 mo old) and mature bulls (between 4 and 6 yr old) were analyzed separately. Semen characteristics generally improved significantly with age of young bulls. Season significantly affected all semen traits in young bulls but did not significantly affect volume and sperm motility of mature bulls. Performance was better in winter than in summer. The highest numbers of motile spermatozoa per ejaculate were obtained with intervals of at least 4 to 5 d between collections. Although the bull handler and semen collector caused less than 10% of the variance, the collection team significantly affected semen volume, number of sperm, and number of motile sperm per ejaculate for both growing and mature bulls. Heritabilities for volume, concentration, sperm motility, number of sperm, and number of motile sperm per ejaculate were, respectively, 0.24, 0.52, 0.31, 0.38, and 0.49 for young bulls and 0.44, 0.36, 0.01, 0.54, and 0.64 for mature bulls. Repeatability of semen traits varied from 0.41 to 0.64. Genetics, management, and environmental factors clearly contribute to semen production in Holstein bulls.

Aging↗

Relationship between maternal plasma progesterone concentration and interferon-tau synthesis by the conceptus in cattle.

The objective of this study was to determine the relationship between maternal progesterone concentration and conceptus synthesis of interferon-tau as an index of conceptus viability at the time of maternal recognition of pregnancy. Heifers of mixed beef breeds were randomly assigned to receive 1 of 2 treatments: 1) intramuscular injection of 1500 IU hCG on Day 5 after artificial insemination (AI; n = 12) or 2) intramuscular injection of saline on Day 5 after AI (n = 17). Ovaries were scanned daily by transrectal real-time ultrasonography. Progesterone concentrations were determined from daily blood samples collected from the jugular vein. Heifers were slaughtered on Day 18 after AI and conceptus tissues were collected. These were incubated individually at 37 degrees C in RPMI medium, and supernatant collected after 24 h. Conceptus secretory products in the supernatant were analyzed for interferon concentration by antiviral assay using vesicular stomatitis virus. Transrectal ultrasonography showed all heifers that received hCG had at least 1 extra corpus luteum (CL) in addition to the spontaneous CL formed from the previous ovulation (10 with 2 CL, 2 with 3 CL). A significant increase in plasma progesterone concentration was detected in pregnant heifers treated with hCG (n = 9) vs pregnant control heifers (n = 11; P < 0.001). There was a tendency for an increase (P = 0.059) in synthesis of interferon-tau by conceptuses from hCG-treated heifers compared to control heifers. Maternal plasma progesterone concentrations were correlated with interferon-tau production by the conceptuses (r = 0.593, P < 0.006), suggesting that higher maternal progesterone may provide a more suitable environment for the developing conceptus.

Journal Article↗

The effect of the Hurnik-Morris (HM) system on sow locomotion, skin integrity, and litter health.

Gilts (n = 187) were randomly assigned to either the Hurnik-Morris housing system (HM) or a conventional gestation crate system (GC) prior to breeding. The Hurnik-Morris system provides housing for sows in small groups. Gilts were synchronized for estrus and bred to Duroc Hampshire commercial crossbred boars. Gilts were reared in their respective housing systems through their gestation period. Housing system during breeding and gestation of sows did not affect their respective piglet mortality and piglet viability levels. The slightly higher feet and leg problem scores for the HM sows at weaning may indicate a necessity for farrowing accommodation that will allow movement of sows during lactation.

Animals↗

Modulation of postthaw motility, survival, calcium uptake, and fertility of bovine sperm by female genital products.

Because the different portions of the female genital tract act in many ways on sperm metabolism, the current study was undertaken to modulate the survival and fertilizing ability of bovine semen by incorporation of products from the oviduct or the follicle in extenders before freezing. Motility rates at 6 h in vitro showed a net positive effect when biological factors from total retentate or from a fraction of bovine follicular fluid (total retentate = 43%; fraction 2 = 54%), oviductal cell culture (total retentate = 43%; fraction 2 = 58%), or granulosa cell culture (total retentate = 43%; fraction 3 = 53%) were added to the extenders compared with the addition of BSA (31%). Fraction 3 of granulosa cell culture retentate also had a significant stimulatory effect on the number of sperm that penetrated mucus of cows in estrous compared with BSA (n = 205 vs. n = 159). The intracellular sperm Ca2+ concentrations were very different across treatments after thawing. Sperm from straws with BSA had the highest concentration. At 4 h, intracellular Ca2+ concentration increased for all treatments, except that for sperm treated with BSA and Ca alone, internal Ca2+ declined. Heparin plus Ca stimulated a greater internalization of Ca2+ than did Ca alone for retentate from bovine follicular fluid, oviductal cell culture, and BSA treatments: glucose consistently and significantly reduced internalization. In vitro fertilization rates were similar, and no significant differences were observed across treatments.

Animals↗

Modulation of postthaw motility, survival, calcium uptake, and fertility of bovine sperm by magnesium and manganese.

Because Mg2+ and Mn2+ are potent stimulators of motility through the stimulation of adenylate cyclase activity, the current study was undertaken to modulate the fertilizing ability of bovine semen by incorporation of various concentrations of those two salts in extenders before freezing. Motility analysis at 6 h in vitro showed a positive effect of MgCl2 in a dose-dependent manner from 0.5 to 5 mM (31 to 50%). Manganese at the concentration of 0.1 mM also supported good sperm motility (53%) compared with that of the control (28%). Although survival was increased, no detrimental effects were seen on the number of sperm that penetrated mucus of cows in estrus. The intracellular Ca2+ concentration of sperm was very different across treatments after thawing; spermatozoa that were extended with 2 mM MgCl2 and 0.5 mM MnCl2 possessed the highest concentrations at thawing. Four hours later, in the presence of Ca, spermatozoa that were extended in 0.1 mM MnCl2 showed the highest uptake. In the presence of Ca and heparin, spermatozoa that were extended in different amounts of Mg showed Ca2+ concentrations that increased in a dose-dependent manner. This effect was negated by glucose. Functional fertilizing capacity was also evaluated by in vitro fertilization, and the different treatments did not show any detrimental effects. In summary, 5 mM MgCl2 and 0.1 mM MnCl2 both have beneficial effects for the maintenance of sperm motility without detrimental effects on mucus penetration and fertilizing ability. Furthermore, these treatments do not prevent subsequent Ca2+ uptake in response to heparin. These in vitro studies are potentially a good sorting system to predict the benefits of extender modifications.

Adenylyl Cyclases↗

Regulation of internal Ca2+ by chilled bull and boar spermatozoa.

Intracellular Ca2+ in ejaculated spermatozoa from five bulls and five boars was determined with indo-1,AM in Ca(2+)-free medium. Aliquots of each ejaculate were then wrapped and held either on ice or at 25 degrees C for 20 min. At this time all samples were returned to 25 degrees C, either Ca(2+)-free medium or medium containing 1 mM Ca2+ was added, and intracellular Ca2+ was determined for an additional 120 min. Before and immediately after chilling, internal Ca2+ in bull spermatozoa did not differ. However, exogenous Ca2+ caused both control and chilled bovine spermatozoa to accumulate Ca2+ over the subsequent 120 min (P < 0.01) with the control spermatozoal rate of Ca2+ intake being faster than that of the chilled (slopes, 29.531 vs 8.374 relative Ca2+ units/min; least squares means (lsmean) x 10(4) +/- 1.901 (pooled SE); P < 0.01). Viability of all spermatozoa was reduced after Ca2+ exposure, and the acrosomal morphology of chilled cells was adversely affected by Ca2+. For boar spermatozoa, chilling caused an immediate increase in internal Ca2+, confirming the greater sensitivity of boar spermatozoa to low temperatures. The presence of exogenous Ca2+ caused greater Ca2+ uptake by both control and chilled porcine cells relative to their Ca(2+)-free analogues (0.939 vs 0.764 and 1.015 vs 0.836 Ca2+ units; lsmean +/- 0.166 (pooled SE); P < 0.01 for control and chilled boar spermatozoa with and without Ca2+ at 150 min). Control spermatozoa, however, accumulated Ca2+ (P < 0.01) at a much greater rate (slope, 20.964 +/- 2.73 relative Ca2+ units x 10(4)/min; P < or = 0.001) than chilled cells, which only tended (P = 0.08) to accumulate Ca2+ (slope, 4.782 +/- 2.73 relative Ca2+ units x 10(4)/min). This study suggest that chilling causes a species-specific effect on Ca2+ regulatory capabilities of spermatozoa that differs from cold shock and that spermatozoa may be able to compensate, at least partially, for such injuries.

Animals↗

Composition and behavior of head membrane lipids of fresh and cryopreserved boar sperm.

Head plasma membranes were isolated from fresh or cryopreserved ejaculated boar spermatozoa and the lipids were extracted for determination of lipid fluidity (n = 6 for fresh and cryopreserved) and for compositional analysis (n = 5 for fresh, 6 for cryopreserved). Composition of the egg yolk extender was also determined. For fluidity determination, the mixed lipids were allowed to form natural liposomes. Bilayer fluidity of these liposomes was analyzed in the presence or the absence of 1 mM Ca2+ with the probes tPNA, which preferentially locates into gel-phase areas, and cPNA, which enters fluid and gel-phase areas equally and thus assesses bulk lipids. Fluidity of liposomes declined significantly during controlled-rate cooling for all samples. Compared to lipids from fresh membranes, gel lipids from cryopreserved cells lost fluidity at a significantly more rapid rate, as did bulk lipids in the presence of Ca2+ (P < 0.001). Fluidity increased during subsequent rewarming (5 to 50 degrees C), again at a slower rate for lipids from fresh cells, with the cryopreservation effect being significant for all probe/Ca2+ combinations (P < or = 0.05). Calcium altered the fluidity characteristics of membrane lipids from fresh but not cryopreserved sperm when analyzed during cooling with cPNA (P < 0.01) and during rewarming with cPNA (P < 0.0001) and tPNA (P < 0.05). Lipids from cryopreserved cells contained significantly less sphingomyelin (14.6 +/- 1.1 vs 22.4 +/- 1.6 mol%) and more phosphatidylcholine (51.5 +/- 2.0 vs 40.5 +/- 2.4%). The octadecanoate (18:0) content in both phosphatidylserine and phosphatidylethanolamine decreased after cryopreservation (P < 0.05). The polyunsaturated fatty acids docosatetraenoate (22:4) and/or arachidonate (20:4) increased in these phospholipids and in sphingomyelin and phosphatidylinositol (P < 0.05). The alterations in the molecular interactions, composition, and Ca2+ sensitivity of membrane lipids may interfere with the normal membrane events of fertilization.

Animals↗