Epididymal maturation and ejaculation are key events for further in vitro capacitation of boar spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation, and the ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to assess the effects of epididymal maturation, ejaculation and in vitro capacitation on sperm viability, acrosome integrity, mitochondrial activity, membrane fluidity, and calcium influx, both as indicators of capacitation status and sperm motility. Results indicated that boar spermatozoa acquired the ability to move in the epididymal corpus; however, their motility was not linear until the ejaculation. Epididymal spermatozoa showed low membrane fluidity and intracellular calcium content; ejaculation led to an increased calcium content, while membrane fluidity showed no changes. Acrosome integrity remained constant throughout the epididymal duct and after ejaculation and in vitro capacitation. The frequency of viable spermatozoa with intact mitochondrial sheath was higher in caput and ejaculated samples than in corpus and cauda samples, whereas the frequency of spermatozoa with high membrane potential was significantly lower in cauda samples. In vitro capacitation resulted in a decreased frequency of viable spermatozoa with intact mitochondrial sheath and an increased frequency of spermatozoa with high membrane potential in ejaculated samples. These results indicated that both epididymal maturation and ejaculation are key events for further capacitation, because only ejaculated spermatozoa are capable of undergoing the set of changes leading to capacitation.     

Geldanamycin augments nitric oxide production and promotes capacitation in boar spermatozoa

Sperm capacitation and the acrosome reaction are fundamentally important to fertilization. Nitric oxide (NO) has been shown to have various functions in male reproduction. This work investigates whether boar sperm can generate NO, as well as the effects of NO and geldanamycin (GA), a heat-shock protein 90 (HSP90)-specific inhibitor, on the capacitation of boar spermatozoa. Observations showed that porcine sperm produced low levels of NO under non-capacitating conditions. However, the NO concentration almost doubled under capacitating conditions (P<0.001). Treatment with NG-nitro-L-arginine methyl ester (L-NAME) reduced the production of NO by 30-40% in capacitating sperm (P<0.05). GA treatment increased it by 23-75% in a dose-dependent manner (P<0.05). L-NAME treatment reduced the percentage of sperm undergoing the acrosome reaction, whereas sodium nitroprusside, an NO-releasing compound, and GA treatment increased the percentage of sperm undergoing the acrosome reaction (P<0.05). GA treatment promoted the production of NO and the acrosome reaction. The increase in NO production by GA treatment was similar to that caused by the calcium ionophore, A23187, suggesting that the GA-induced acrosome reaction may be triggered by an increase of the intracellular calcium concentration. The signaling pathway involved in GA-mediated NO production and its biological function in fertilizing boar spermatozoa will be elucidated in further studies.     

Role of nitric oxide on quality of freshly ejaculated bull spermatozoa during heparin-induced in vitro capacitation

The objective of this study was to assess the effects of nitric oxide (NO) on heparin-induced capacitation in vitro of fresh bull sperm, through the addition of Nω-nitro-l-arginine methyl ester (L-NAME, a NO-synthesis inhibitor) and l-arginine (L-Arg, a NO-synthesis precursor) to the capacitation medium. In Experiment 1, different concentrations of L-NAME (0.1, 1, 10 mM) and of L-Arg (10 mM) were added to the capacitation medium. Sperm motility and vigor were subjectively appraised using direct light microscopy; sperm membrane integrity was examined using a 2% Trypan blue solution while the concentration of nitrate/nitrite (NO₃⁻/NO₂⁻) was determined by using the Griess method over a 5 h capacitation period. The addition of 10 mM L-NAME has inhibited NO synthesis, sperm progressive motility, sperm vigor and sperm membrane integrity (P < 0.05) as compared to control. The addition of 10 mM L-Arg to the capacitation medium increased all variables evaluated in comparison to the control (P < 0.05). In Experiment 2, mitochondrial activity was assessed through the MTT test (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and sperm capacitation was assessed through the test of penetration in homologous oocytes after addition of the 10 mM L-NAME, and of the 10 mM L-Arg. The addition of 10 mM L-NAME caused mitochondrial activity (40%) and the percentage of oocytes penetrated (77%) to decrease in relation to the control (P < 0.05). After addition of 0.6 mM L-Arg + 10 mM L-NAME, partial reversal of mitochondrial activity did occur (only 20%). The addition of 10 mM L-Arg increased the percentage of oocytes penetrated as compared to control (21%) (P < 0.05). These results indicate that: (1) NO is involved in control of progressive sperm motility, vigor, membrane integrity, and mitochondrial activity along the period of heparin-induced capacitation of fresh bovine sperm via NOS/NO; (2) adequate L-Arg/NO concentrations into the capacitation medium can potentiate heparin action or act independently for increasing the number or the quality of capacitated sperm.     

Ginger and echinacea extracts improve the quality and fertility potential of frozen-thawed ram epididymal spermatozoa

The current study examined the impact of the supplementation of ginger and echinacea extract, as natural antioxidant agents, in freezing extender on the quality and fertility potential of ram epididymal spermatozoa after cryopreservation. Epididymal spermatozoa isolated from Forty testicles, obtained from 20 rams, with motility >80% and total morphological abnormalities <10% were pooled, divided into 7 aliquots and used for cryopreservation. The semen samples were re-suspended with basic Tris egg yolk diluent containing ginger and echinacea extracts (5, 10 and 20 mg/l). The control diluent comprised of only extender and lacked any antioxidant agent. For the determination of sperm quality, frozen straws were thawed after 7–10 days, and then the sperm characteristics were assessed. The supplementation of ginger at a concentration of 10 mg/l, as well as the addition of 10 and 20 mg/l echinacea extract significantly improved total motility and velocity parameters. The status of acrosome integrity and lipid peroxidation significantly improved in spermatozoa when supplemented with 10 mg/l ginger and 20 mg/l echinacea extract. Also, 5 mg/l ginger extract and 20 mg/l echinacea extract significantly improved mitochondrial activity. The highest ratio of the dispersion of sperm chromatin was observed in spermatozoa treated with 10 mg/l ginger extract. The cleavage rate was markedly higher in matured oocytes that were fertilized with frozen spermatozoa treated with 20 mg/l ginger extract and 10 mg/l echinacea. The application of ginger and echinacea extract resulted in improvement in the quality and fertility of frozen-thawed spermatozoa. However, future studies are wanted to elucidate how the active components in these extracts prevent cryo-damages in spermatozoa.     

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.     

Some biochemical characteristics and preservation of epididymal camel spermatozoa (Camelus dromedarius)

Testicles were isolated from thirty five apparently healthy dromedary camels (Camelus dromedarius), aged between 5 to 18 years, in a local slaughterhouse during the rutting season. Epididymal fluid was collected from one epididymis for determination of twelve biochemical and antioxidant parameters using ELISA commercial kits. Spermatozoa were harvested from each region of the other epididymis (head, body and tail) and stored in SHOTOR^®, Green buffer^® + 20% egg yolk and INRA-96^® extenders at 5 and 30 °C. Results revealed that, in the epididymal fluid, concentrations of testosterone, glucose, albumin, total protein, cholesterol, fatty acids, iron, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were 5.19 ± 1.69 ng/mL, 3.10 ± 0.41 mmol/L, 6.26 ± 1.26 g/dL, 0.50 ± 0.07 mg/dL, 1.74 ± 0.09 mmol/L, 6.62 ± 0.81 nmol/ul, 926.20 ± 100.18 ug/dL, 51.17 ± 7.74 mIU/ml, and 143.16 ± 18.67 mIU/ml, respectively. The antioxidants activities of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymal fluid were 121.55 ± 6.57 nmol/min/ml, 59.35 ± 10.98 nmol/min/ml and 0.18 ± 0.03 U/ml, respectively. Epididymal sperm motility and concentration were higher (P < 0.05) in the body and tail than the head. The viability indices of total and forward sperm motility, at 5 and 30 °C, obtained from the tail region were superior (P < 0.05) in both SHOTOR^® and INRA-96^® extenders than Green buffer extender. It may be concluded that INRA-96^® extender is the best for storing dromedary epididymal spermatozoa at 5 and 30 °C.     

Cryopreservation of epididymal sperm from ibexes (Capra pyrenaica) using short equilibration time with glycerol

Two experiments were conducted to study the effect of shortening the equilibration time with the cryoprotectant glycerol before freezing epididymal sperm recovered postmortem from Iberian ibex. In the first experiment, the standard equilibration time of 3 hours was compared with 2 hours, and subjective sperm motility and quality of movement were greater (P < 0.05) in the latter group. In the second experiment, reducing the equilibration time from 2 hours to 15 minutes did not affect sperm motility (evaluated subjectively and objectively), viability, acrosomal integrity, or membrane functional integrity. In conclusion, shortening the equilibration time can be used as a technique to simplify the cryopreservation process and this provides practical advantages under field conditions.     

Sperm characteristics and heterologous in vitro fertilisation capacity of Iberian ibex (Capra pyrenaica) epididymal sperm,frozen in the presence of the enzymatic antioxidant catalase

The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris–citric acid–glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200 IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P < 0.01). The percentage of cleaved embryos at 48 hpi was higher (P < 0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200 IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates.     

Early puberty in local Naga boar of India: assessment through epididymal spermiogram and in vivo pregnancy

Male Naga pig of India, a miniature breed is known for its meat quality and early puberty. No scientific efforts were made to verify the farmers' view that this breed reaches puberty at around 2 months of age. A preliminary study was, therefore, conducted with the objectives: (a) to find out the age at puberty based on mature spermiogram and in vivo pregnancy and (b) to record the sperm morphology in different parts of the epididymis. Animals were selected from two different age groups: group I aged 53 days and 2.4 kg and group II of 85 days and 3.0 kg. Semen samples collected from different sections of epididymis were analyzed for sperm motility, live spermatozoa, and morphological abnormalities. Motility increased (P<0.01) and live spermatozoa and total morphological abnormalities decreased (P<0.001) from caput through cauda epididymis in both the groups. Sperm motility, live spermatozoa and morphologically normal spermatozoa in each section of the epididymis were higher (P<0.01) in group II than I. Boars with >60% progressive motility, >70% live spermatozoa, <15% total morphological abnormalities and <10% abnormal acrosomes in cauda epididymal spermatozoa were considered mature spermiogram. As per this definition, pigs of group II had only mature spermiogram. In vivo pregnancy confirmation indicated that Naga boar could impregnate female as early as 90 days of age. In conclusion, Naga boar attained puberty by not later than 3 months with 3.0 kg, which is the lowest body weight at puberty in this species reported so far, as reflected by mature epididymal spermiogram and in vivo pregnancy confirmation.     

Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis. © 1993 Wiley-Liss, Inc.     

Computer automated motion analysis of crossbred bull spermatozoa and its relationship with in vitro fertility in zona-free hamster oocytes

The objective of this study was to determine the effective relationship between different motion characteristics of bull spermatozoa assessed by computer assisted semen analyzer (CASA) and in vitro fertilization percentage in zona-free hamster oocytes. A total of 64 frozen semen samples from 16 different crossbred bulls (Bos taurusxBos indicus) with four ejaculates from each bull were taken for analysis. Various motion characteristics of spermatozoa like progressive motility, path velocity, progressive velocity, beat cross frequency, straightness and linearity were recorded. Hypo-osmotic swelling test and sperm penetration bioassay were conducted to assess the membrane integrity and fertilization percentage of spermatozoa respectively. Significant positive correlation (P<0.01) was found between fertilization percentage and progressive motility (r=0.791) and between velocity parameters (VAP; r=0.612 and VSL; r=0.625) and fertilization percentage. Among different CASA variables, progressive motility alone contributed to 62.6% variation in the fertilization percentage. The velocity measurements (VAP and VSL) together with progressive motility and %HOS spermatozoa contributed to 66.1% of variation in fertilization percentage which was found to be significant (P<0.05).     

Tyrosine phosphorylation of cytochrome c as a signaling event in frozen thawed buffalo spermatozoa at the cross-roads of capacitation and apoptosis

Considering the importance of cytochrome c in both life and death, it was of significant interest to investigate the expression of cytochrome c, its tyrosine phosphorylation status and immunolocalization patterns in a frozen-thawed buffalo sperm cell in comparison to in vitro capacitated [heparin (10 μg/ml) induced, for 4 h] and stress [apoptotic (10 μM staurosporine), oxidative (25 μM H₂O₂) and osmotic (180 mM NaCl) for 4 h] induced conditions. Proteins were subjected to immunoblotting and probed by using monoclonal anti-phosphotyrosine antibodies. A significant (p < 0.05) increase in expression of tyrosine phosphorylated cytochrome c was observed in capacitated buffalo sperm in comparison to frozen-thawed samples. cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent tyrosine phosphorylation of cytochrome c was found during in vitro capacitation of buffalo spermatozoa. Localized increase in cytochrome c and tyrosine phosphorylated proteins were observed in frozen thawed and capacitated sperm. The information generated in this study can be used to understand the molecular mechanism of regulation of an apoptotic protein (cytochrome c) by tyrosine phosphorylation (a capacitation marker) in a frozen thawed sperm cell which could be a good target to combat apoptosis.     

Hormonal regulation of zona-binding ability and fertilizing ability of rat epididymal spermatozoa

Rat spermatozoa from the proximal caput, the proximal corpus, the middle corpus, and the distal cauda epididymidis were examined for their ability to bind to the zona pellucida after a 1-, 2.5-, or 4.5-h incubation at 34°C with rat eggs in cumulus. Caput spermatozoa did not bind to the zona after 1, 2.5, or 4.5 h of incubation. Corpus spermatozoa did bind to the zona, but the percentage of eggs with bound spermatozoa and number of bound spermatozoa per egg increased with the length of incubation. Cauda spermatozoa bound readily to the zona pellucida, and their zona binding ability did not change with longer incubations. It thus appears that rat spermatozoa gradually acquire the ability to bind to the zona pellucida in the corpus epididymidis. The zona-binding capacity of cold immobilized cauda spermatozoa, defined as the percentage of eggs with bound spermatozoa, increased with the number of spermatozoa incubated and reached a plateau characteristic of the endocrine status of the animal. After castration, zona-binding ability is progressively lost from day 3 until day 10 where it is nil. Testosterone supplementation maintains zona-binding ability to control levels. Similarly, fertilizing ability declines from day 5 after castration until day 10. Testosterone prevents this loss of fertilizing ability. It thus appears that the development of zona-binding ability during epididymal transit is, like the development of fertilizing ability, under androgen regulation. The close correlation between the onset of fertilizing ability and zona-binding ability during maturation, the loss of fertilizing ability and zona-binding ability after castration, and the recovery of both fertilizing ability and zona-binding ability with testosterone treatment suggests that the androgen-dependent development of zona-binding ability is an important component of the acquisition of sperm fertilizing ability during epididymal transit.     

The zymogen form of acrosin in testicular,epididymal, and ejaculated spermatozoa from ram

The sperm-specific proteinase acrosin (EC 3.4.21.10) is found in spermatozoa as a zymogen. We have looked for different forms of this zymogen in testicular, epididymal, and ejaculated spermatozoa from ram and have compared total sperm extracts made immediately after cell disruption with extracts made later from isolated sperm heads. We have concluded that the autoactivatable zymogen form, known generally as proacrosin, is the only form of acrosin within intact mature ram spermatozoa; no other zymogen form was detected, although lower levels of proacrosin were found in some samples of testicular spermatozoa. From studies of the activation process, it appears that ram proacrosin is truly autoactivatable; no evidence could be found for the involvement of any auxiliary enzyme. Estimations of the molecular weight of proacrosin using gel chromatography (60,000) and SDS-polyacrylamide gel electrophoresis (51,300) indicated that the zymogen is monomeric. Comparison with the molecular weight of ram acrosin (44,000 or 40,000, using the two respective methods) indicated that a single acrosin molecule is derived from each zymogen molecule. The sperm acrosin inhibitor (molecular weight 11,000 or 8,000) was present in testicular spermatozoa as well as in ejaculated spermatozoa; there was no evidence that it was produced as a result of zymogen activation.     

A comparison of the protective action of chicken and quail egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa

Egg yolk-based diluents provide adequate cryoprotection for the sperm of several mammalian species. Traditionally, chicken egg yolk has been used as additive for the freeze preservation of spermatozoa because of its wide availability. Variations in the chemical composition of the egg yolk of different avian species appear to influence the protection afforded during cooling, freezing, and thawing. The aim of the present study was to assess the use of quail egg yolk as a novel additive for the epididymal spermatozoa of a threatened wild ruminant species—the Spanish ibex—and to compare its efficacy with chicken egg yolk. Epididymal spermatozoa were diluted using a Tris–citric acid–glucose medium (TCG) composed of 3.8% Tris (w v⁻¹), 2.2% citric acid (w v⁻¹), 0.6% glucose (w v⁻¹), 5% glycerol (v v⁻¹), and 6% egg yolk (v v⁻¹). Sperm masses from the right epididymes were diluted with TCG-6% chicken egg yolk medium, while those from the left were diluted with TCG-6% quail egg yolk. The thawed spermatozoa preserved with TCG-6% quail egg yolk extender exhibited lower motility (P < 0.001), membrane integrity (P < 0.001), and viability (P < 0.01) than those diluted with the TCG-6% chicken egg yolk extender. The fertility of spermatozoa frozen in TCG-6% chicken egg yolk tended to be higher than in those frozen with TCG-6% quail egg yolk (63.3% vs 36.4%, P = 0.19). These results show that quail egg yolk offers no advantages over chicken egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa.     

Influence of recovery method and microbial contamination on the response to freezing–thawing in ibex (Capra pyrenaica) epididymal spermatozoa

The method of sperm recovery may influence the initial quality of sperm samples and their response to freezing–thawing. The aim of the present work was to compare two methods for collecting epididymal spermatozoa in order to improve the quality of recovered sperm and reduce possible contamination. Testes were obtained from 23 legally hunted, adult ibex males. The sperm mass of the right epididymis was collected by small longitudinal and transverse cuts made in the cauda epididymidis. The sperm mass of the left epididymis was collected by retrograde flushing from the vas deferens to the cauda epididymidis (using a cannula), employing a Tris, citric acid, glucose, egg yolk-based medium. The flushing method recovered more spermatozoa (P < 0.001) than the cutting method. After freezing–thawing, greater acrosomes damage (P < 0.001) and more morphological abnormalities (P < 0.05) were seen among the sperm cells recovered by the cutting method than among those obtained by retrograde flushing. The method of sperm recovery did not, however, influence the microbial contamination rate. In frozen–thawed samples that were microbially contaminated, motility was significantly reduced (P < 0.05) and membrane integrity tended to be poorer (P = 0.06). In conclusion, retrograde flushing is recommended for ibex sperm collection since it would appear that microbial contamination is no more of a problem than that encountered with the cutting method, while a larger number of sperm cells more resistant to freezing–thawing can be obtained.     

Sperm capacitation in the domestic cat (Felis catus) and leopard cat (Felis bengalensis) as studied with a salt-stored zona pellucida penetration assay.

The ability of domestic cat or leopard cat spermatozoa to penetrate zonae pellucidae (ZP) of salt-stored, domestic cat oocytes was examined as an assay for sperm capacitation. Ovarian oocytes were recovered after ovariectomy and matured in vitro for 18-36 h. Following removal of cumulus cells, the oocytes were used fresh, or stored (4 degrees C, 0.5-24 weeks) in a HEPES-buffered hypertonic salt solution. Electroejaculated, washed sperm (2-4 x 10(6) sperm/ml) were preincubated for 1.0 h (38 degrees C, 5% CO2 in air) and then co-incubated (2 x 10(5) sperm/ml) with fresh or stored oocytes for 6.0 h. Gametes were incubated in a protein-free, modified Tyrode's solution (TLP-PVA) or in the same medium containing 4.0 mg/ml bovine serum albumin (BSA; TALP-PVA). Treatments were compared for percentage ZP penetration (defined as sperm heads reaching more than halfway through the ZP) as an index of sperm capacitation. In both the domestic cat and leopard cat, there was no difference (P greater than 0.05) in sperm penetration of fresh ZP (domestic cat, 42.5 +/- 5.4%; leopard cat, 38.6 +/- 2.8%) or stored ZP (domestic cat, 32.4 +/- 4.2%; leopard cat, 27.6 +/- 2.3%). Sperm incubated in protein-free medium (TLP-PVA) were less capable (P less than 0.05) of ZP penetration (domestic cat, 14.6 +/- 5.9%; leopard cat, 7.9 +/- 3.0%) than sperm incubated in medium TALP-PVA containing BSA (domestic cat, 60.3 +/- 5.9%; leopard cat, 58.4 +/- 3.0%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of porcine pre-ovulatory oviductal fluid on boar sperm function

Sperm storage within the oviductal isthmus prior to ovulation typically involves binding to oviductal epithelial cells, which are thought to modulate sperm functions including internal calcium concentration, membrane fluidity, and motility. Around the time of ovulation the spermatozoa are gradually released so that they eventually encounter the oocytes within the oviductal ampulla. Previous studies have shown that the oviductal epithelial cells selectively sequester high quality spermatozoa, but the role of oviductal fluid as a selective modulator of sperm function has been investigated to a lesser extent. Here we address the hypothesis that oviductal fluid is also likely to modulate sperm function. Using samples of porcine oviductal fluid collected in the follicular phase of the estrus cycle, we show that short exposure (20 min to 50 μg/mL of oviductal fluid proteins) to either of two separate proteins fractions (> or < 100 kDa) promotes boar sperm viability and acrosomal integrity, decreases sperm plasma membrane fluidity (measured using merocyanine S540), and increases zona binding and polyspermy during in vitro fertilization. Exposure to the lower molecular fraction significantly inhibited, but did not abolish, the bicarbonate-induced stimulation of motility. The results show that subpopulations of spermatozoa respond differentially to oviductal fluid, and suggest that exposure to oviductal fluid in vivo could exert a further level of functional sperm selection.     

In vitro evaluation of fresh sperm quality in tomcats: A comparison of two collection techniques

The collection of semen from tomcats by urethral catheterization (CT) after medetomidine administration offers a novel and easy approach to obtain good quality sperm for in vitro fertilization. This study was designed to compare the sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in seventeen adult cats by urethral catheterization, after which the cat was orchiectomized. Motility, morphology, plasma membrane integrity, acrosomal status, and in vitro fertilizing capacity of both fresh CT and EP samples were evaluated. The results showed that both total and progressive motility, as well as the percentage of normal spermatozoa, were higher for EP sperm than for CT sperm (P < 0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P < 0.01), while CT sperm contained more spermatozoa with tail abnormalities (P < 0.01). Other morphological parameters, as well as plasma membrane integrity, did not differ (P > 0.05) between CT and EP sperm. Nevertheless, no difference (P > 0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the two different methods was found. In conclusion, semen collection by means of urethral catheterization after medetomidine administration yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Since this novel catheterization technique is repeatable, is easy to perform and facilitates semen preparation protocols, it may be preferable for routine IVF experiments with fresh spermatozoa.     

Glass wool filtration of bull cryopreserved semen: a rapid and effective method to obtain a high percentage of functional sperm

Frozen-thawed bull sperm are widely used in assisted reproductive technologies, but cryopreservation negatively affects semen quality. Several sperm selection techniques have been developed to separate motile sperm from non-motile cells. The aim of the present study was to evaluate the effectiveness of the glass wool column filtration to select functional sperm from frozen-thawed bull semen samples. Frozen semen from six Holstein bulls was thawed and filtered through a glass wool column, followed by assessment of routine and functional sperm parameters. In a set of experiments, sperm aliquots were also processed by swim up to compare both selection methods. Samples recovered in the glass wool filtrate had high percentages of viable (94 ± 3%, mean ± SD), progressively motile (89 ± 4%), acrosome-intact (98 ± 1%), and non-capacitated (80 ± 10%) sperm; these values were higher (P < 0.05) than those obtained after performing the swim up procedure. Moreover, the glass wool filtration yielded 67 ± 19% motile cells, in comparison with 18 ± 8% obtained with swim up (P < 0.05), calculated as the concentration of progressively motile cells selected relative to their concentration in the sample before the selection procedure. Glass wool-filtered sperm were able to undergo capacitation-related events, based on the increase in the percentage of cells classified as capacitated by CTC staining (B-pattern) after incubation with heparin (50 ± 5%) in comparison with control conditions with no heparin (17 ± 4%) or heparin + glucose (16 ± 2%; P < 0.05). Moreover, they underwent acrosomal exocytosis in response to pharmacologic (calcium ionophore A23187 and lysophosphatidylcholine) and physiological (follicular fluid) stimuli, and they fertilized in vitro matured cumulus-oocyte complexes and denuded oocytes (two-cell embryos: 72 ± 4% and 52 ± 6%, respectively). We conclude that glass wool filtration is a low-cost, simple, and highly effective procedure to select functionally competent sperm for reproductive technologies in the bull, which may be useful for other domestic and farm animals, as well as for endangered species.