Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

Effect of Chilling Duration on Post‐Thaw Characteristics of Sperm from the North American bison (Bison bison)

The objective of this study was to determine the duration for which sperm from the North American bison (Bison bison) could be chilled prior to being cryopreserved, without compromising post‐ thaw sperm quality. This would permit transport of samples collected remotely, to the laboratory (at 4°C) for cryopreservation. Epididymal sperm from plains bison (n = 11) and ejaculated sperm from wood bison (n = 3) were collected, extended and held at 4°C for extended periods of time. At intervals, an aliquot was cryopreserved. Post‐thaw sperm motion characteristics were evaluated by computer assisted sperm analysis. Representative plains bison sperm samples (n = 3) were evaluated for their in vitro fertilizing ability in a heterologous system using bovine oocytes. There was no statistical difference in total and progressive motility of plains bison epididymal sperm when cryopreserved after chilling for 24, 48 or 72 h. For wood bison ejaculated sperm, there was no difference in total and progressive motility for sperm cryopreserved following 24 or 48 h of chilling. However, one of the three bulls showed significantly poorer fertilization (based on cleavage rate) with sperm chilled for 72 compared to 24 and 48 h prior to freezing. In conclusion, plains bison epididymal sperm can be chilled for 72 h and wood bison ejaculated sperm can be chilled for at least 48 h prior to cryopreservation without compromising post‐thaw sperm motility, while heterologous in vitro fertilization (IVF) assay indicated a between‐bull variation in the in vitro fertilizing ability of sperm chilled for an extended duration before cryopreservation.     

Changes in fertilization medium viscosity using hyaluronic acid impact bull sperm motility and acrosome status

The female reproductive tract, in particular the composition of the uterine and oviduct fluids, is responsible, at least in part, for triggering sperm cell modifications, essential for the acquisition of fertilization ability. Hyaluronic acid (HA) is a glycosaminoglycan present in these fluids, and its role in the fertilization process and sperm functionality is still barely understood. This work was designed to (a) determine the rheological characteristics of the fertilization medium by the addition of HA and (b) determine the HA influence on sperm motility and functional status. To that end, the in vitro fertilization medium was supplemented with 4 doses of HA (6, 60, 600 and 6,000 µg/ml) and analysed for viscosity and adhesion strength characteristics. Then, thawed semen from 6 bulls were incubated in these media and assessed at 4 different moments for morphological and functional parameters (plasma and acrosomal membrane integrities, mitochondrial membrane potential, capacitation, acrosomal reaction, and motility). The rheological evaluation showed that the addition of HA was able to increase both the viscosity and the adhesion strength of the fertilization medium, especially in the 6,000 µg/ml group in which the effect was more pronounced. No influence of HA could be observed on mitochondrial potential, and acrosomal and plasma membrane integrities. However, HA supplementation, at lower doses, led to an increase in the number of reacted sperm, as well as changes in motility parameters, with increase in the number of motile, rapid and progressive spermatozoa. In conclusion, the addition of HA alters the rheological properties of the fertilization medium and leads to the improvement of the properties related to sperm motility and capacitation, without compromising other functional aspects of the cell.     

Effect of male accessory gland secretions on sensitivity of porcine sperm acrosomes to cold shock, initiation of motility and loss of cytoplasmic droplets

Hyaluronidase release was used as an index of acrosomal membrane damage during cold shock of epididymal boar sperm and ejaculated sperm from intact and vesiculectomized boars. Sperm were also incubated with seminal plasma from intact and vasectomized boars to examine the contributions of male accessory gland secretions. Acrosomal membranes of epididymal sperm were more resistant to cold shock than those of ejaculated sperm. Only 36% of the hyaluronidase released by ejaculated sperm was released by the epididymal sperm in spite of similar hyaluronidase content of the sperm. Preincubation of epididymal sperm in seminal plasma from both intact and vasectomized boars increased resistance to cold shock by 60 to 80%. Initial dilution of epididymal sperm with seminal plasma, rather than Ringer-fructose buffer, was associated with low progressive motility and with retention of cytoplasmic droplets. In contrast, acrosomal membranes of ejaculated sperm from intact and vesiculectomized boars exhibited similar sensitivity to cold shock, releasing hyaluronidase capable of forming .20 and .19 mumol N-acetylglucosamine from hyaluronic acid/10(8) sperm in 8 min. Moreover, seminal plasma from vasectomized boars had no effect on acrosomal sensitivity to cold shock of ejaculated sperm from vesiculectomized boars.     

Disappearance of the PHA-E lectin binding site on the surface of ejaculated sperm and sperm capacitation in the dog

Ejaculated semen and cross sections of the cauda epididymides collected from 3 normal dogs were smeared or stamped on glass slides, and the sperm on the slides were stained with 7 different FITC-lectins (Con A, DBA, GS-1, PHA-E, PSA, UEA-1, WGA) to examine the relation between sperm-binding glycoprotein derived from the canine prostate and sperm capacitation. The only lectin that stained the ejaculated sperm but not the cauda epididymal sperm was PHA-E. The sperm ejaculated by 5 other dogs were incubated for 4 hr in fluid flushed from the uterine horns or oviducts of estrous bitches, and then the percentages of actively motile sperm and hyperactivated sperm (HA-sperm) were determined. The percentages of PHA-E-labeled sperm and sperm labeled with fluoresceinated Ca indicator to assess the influx of Ca into the sperm were also evaluated. The mean percentages of actively motile sperm, HA-sperm, and Ca-labeled sperm after 4 hr of incubation in the uterine flush fluid and oviductal flush fluid were significantly higher than in control medium (P<0.05, 0.01), but the mean percentages of PHA-E-labeled sperm were lower (both P<0.01). The percentages of PHA-E-unlabeled sperm correlated with the percentages of both HA-sperm and Ca-labeled sperm (r(2)=0.787 and 0.812, respectively). The results indicate that loss of the glycoprotein secreted by the canine prostate on the sperm surface induces the influx of Ca into the sperm, and then hyperactivation of the sperm.     

Tris–Egg Yolk–Glycerol (TEY) Extender Developed for Freezing Dog Semen is a Good Option to Cryopreserve Bovine Epididymal Sperm Cells

Cryopreservation of epididymal spermatozoa is often performed after shipping the excised testis–epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze–thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed^® or a Tris–egg yolk (TEY)‐based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin–nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post‐thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed^®. Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed^®. We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post‐thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial extender AndroMed®.     

Exogenous cholesterol prevents cryocapacitation-like changes,membrane fluidity,and enhances in vitro fertility in bubaline spermatozoa

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 10⁶ spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca²⁺, capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca²⁺ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 10⁶ spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 10⁶ spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 10⁶ spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca²⁺); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca²⁺, low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca²⁺, medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 10⁶ spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing–thawing-associated damages in bubaline species.     

Artificial insemination of frozen epididymal sperm in beagle dogs

Freeze-storage of epididymal sperm is an important technique for the preservation of gametes in animals, including those becoming extinct. We froze canine sperm recovered from the cauda epididymis and investigated the fertility. The qualities of sperm from the cauda epididymis before freezing were: mean sperm motility, 89.4 +/- 1.6 (SE) %; sperm viability, 89.1 +/- 1.1%; and these were significantly higher than those of sperm from the caput-corpus epididymis (P<0.01, P<0.05). The number of sperm recovered from both cauda epididymides varied among animals: 6.3-122.3 x 10(7), mean 61.5 +/- 10.0 x 10(7). Freezing was used only for sperm recovered from the cauda epididymis. The sperm motility and viability after thawing were 19.5 +/- 2.5% and 53.1 +/- 3.3%, respectively. These were slightly lower than those of frozen-thawed ejaculated sperm, but the differences were not significant. When 2 x 10(8), 3 x 10(8), or 4 x 10 (8) sperm were inseminated in the unilateral uterus, only one animal inseminated with 3 x 10(8) sperm was fertilized (1/16, 6.3%). When 1 x 10(8) sperm were inseminated in the bilateral uterine tubes, one of six animals (16.7%) was fertilized. Therefore, although the qualities of epididymal sperm after thawing were similar to those of ejaculated sperm, the conception rate obtained with frozen-thawed epididymal sperm was low in beagle dogs. It is necessary to investigate the differences in damage between epididymal sperm after thawing and ejaculated sperm and to develop a method for improving the conception rate.     

Sperm Mitochondrial Function is Affected by Stallion Age and Predicts Post-Thaw Motility

Sperm motility and quality decline with stallion age and sperm preservation, but the mechanisms of functional deficit have not been explained. We tested the hypothesis that mitochondrial deficits underlie age- and cryopreservation-related deficits in stallion fertility by measuring mitochondrial function, motility, and reactive oxygen species (ROS) in 88 frozen-thawed ejaculates from 19 stallions of varying ages. As expected with increasing age, total sperm motility, progressive motility, and average path velocity decreased, and ROS production increased. For every unit increase in oxygen consumption, there was a 77% increase in the odds of sperm movement (P < .05), confirming the link between mitochondrial functionality and motility. In addition, the rate of mitochondrial oxygen consumption increased from 4 years of age to a peak at 12 years of age and decreased steadily thereafter (P < .05). This confirms the importance of mitochondrial functionality for overall sperm health and motility, implicating mitochondrial dysfunction as a major contributor to sperm aging and cryopreservation damage.     

Methyl‐β‐Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida‐Binding Ability of Frozen/Thawed Bovine Spermatozoa

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein‐free media. However, the additive capacitating effect of methyl‐β‐cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen–thawed bovine spermatozoa in a BSA‐containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose‐dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre‐incubation of spermatozoa in MβCD‐supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)‐binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen–thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.     

Effect of Foetal Bovine Serum on sperm motility,acrosome reaction and spermatic interaction to zona pellucida in alpacas (Vicugna pacos)

The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm‐zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.     

Pentoxifylline induces capacitation and acrosome reaction and improves quality of motility in canine ejaculated spermatozoa

To evaluate effects of different concentrations of pentoxifylline, as phosphodiesterase inhibitor, on quality of motility, capacitation and acrosome reaction, Ejaculated spermatozoa were collected from crossbred dogs. The sperm were incubated at concentrations of 0.1, 1, 10 and 100 mM pentoxifylline for 2 h. Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Capacitation and acrosome reaction were also evaluated by chlortetracycline fluorescence staining. SMI as quality index of sperm was significantly increased in concentrations of 10 and 100 mM pentoxifylline during 1 and 2 h compared to control. The number of capacitated or acrosome reacted spermatozoa significantly (P < 0.05) were higher than controls at high concentrations of pentoxifylline (10 and 100 mM) during 1 and 2 h. In conclusion, high concentration of pentoxifylline is able to induce capacitation and acrosome reaction and improves quality of motility in canine ejaculated spermatozoa.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Detection,Localization and Tyrosine Phosphorylation Status of Ser/Thr Protein Phosphatase1γ in Freshly Ejaculated,In Vitro Capacitated and Cryopreserved Buffalo Spermatozoa

Several recent studies have indicated the important roles of Ser/Thr protein phosphatase1γ (PP1γ) in regulating the motility and capacitation of mammalian spermatozoa. Here, we report the presence and distribution of PP1γ protein in freshly ejaculated, in vitro capacitated and cryopreserved buffalo spermatozoa. The presence of PP1γ and its distribution were assessed by Western blotting and indirect immunofluorescence techniques, whereas the isoforms of PP1γ and their tyrosine phosphorylation status were identified by using 2D electrophoresis. The number of isoforms and the status of tyrosine phosphorylation of PP1γ were increased in capacitated spermatozoa when compared with freshly ejaculated spermatozoa. Differential pattern of expression and tyrosine phosphorylation of PP1γ were observed in cryopreserved spermatozoa, wherein some isoforms were degraded and some were tyrosine phosphorylated. In addition, immunofluorescence technique revealed that PP1γ was localized to principle, mid‐piece, post‐acrosomal and equatorial regions of buffalo spermatozoa. Differential distribution of tyrosine‐phosphorylated proteins were observed in fresh, capacitated and cryopreserved spermatozoa. The tyrosine phosphorylation of several proteins (20, 37, 38, 52, 60, 79 and 100 kDa) were increased when sperm cells were incubated with PP1γ inhibitor, okadaic acid. Together, our results suggest that buffalo spermatozoa express different isoforms of PP1γ protein. The protein expression and tyrosine phosphorylation of PP1γ were increased during capacitation. Furthermore, the differential pattern of expression and tyrosine phosphorylation of PP1γ were observed in cryopreserved spermatozoa. In addition, the inhibition of PP1γ protein increases protein tyrosine phosphorylation in capacitation.     

Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z‐VAD‐FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO₂ and evaluated motility and kinetic parameters by computer‐assisted semen analysis (CASA) and viability (propidium iodide), apoptotic‐like membrane changes (YO‐PRO‐1), acrosomal status (PNA‐FITC), intracellular calcium (FLUO‐3), membrane fluidity (M540) and ROS production (CM‐H₂DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO‐PRO‐1+ and acrosome‐reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca²⁺ levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.     

Effect of caffeine on metabolic activity of ejaculated and epididymal spermatozoa of buffalo (Bubalus bubalis)

Split fractions of 25 ejaculated semen samples and spermatozoa from 5 caudae epididymides were used to study the effect of different levels of caffeine on the motility and fructolytic activity. During the first hour of incubation at 37°C, addition of caffeine to unwashed ejaculated buffalo sperm significantly increased the percentage of motility and amount of fructose utilized. In presence of 2–8 mM caffeine, sperm maintained their initial motility at least 2 hours at 37°C. Maximal stimulation of fructolytic activity was obtained with 2 mM whereas the minimal stimulation was found with higher concentration of 8–10 mM caffeine. Compared to ejaculated spermatozoa, epididymal sperm appeared to be more influenced by caffeine. Fructolytic activity was stimulated at least 1.5 times in the presence of 2 mM caffeine. Nearly during all incubation periods, the amounts of fructose utilized by caffeine-treated sperm were greater than in control samples. Data on epididymal sperm motility demonstrated that caffeine (2–10 mM) significantly stimulated and maintained the initial motility at least 3 hours at 37°C .     

An In Vitro Evaluation of Biochemical Processes Involved in Lead‐Induced Changes on Ram Spermatozoa

Lead (Pb²⁺) is a toxic heavy metal which interferes with several physiological processes regulated by Ca²⁺, including those characterized by changes of the membrane stability and the motility of spermatozoa necessary for the fertilization of the oocyte. In this study, ejaculated sperm from six rams (Ovis aries) have been incubated in vitro with or without 50 ng Pb²⁺/ml during 30 min and in the presence or absence of three different potential modulators of the effects of Pb²⁺ on changes in the sperm membrane before fertilization: charybdotoxin, quinacrine and staurosporine. Sperm samples incubated with Pb²⁺ have shown significant reductions in acrosome integrity and sperm viability and an increase in progressive movement. None of the studied potential modulators had a protective effect against Pb²⁺ action. On the contrary, Pb²⁺‐incubated sperm in the presence of staurosporine had lower acrosome integrity, and lower sperm viability was observed when spermatozoa were incubated with Pb²⁺ + charybdotoxin. Quinacrine was the only tested substance capable of increasing the concentration of Pb²⁺ in spermatozoa; thus, the enhancement of Pb²⁺ effects produced by staurosporine and charybdotoxin was not produced by an increased uptake of Pb²⁺ by spermatozoa. However, the increase of intracellular Pb²⁺ in those spermatozoa incubated with quinacrine did not result in an adverse effect on sperm motility or viability although the acrosome integrity was negatively affected.     

Effects of hyaluronic acid on Ca(2+) influx, lactate dehydrogenase activity, and cyclic AMP synthesis in canine ejaculated sperm during In Vitro capacitation

The effects of hyaluronic acid, which comprises the cumulus intercellular matrix, on Ca(2+) influx, lactate dehydrogenase (LDH) activity, and cyclic AMP synthesis in canine sperm during capacitation was investigated. Ejaculated sperm were collected from 10 Beagle dogs and the sperm were incubated for 4 hr in Eagle's MEM containing 10 microg/ml of hyaluronic acid. The percentages of actively motile sperm, hyperactivated sperm (HA-sperm), acrosome-reacted sperm (AR-sperm), and sperm labeled with fluoresceinated Ca(2+) indicator (Ca(2+)-labeled sperm) were evaluated to assess Ca(2+) influx into the sperm. LDH activity and cAMP concentration were measured in homogenized sperm. The mean percentages of motile sperm, HA-sperm, and Ca(2+)-labeled sperm in the MEM containing hyaluronic acid were higher than in the control medium (P<0.05, 0.05, and 0.01, respectively), but there was no difference between the percentages of AR-sperm. Mean LDH activity and mean cAMP concentration were also significantly higher than the control values (P<0.05). The percentages of HA-sperm correlated with those of Ca(2+)-labeled sperm (r(2)=0.810). The results indicate that hyaluronic acid increases Ca(2+) influx, LDH activity, and cAMP synthesis in canine ejaculated sperm during capacitation in vitro.     

A comparison of bovine seminal quality assessments using different viewing chambers with a computer-assisted semen analyzer

Computer-assisted sperm analysis of fresh and frozen-thawed bovine sperm requires proper handling and preparation, and the type of slide used in the assessment is critical if the resultant data are to be useful quality control measurements. In the present study, 4 different slide viewing chambers, a Makler chamber, a clean slide-coverslip, or a 2- or 4-cell chamber Leja slide, were compared with assess their utility in providing reliable measurements of sperm motility variables. A Hamilton-Thorne IVOS Computer-Assisted Semen Analyzer (CASA) was the instrument used to determine sperm measurements utilizing the 4 different chambers. Fifty-eight different freeze batches of bovine semen that had been collected from 47 bulls at 7 sites that sex-sort sperm using Sexing Technologies sorting criteria were incorporated into the trial. Neither the percentage of motile sperm nor the percentage of progressively motile sperm differed for the Makler chamber vs. slide-coverslip comparisons. Similarly, total and progressively motile sperm did not differ between the 2- and 4-cell chambered Leja slides. However, total and progressive motility of sperm determined with the Makler chamber and slide-coverslip were greater (P < 0.0001) than motilities recorded by the 2- or 4-cell chambered Leja slides. Based on the results, the type of viewing chamber can affect the range of sperm motility values when CASA is used for quality control evaluations of thawed, cryopreserved sex-sorted sperm samples.     

葡萄糖在牛体外受精及胚胎发育中的影响

在牛精子获能液中添加不同浓度的葡萄糖,应用体外培养技术对牛精子体外获能及早期胚胎发育进行研究,通过观察顶体反应、超激活、活率和早期胚胎发育率,筛选出获能液中最适葡萄糖浓度及其有利于牛早期胚胎发育的最佳浓度.结果表明:葡萄糖是精子获能和维持超激活运动的主要能源物质,其代谢过程中产生的活性氧在牛精子体外获能、受精过程中起重要作用,高浓度(超过9.15 mM)葡萄糖有利于获能的完成;但是对早期胚胎发育不利,对早期胚胎发育来说其最适添加量为6.10 mM,此时的囊胚率最高.