Molecular characteristics of horse phospholipase C zeta (PLCζ)

A sperm‐specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca²⁺]_i) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT‐PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long‐lasting [Ca²⁺]_i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca²⁺]_i oscillation‐inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.     

Inositol-1,4,5-Trisphosphate Receptor-1 and -3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca2+ During Quail Egg Activation

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca²⁺ ([Ca²⁺]i): transient elevations in [Ca²⁺]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca²⁺ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca²⁺]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca²⁺ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca²⁺]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca²⁺ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate in creases in [Ca²⁺]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca²⁺ wave and spiral-like Ca²⁺ oscillations, respectively.     

The addition of calcium ion chelator,EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation‐like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal‐derived materials. In this study, we focused on the increase of intracellular Ca²⁺ ([Ca²⁺]_i) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca²⁺]_i indicator, Fluo‐3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca²⁺ chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post‐thawed sperm motility. When the frozen–thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca²⁺]_i and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen–thawed boar sperm.     

Mechanisms involved in the cytosolic calcium ion elevation induced by activating factors in chicken and rat phagocytes

The aim of the present study was to determine the mechanism of cytosolic calcium ion concentration [Ca²⁺]_i elevation in chicken and rat phagocytes stimulated with phorbol myristate acetate (PMA), leukotriene B₄ (LTB₄), formyl‐methionyl‐leucyl‐phenylananine (fMLP) and Saccaromyces cerevisiae culture supernatant (SCS). Pretreatment with EGTA completely suppressed the PMA‐induced [Ca²⁺]_i elevation in rat and chicken phagocytes, suggesting that all the [Ca²⁺]_i elevation induced in the PMA‐stimulated rat and chicken phagocytes was attributable to the influx of extracellular Ca²⁺. On the other hand, the elevation of LTB₄‐, FMLP‐ and SCS‐induced [Ca²⁺]_i was only partially suppressed by ethyleneglycol‐bis (β‐aminoethyl)‐N,N,N′,N′‐tetraacetic acid ethylene (EGTA) pretreatment of phagocytes. The results indicated that two pathways of [Ca²⁺]_i elevation, recruitment from the intracellular Ca²⁺ store and influx of extracellular Ca²⁺, are involved in the [Ca²⁺]_i elevation of LTB₄‐, fMLP‐ and SCS‐stimulated phagocytes. In fMLP‐stimulated rat neutrophils, [Ca²⁺]_i elevation showed a two‐phase pattern in which the time lag between the first and second phase was approximately 1 min. The EGTA treatment of the fMLP‐stimulated cells induced a reduction of the first phase level and a disappearance of the second phase. The reason for the special influence of EGTA observed in fMLP‐stimulated cells is unknown, but the disappearance of the second phase of the [Ca²⁺]_i may be elicited by the EGTA‐induced decrease of the first phase [Ca²⁺]_i elevation that depends on IP₃ and diacylglycerol induced by fMLP.     

The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility

Pregnancy rates in managed horse populations depend on the innate fertility of the mares and stallions involved and on the quality of breeding management. Of course, because a single stallion usually mates many mares, stallion fertility is a critical factor in the overall success of a breeding program. Unfortunately, accurate evaluation of stallion fertility per se requires a large number of normal mares to be mated and is necessarily retrospective. Rather, the ideal is to predict fertility in advance of the stallion's breeding career, and this is currently attempted by way of a thorough physical examination and a routine analysis of semen quality. However, while such a ‘breeding soundness examination’ identifies stallions that clearly lack the capacity for adequate fertility, it is of limited use for predicting the level of fertility and fails to identify some seriously sub‐fertile animals. Similarly, while various sperm function tests (e.g., sperm head morphometry, the hypoosmotic swelling test, glass wool‐sephadex filtration, progesterone receptor exposure) have been shown to correlate fairly well with fertility in the field, most examine only a single or a narrow range of the attributes that a sperm must possess if it is to fertilize an oocyte in vivo, and are thus more useful for identifying specific causes of sub‐fertility than for predicting the level of fertility. On the other hand, combining the results of the various sperm function tests does improve the reliability of fertility estimation and current research is therefore concentrated on identifying a range of tests that covers as many important sperm attributes as possible but that can be performed rapidly and cheaply. In this respect, flow‐cytometry has proven to be an ideal tool because it allows the objective, rapid and simultaneous analysis of a number of properties in a large number of sperm. Moreover, stains are available for an increasing range of sperm characteristics including viability, capacitation and acrosome status, mitochondrial activity and chromatin integrity. Flow‐cytometric analysis of sperm with appropriate probes thus offers considerable promise for the prediction of stallion fertility.     

Growth hormone release induced by an amino acid mixture from primary cultured anterior pituitary cells of goats

The effects of amino acids on growth hormone (GH) release and cytosolic calcium concentration ([Ca²⁺]_i) were investigated in caprine anterior pituitary cells cultured for 3 d in Dulbecco modified Eagle medium. The addition of an amino acid mixture consisting of seven nonessential amino acids (NEAA: l-Asp, Gly, l-Ala, l-Ser, l-Pro, l-Asn, and l-Glu; concentration of each 12.5–200 μmol/l) in the medium significantly raised GH release from the cultured cells in a concentration-dependent manner with the maximum release at 200 μmol/l NEAA. Although an addition of l-Asp (0.1–100 μmol/l) caused a significant rise in GH release in a concentration-dependent manner, neither the individual amino acids contained in NEAA except l-Asp nor others (l-Leu, l-Phe, l-Gln, l-Met, and l-Arg) caused a rise in GH release when added alone to the medium. The rise in GH release induced by NEAA (200 μmol/l) and GH-releasing hormone (GHRH, 10 nmol/l) was significantly reduced by the addition of EGTA (l.8 mmol/l) and nifedipine (1 μmol/l) to the medium, respectively. The addition of NEAA (200 μmol/l) caused a rapid and transient [Ca²⁺]_i increase, followed thereafter by a steady increase. The prior addition of nifedipine (1 μmol/l), which itself significantly reduced the basal [Ca²⁺]_i, completely abolished the response induced by NEAA or GHRH. From these findings, we conclude that: 1) NEAA raises GH release and [Ca²⁺]_i in cultured caprine anterior pituitary cells, and 2) Ca²⁺ influx from the medium may be responsible for the cellular action of NEAA.     

Involvement of the Ca2+ signaling pathway in osteoprotegerin inhibition of osteoclast differentiation and maturation

The purpose of this study was to determine whether the Ca²⁺ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca²⁺ concentration [Ca²⁺]_i and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca²⁺]_i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca²⁺ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca²⁺ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.     

pH-dependent modulation of intracellular free magnesium ions with ion-selective electrodes in papillary muscle of guinea pig

A change in pH can alter the intracellular concentration of electrolytes such as intracellular Ca²⁺ and Na⁺ ([Na⁺]_i) that are important for the cardiac function. For the determination of the role of pH in the cardiac magnesium homeostasis, the intracellular Mg²⁺ concentration ([Mg²⁺]_i), membrane potential and contraction in the papillary muscle of guinea pigs using ion-selective electrodes changing extracellular pH ([pH]_o) or intracellular pH ([pH]_i) were measured in this study. A high CO₂-induced low [pH]_o causes a significant increase in the [Mg²⁺]_i and [Na⁺]_i, which was accompanied by a decrease in the membrane potential and twitch force. The high [pH]_o had the opposite effect. These effects were reversible in both the beating and quiescent muscles. The low [pH]_o-induced increase in [Mg²⁺]_i occurred in the absence of [Mg²⁺]_o. The [Mg²⁺]_i was increased by the low [pH]_i induced by propionate. The [Mg²⁺]_i was increased by the low [pH]_i induced by NH₄Cl-prepulse and decreased by the recovery of [pH]_i induced by the removal of NH₄Cl. These results suggest that the pH can modulate [Mg²⁺]_i with a reverse relationship in heart, probably by affecting the intracellular Mg²⁺ homeostasis, but not by Mg²⁺ transport across the sarcolemma.     

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Effect of BAPTA-AM on Thawed Stallion Spermatozoa Extended in INRA 96 or Tyrode's Medium

We studied the effect of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) on the outcome of cryopreservation of stallion spermatozoa and whether reextension of thawed sperm in a more physiological and Ca²⁺-containing medium might improve the characteristics of thawed stallion spermatozoa. Individual ejaculates from six stallions were collected and split into three subsamples. The first two samples were supplemented with the membrane-permeable Ca²⁺ chelator BAPTA-AM at final concentrations of 5 and 10 μM, respectively, while the third subsample served as control. After 4 weeks of storage, samples were thawed in a water bath at 37°C and evaluated using flow cytometry and computer-assisted sperm analysis (CASA). In a second experiment, in order to determine whether restoring Ca²⁺ could improve sperm quality after cryopreservation, thawed semen was washed by centrifugation and resuspended in Tyrode's complete medium. BAPTA-AM supplementation did not modify the outcome of cryopreservation; however, changing the spermatozoa from INRA 96 to Tyrode's complete medium resulted in significant improvements in the percentages of live sperm and total motility post thaw.     

Activation of Mouse Oocytes Following Intracytoplasmic Injection of Chicken Sperm Extract

The cytosolic factor from the sperm of a wide variety of species has been reported to induce [Ca²⁺]i increase and/or activation in oocytes. Although many species have been studied, there is no data available on the chicken sperm factor responsible for activation of oocytes. This study was aimed to verify the activation of mouse oocyte by intracytoplasmic injection of chicken sperm extract (CSE). Survival rate of oocytes without rupture or lytic degeneration following injection was greater than 80% regardless of the presence or absence of CSE. Among the intact oocytes, activation rates following injection were 27.5% (11 of 40) in the sham operation group. Injection of CSE resulted in significantly higher activation rates in the 1/10 dilution group (57.8%, 26 of 45) compared with the sham operation group (p = 0.0045). Calculated amount of injected sperm extract of 1/10 dilution of CSE was equivalent of 12 chicken sperm. When the 1/10 diluted mouse sperm extract (MSE) was injected, survival rate of injected oocytes and activation rate of the survived oocytes was 82% (41 of 50) and 78% (32 of 41), respectively. Activation rate of MSE‐injected oocytes was a little but significantly higher than that of CSE‐injected oocytes (p < 0.037). In conclusion, it suggests that CSE can activate the mouse oocyte and that oocyte activating sperm factor(s) may be common between avian and mammal.     

Phosphorylation of the MAPK Pathway has an Essential Role in the Acrosome Reaction in Miniature Pig Sperm

In almost all animal species, sperm acrosome reaction (AR) is a crucial step for fertilization. The step is a Ca²⁺‐dependent secretory event that must be completed before fertilization. Many researchers have reported that several chemicals (such as ionomycin, thapsigargin and caffeine) artificially induce this step by increasing [Ca²⁺]_i. However, little information has been known on events that occur following Ca²⁺ induced initiation of the sperm AR. We show here for the first time that phosphorylation of the mitogen‐activated protein kinase (MAPK) pathway is required for the AR in miniature pig sperm. Following caffeine treatment artificially inducing the AR in miniature pig sperm, Raf was phosphorylated and then MAP kinase kinase (MEK) and extracellular‐signal regulated kinase1 (ERK1) were also phosphorylated in a time‐dependent manner. However, the total ERK1 level did not change during the culture. Pre‐treatment of sperm with U0126, a MEK inhibitor, significantly suppressed both the AR and phosphorylation of MEK/ERK1 in a dose‐dependent manner. Additionally, pre‐incubation of the sperm with seminal vesicle (SV) fluid, which is known to contain a decapacitation factor, suppressed both the AR and MEK/ERK1 phosphorylation. These results suggest that phosphorylation of MAPK pathway plays an important role in the AR in miniature pig sperm. Moreover, the SV fluid may have an inhibitory effect on the AR via the suppression of the MAPK pathway.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

Influence of Semen Storage and Cryoprotectant on Post-thaw Viability and Fertility of Stallion Spermatozoa

The aim of the current study was to verify that stallion spermatozoa could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryopreservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio^® extender containing one of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5°C for 20 minutes, then frozen in nitrogen vapor. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty-three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine semen for 24 hours before freezing, while maintaining sperm viability and fertility, is possible.     

Diagnostic Methods for Evaluation of Stallion Subfertility: A Review

Classically, evaluation of the breeding stallion for reduced fertility has relied on physical examination of the reproductive system, as well as evaluation of sperm number, motility, and morphology. Over the past 20 years, a number of other diagnostic methods have become available to facilitate reproductive evaluation of the stallion. Specifically, ultrasound imaging has provided much-improved diagnostic methods for evaluation of the external and internal genitalia of the stallion, and these methods have now become routine in evaluation of the stallion. Biochemical analyses of semen can provide useful information for diagnosis of azoospermia (determination of alkaline phosphatase), detection of urine contamination, or changes in pH. Numerous sperm function assays provide information concerning subcellular compartments of the sperm including the plasma membrane, DNA, acrosome, and mitochondria. Data correlating these functional assays with fertility in the stallion are limited in most cases, with the exception of the sperm chromatin structure assay. Finally, the recent sequencing of the equine genome offers the possibility of both marker-assisted selection for fertility traits and more specific information about genetic mutations that may be associated with differing levels of fertility in the stallion.     

Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca²⁺-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca²⁺]_i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPases (SERCAs), “thapsigargin.” Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca²⁺-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.     

The concentration of ionized magnesium in serum during the periparturient period of non-paretic dairy cows

Ion-selective electrodes have recently been designed for determining the ionized concentration of magnesium (Mg²⁺) in serum. This development may allow new insights into some metabolic diseases of cattle. For this report, the concentrations of Mg²⁺, total magnesium (Mg_tot), ionized calcium (Ca²⁺), total calcium (Ca_tot), and inorganic phosphate (P_i) were determined in sera from seventeen 3-to 16-year-old Brown Swiss and crossed Simmental/Red Holstein cows during the periparturient period. In each animal, a transient increase of Mg²⁺ and Mg_tot serum concentrations was observed in association with the transient decrease in serum concentrations of Ca²⁺, Ca_tot and P_i after parturition. On average, throughout the study, the serum Mg²⁺ concentrations were 68.5% of those of Mg_tot, whereas the serum Ca²⁺ concentrations were 52% of those of Ca_tot. The possible mechanisms involved in the transient increase of Mg²⁺ and Mg_tot serum concentrations are discussed.Abbreviations Ca²⁺ ionized calcium - Ca_tot total calcium - Mg²⁺ ionized magnesium - Mg_tot total magnesium - P_i inorganic phosphate - PTH parathyroid hormone - PTHrP parathyroid homrone related protein