Neonatal estrogen exposure of male rats alters reproductive functions at adulthood

The effects of neonatal exposure to different doses of diethylstilbestrol (DES) on the reproductive functions of male rats at adulthood were evaluated. Sprague-Dawley rats (5-8/group) received sc injections of 25 microl olive oil containing DES (Sigma Chemical Co., St. Louis, MO) at a dose of 10 microg, 1 microg, 100 ng, 10 ng, or 1 ng per rat on alternate days from Postnatal Days 2-12. Control animals received olive oil only. All animals were allowed to develop until 83-91 days of age; however, when they were 70 to 80 days old, four male rats each from the 10 microg, 1 microg, 100 ng, and control groups were cohabited with untreated 60- to 70-day-old females (1:1) for 12 days. At the end of cohabitation, both mated and unmated male rats were weighed, and blood and tissue samples were collected and processed. Results revealed that although sperm motility patterns and sperm morphology were adversely affected in the 10- microg group, other reproductive parameters, including 1). daily sperm production (DSP)/testis; 2). absolute and relative weights of the testis, epididymis, and seminal vesicle; and 3). sperm numbers in both regions of the epididymis declined significantly in a dose-dependent manner in the 10- and 1- microg groups. Conversely, in the <1- microg groups, none of these parameters (except DSP/testis and weight of the epididymis in the 100-ng group, and sperm numbers in the epididymis of the 100- and 10-ng groups) was different from controls. Generally, plasma testosterone levels decreased in the 10- and 1- microg groups, FSH level increased in the 10-microg group, and prolactin and LH levels were unaltered. In the fertility study, although each male in the 1-microg, 100-ng, and control groups produced a copulatory plug and impregnated a female, none could do so in the 10-microg group. The mean number of pups per litter was reduced to eight in the 1-microg group, in contrast to 15 each in the 100-ng and control groups. In conclusion, exposure of neonatal male rats to DES altered sperm motility patterns, sperm fertility (as evident from the reduced number of pups in the 1-microg group), and sexual behavior (as evident from the absence of copulatory plugs in the 10-microg group) and reduced weights of reproductive organs, DSP/testis, and sperm numbers in the epididymis. Whether these alterations/reductions persist in older rats (6-8 mo of age) is under investigation.     

Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.     

The effects of combined conventional treatment, oral antioxidants and essential fatty acids on sperm biology in subfertile men

We evaluated the effects of combined conventional treatment, oral antioxidants (N-acetyl-cysteine or vitamins A plus E) and essential fatty acids (FA) on sperm biology in an open prospective study including 27 infertile men. The evaluation included sperm characteristics, seminal reactive oxygen species (ROS), FA of sperm membrane phospholipids, sperm oxidized DNA (8-OH-dG), and induced acrosome reaction (AR). Treatment did not improve sperm motility and morphology, nor decrease the concentration of round cells and white blood cells in semen. Sperm concentration increased in oligozoospermic men (7.4+/-1.3 to 12.5+/-1.9 million/ml). Treatment significantly reduced ROS (mean+/-SEM) (775.3+/-372.2 to 150.3+/-105.2 x 10(3)counts/10 second) and 8-OH-dG (45.3+/-10.4 to 16. 8+/-3.3 fmol/microg DNA). Treatment increased the AR (55.1+/-2.2 to 71.6+/-2.2%), the proportion of polyunsaturated FA of the phospholipids, and sperm membrane fluidity. The overall pregnancy rate was 4.5% in 134 months. The per month pregnancy rate tended to be higher in partners of (ex)-smokers (7.15%, n=14,70 months) than in never-smokers (1.6%, n=13,64 months) (OR:4.57, 95% Cl:0.55-38.1).     

The effect of seminal sperm concentration on sperm transport in the reproductive tract of female rabbits treated with progesterone or diethylstilbestrol

Forty-two mature Baladi female rabbits were used in a randomized 3x2 factorial experiment to determine the effects of three treatments (control, progesterone injection: 2 mg/doe and DES injection: 0.1 mg/doe) and two semen sperm cell concentrations (1x10(6) and 60x10(6) sperm/0.25 ml semen on sperm transport and distribution in the female reproductive tract. The injections were given for three consecutive days after which rabbits were injected with 5 IU HCG and inseminated with 0.25 ml semen. The does were sacrificed 10 hrs after insemination and the sperm were recovered and counted from the oviducts, uterine horns, cervices and vagina. Total spermatozoa recovered was high when rabbits were inseminated with 60x10(6) sperm as compared to those inseminated with 1x10(6) sperm. When rabbits were injected with progesterone or DES, the number of sperm recovered relative to the total number of sperm inseminated was high in rabbits inseminated with 1x10(6) sperm, in comparison to those inseminated with 6x10(6) sperm. The number of sperm recovered was highest from cervix which was followed by vagina, uterus and oviducts. DES increased the number of the total sperm recovered while progesterone decreased the number as compared to control. This trend was also observed within the different segments of the reproductive tract and with groups inseminated with 1x10(6) or 60x10(6) sperm/0.25 ml semen. The effect of DES was more obvious with does inseminated with low sperm numbers. Significant correlation coefficients were found between the sperm numbers recovered in the uterus and oviducts and in the cervix and uterus of all groups of rabbits.     

Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm

The objective was to determine the optimal concentration of heparin for sperm capacitation, as well as the optimal sperm concentration for in vitro fertilization using flow cytometrically-sorted sperm from individual bulls. A total of 5327 bovine oocytes and sperm from four bulls were examined. Oocytes from slaughterhouse ovaries were matured in TCM199 for 22-24 h. Flow cytometrically-sorted sperm as well as unsorted control sperm from the same bulls were cryopreserved. For sperm from each of the four bulls, oocytes were inseminated in a three-by-three factorial design plus one control group (three heparin concentrations: 0, 2, and 10 microg/ml and three sperm concentrations: 0.5 x 10(6), 1.5 x 10(6), and 4.5 x 10(6) ml(-1); 10 microg/ml of heparin and 1.5 x 10(6) ml(-1) of sperm were used for the unsorted control). Presumptive zygotes were cultured in chemically defined media, CDM-1 and CDM-2 for 52-54 h and 96 h, respectively. Samples of about 10 oocytes from each of the 10 treatment groups per replicate were fixed at 18-20 h after insemination to determine sperm pronuclei formation and polyspermy. Increased polyspermy resulted as heparin and sperm concentrations increased (P < 0.05). A higher rate of polyspermy was found in oocytes inseminated with unsorted control sperm compared with sorted sperm (P < 0.05). Sperm of one of four bulls tested required no heparin and lower concentration (0.5 x 10(6) ml(-1)) to obtain optimal cleavage and blastocyst rates while optimal parameters for another bull were higher heparin (10 microg/ml) and sperm concentrations (4.5 x 10(6) ml(-1)). Optimal parameters for the other two were intermediate levels of heparin and sperm. Sperm appeared to be partially capacitated during the flow cytometric-sorting process used for sex pre-determination. When heparin and sperm concentrations were optimized for individual bulls, blastocyst production per oocyte was similar for sorted and unsorted sperm for three of the four bulls studied.     

Generation of reactive oxygen species in sperms of rats as an earlier marker for evaluating the toxicity of endocrine-disrupting chemicals

Abstract

Bisphenol A (BPA) and diethylstilbestrol (DES) have been reported to cause sperm toxicity. To identify an earlier marker of toxicity of environmental substances or food additives, this study determined whether the levels of reactive oxygen species (ROS) in sperms could serve as indices for the prediction of sperm toxicity and quality. Male Wistar rats were given drinking water containing various doses of BPA or DES for 8 weeks. Some rats were treated with 0.45% N-acetyl cysteine (NAC) for 2 days prior to the administration of DES or BPA. Administration of BPA or DES to rats for 1 week dose-dependently increased the production of ROS, even at doses and time points which had no effect on sperm motility. 4-Hydroxy-2-nonenal modified proteins increased in sperms 8 weeks after BPA or DES treatment. NAC reversed oxidative stress and prevented the loss of sperm function in the DES or BPA-treated group. During observation, changes in the sperm motility, sperm count and morphology were not correlated to the increase in ROS levels. These results suggest that ROS levels may be used as an early indicator of sperm count and quality decreases which result from chronic toxicity.     

Duration of spermatogenesis and daily sperm production in the jaguar (Panthera onca)

The jaguar, like most wild felids, is an endangered species. Since there are few data regarding reproductive biology for this species, our main goal was to investigate basic aspects of the testis and spermatogenesis. Four adult male jaguars were utilized; to determine the duration of spermatogenesis, two animals received an intratesticular injection of H(3)-thymidine. Mean (+/-SEM) testis weight and the gonadosomatic index were 17.7+/-2.2g and 0.05+/-0.01%, respectively, whereas the seminiferous tubules and the Leydig cells volume density were 74.7+/-3.8 and 16.7+/-1.6%. Eight stages of spermatogenesis were characterized, according to the tubular morphology system and acrosome development. Each spermatogenic cycle and the entire spermatogenic process (based on 4.5 cycles) lasted approximately 12.8+/-0.01 and 57.7+/-0.07 d. The number of Sertoli and Leydig cells per gram of testis was 29+/-4x10(6) and 107+/-12x10(6). Based on the number of round spermatids per pachytene spermatocyte (2.8+/-0.3:1; meiotic index); significant cell loss (30%) occurred during the two meiotic divisions. There were approximately eight spermatids for each Sertoli cell (Sertoli cell efficiency), whereas the daily sperm production per gram of testis was 16.9+/-1.2x10(6). We expect that in the near future, the knowledge obtained in the present investigation will facilitate, utilizing germ cell transplantation, preservation of the germinal epithelium and the ability to generate sperm from jaguars in testes of domestic cats.     

Effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 degrees C

The objectives of this study were (1) to determine the changes in structural, functional and motility parameters of ram-lamb semen stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender and (2) to determine the effect of breed of ram-lambs on the changes in structural, functional and motility parameters of ram-lamb semen from different breeds stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender. Two different concentrations suitable for laparoscopic and cervical insemination were employed in this experiment. A total of 14 ram-lambs (Polled Dorset-5, Suffolk-5, Katahdin-4) with satisfactory breeding potential were selected. Semen samples were collected by electro-ejaculation. Semen samples were extended to 50 and 200 million sperm per ml with a commercial egg yolk based extender (Triladyl, Minitube of America, Verona, WI, USA) at room temperature and were stored at 4 degrees C. The sperm DNA fragmentation index (DFI), percentages of high mitochondrial membrane potential (hMMP) and plasma membrane integrity (PMI) were assessed using flow cytometry as part of structural and functional parameters on Days 0, 1, 4, 6, and 8. A computer assisted sperm analyser (HTM-IVOS, Version 10.8, Hamilton Thorne Research, Beverly, MA, USA) was used to assess the sperm motility parameters on Days 0, 1, 4, 6, and 8. PROC MIXED procedure was used to determine the effect of days of storage, concentration and breed. The concentration and days of storage significantly affected the sperm structural, functional and motility parameters (P<0.0001). Significant concentration x days of storage interaction was found for all structural and functional parameters. There was a significant concentration x days of storage interaction for average path velocity, curvilinear velocity, straightness and linearity. Overall changes in the sperm structural, functional and sperm motility parameters over the storage period were less dramatic in the 200 x 10(6) ml(-1) concentration when compared to 50 x 10(6) ml(-1) concentration. The hMMP and total progressive motility were influenced by breed. In conclusion, the quality of structural, functional and motility parameters declined as days of storage were increased and the magnitude of changes in the parameters was less dramatic at the higher concentration.     

Semen characteristics and testosterone profiles in ferrets kept in a long-day photoperiod, and the influence of hCG timing and sperm dilution medium on pregnancy rate after laparoscopic insemination

Five domestic ferrets previously maintained for 12 weeks under a 16L:8D photoperiod were electroejaculated weekly for 15-65 weeks while continuing to be exposed to the prolonged light cycle. Two ferrets sustained spermatogenesis for 20 and 26 weeks, while sperm production in the remaining males either was sporadic or decreased, remained depressed and then increased to peak levels observed in other males. Regardless of the temporal spermatogenesis patterns within males, the number of electroejaculated spermatozoa with residual cytoplasmic droplets or abnormal acrosomes increased in all ferrets over time. Diluted ejaculates meeting artificial insemination criteria were deposited intravaginally or by transabdominal laparoscopy into the uterine horns of females treated 0 or 24 h earlier with 90 i.u. hCG. Vaginal insemination was ineffective (0 pregnancies in 10 attempts), but 17/24 ferrets (70.8%) inseminated laparoscopically became pregnant and delivered live young (mean litter size, 5.2 kits). Number of motile spermatozoa deposited in utero (1.6-10.0 x 10(6) cells), presence of glycerol in the sperm dilution medium (0 versus 4%) and time of hCG administration (0 versus 24 h before insemination) had no effect on pregnancy results or litter size.     

Semen of Perca fluviatilis L.: sperm volume and density, seminal plasma indices and effects of dilution ratio, ions and osmolality on sperm motility

The objectives of the present study were to characterize sperm volume and density, seminal plasma indices (ionic contents and osmolality) and to study the effects of dilution ratio, ions and osmolality on sperm motility parameters (percentage of motile sperm and sperm velocity) in farmed European perch (Perca fluviatilis L.). The means of sperm volume (ml), sperm density (x10(9)spermml(-1)) and total number of sperm (volumexdensity) per fish were 2.75+/-0.51, 29.19+/-3.15 and 82.19+/-15.26. The seminal plasma osmolality (mOsmkg(-1)), sodium, chloride, potassium and calcium ions concentrations (mM) were measured to be 298.07+/-5.09, 130.97+/-2.19, 106.75+/-2.37, 10.70+/-0.61 and 2.41+/-0.09, respectively. At 15s post-activation of stripped sperm, the percentage of motile sperm (%) and sperm velocity (mums(-1)) were 91.90+/-1.27 and 115.54+/-1.25, respectively, and decreased significantly following sperm activation (P<0.05). The optimal sperm motility was observed when the sperm was prediluted in immobilizing solution (IS) at a ratio 1:50. Prediluted sperm showed the maximum velocity when activated in 2.5mM Ca(2+), 50mM K(+) and sucrose with osmolality 100mOsmkg(-1). Neither Ca(2+) nor K(+) showed a significant effect on the percentage of motile sperm at 15s post-activation. Osmolality higher than 200mOsmkg(-1) significantly decreased the percentage of motile sperm, while osmolality of 300mOsmkg(-1) or above totally suppressed sperm motility.     

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.     

Cigarette smoking and human sperm quality assessed by laser-Doppler spectroscopy and DNA flow cytometry

The sperm qualities of 350 men under fertility investigation were compared in relation to their smoking habits. The sperm variables included number, motility, morphology and vitality. Sperm motility was assessed objectively by laser-Doppler spectroscopy. In a randomly selected group, sperm samples were subjected to flow cytometry to assess the levels of DNA condensation. No significant differences (Kruskal-Wallis' test) in any aspect of sperm quality including DNA distribution could be demonstrated between non-smokers, moderate smokers (1-14 cigarettes/day) and heavy smokers (15-40 cigarettes/day). This was true when the data were pooled and when oligozoospermic/hypozoospermic ejaculates (1-39 x 10(6)/ml) and asthenozoospermic ejaculates (less than 25% of sperm cells with progressive movement) were analysed separately. The distribution of non-smokers, moderate and heavy smokers was the same in groups of men with normal sperm quality as those with impaired quality. The present study does not provide support for the contention that smoking has deleterious effects on sperm quality, at least using conventional parameters.     

Rapid evolution of testis size relative to sperm morphology suggests that post‐copulatory selection targets sperm number in Anolis lizards

Post‐copulatory sexual selection is thought to be responsible for much of the extraordinary diversity in sperm morphology across metazoans. However, the extent to which post‐copulatory selection targets sperm morphology versus sperm production is generally unknown. To address this issue, we simultaneously characterized the evolution of sperm morphology (length of the sperm head, midpiece and flagellum) and testis size (a proxy for sperm production) across 26 species of Anolis lizards, a group in which sperm competition is likely. We found that the length of the sperm midpiece has evolved 2–3 times faster than that of the sperm head or flagellum, suggesting that midpiece size may be the most important aspect of sperm morphology with respect to post‐copulatory sexual selection. However, testis size has evolved faster than any aspect of sperm morphology or body size, supporting the hypothesis that post‐copulatory sexual selection acts more strongly upon sperm production than upon sperm morphology. Likewise, evolutionary increases in testis size, which typically indicate increased sperm competition, are not associated with predictable changes in sperm morphology, suggesting that any effects of post‐copulatory selection on sperm morphology are either weak or variable in direction across anoles. Collectively, our results suggest that sperm production is the primary target of post‐copulatory sexual selection in this lineage.     

Characterization of gossypol-induced sperm abnormalities in bulls

To characterize sperm abnormalities induced by gossypol in cattle, young Brahman bulls (n=8) received either cottonseed meal delivering 8.2 g free gossypol/bull/d (treatment, n=4) or isonitrogenous soybean meal (control, n=4) for 11 wk. At slaughter, semen was collected from the following extragonadal sites: mediastinum/rete testis (Site 1), caput (Site 2), corpus (Site 3) and cauda epididymis (Site 4), and ductus deferens (Site 5). At least 200 fixed spermatozoa per site were examined via differential-interference-phase contrast (DIC) microscopy, with electron microscopy (EM) being employed with select samples. Sperm midpiece abnormalities were categorized as aplastic, fragile or asymmetric, with detached sperm heads being tabulated separately. Of these, aplastic defects were considered most likely to occur during spermatogenesis. Bull sperm midpiece lesions induced by gossypol were ultrastructurally similar to those observed in other, nonruminant, species. Combined midpiece abnormalities generally increased with extragonadal passage (EGP) in the treated bulls, as did fragile and asymmetric but not aplastic midpieces, or detached sperm heads. This pattern of EGP changes in bull sperm morphology following gossypol spermatoxicity suggests that structural weakness induced during spermatogenesis leads to secondary spermatozoal changes during EGP, possibly due to the imposition of motility stressors upon prior weakened structures.     

Effects of dietary gossypol on aspects of semen quality, sperm morphology and sperm production in young Brahman bulls

Eight young reproductively normal Brahman bulls (average age and bodyweight 20 months and 500 kg, respectively) received either cottonseed meal delivering 8.2 g free gossypol/bull/d (treatment group, n=4) or soybean meal (control group, n=4) for 12 wk. After adjustment (1 wk), weekly procedures (11 wk) included blood collection, scrotal circumference measurement and electroejaculation. Semen assessments included sperm motility, percentage of live spermatozoa, general sperm morphology (using brightfield microscopy), and midpiece morphology (using DIC microscopy). After sacrifice (Week 12), sperm production rates (daily and per gram testicular parenchyma) were determined. Treated bulls did not differ from controls in scrotal circumference or the percentage of live spermatozoa. Sperm motility differed at Weeks 9 (P<0.05), 10 and 11 (both P=0.06). Treated bulls had fewer normal spermatozoa at Weeks 5 (P<0.05), 6 (P<0.01) and 7 thru 11 (P<0.001). Beginning from Week 3, treated bulls showed an increased proportion of sperm midpiece abnormalities (P<0.05) which stabilized at 52 to 62.5% between Weeks 5 and 11 (P<0.01 or P<0.001). Treated bulls also had lower sperm production than untreated bulls, both on a daily (P<0.01) and per gram testicular parenchyma (P<0.001) basis. A cottonseed supplement providing 8.2 g of free gossypol per bull per day had adverse effects upon both sperm morphology and spermatogenesis in young Brahman bulls, with the former being first evident within 3 to 4 weeks of feeding of cottonseed meal.     

Role of the major ecto-phosphoprotein in sperm flagellar motility using a cell electroporation method

Previous studies from our laboratory have identified MPS, a 100-kDa protein, as the major phosphoprotein substrate of caprine sperm ecto-cyclic AMP independent protein kinase. In this study the isolated (32)P-labelled MPS has been incorporated into mature caprine (Capra indicus) cauda-epididymal spermatozoa with the help of cell electroporation technique to investigate the effect of MPS on sperm flagellar motility. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2 x 10(8)/ml) to external electric field of intensity 1.5 kV/cm and capacitation of 25 microF at 4 degrees C and post-pulse incubation at 37 degrees C for 1 hr. when nearly 50% of the cells lost motility. Scanning electron micrographs (SEM) demonstrate the formation of micro-pores and local osmotic swelling in the electroporated spermatozoa. MPS incorporation was maximal when its concentration was 30 microg/ml (300 pmol) in the medium and when the post-pulse incubation time was 60 min. At maximum (75%) MPS incorporation, total and forward motility increments were also maximum: 34% (P < 0.01) and 32% (P < 0.01), respectively. The subcellular fractionation data show that major portion of the introduced MPS was bound to the plasma-membrane of spermatozoa. The 32P-labelled electrophoresed intact spermatozoa lost radioactivity due to the action of the endogenous ecto-phosphoprotein phosphatase. Therefore MPS is primarily localised on the sperm external surface leaving its phosphate group(s) oriented in the extracellular medium. The data provided further evidence to strengthen the view that MPS is an ecto-phosphoprotein and that it plays an important role in the regulation of sperm flagellar motility.     

Measurement of boar sperm motility by the trans-membrane migration method

The conventional microscopic methods for evaluating sperm motility of domestic animals are mostly inadequate due to their subjectivity and lack of precision. Recently, a trans-membrane migration method, originally developed for the examination of human sperm motility, has substantially overcome these problems. This study investigated the applicability of the method to boar sperm motility measurement. The apparatus used was simple and consisted only of syringe plungers, poriferous membranes, and modified multi-well culture plates. It measured the proportion of sperm in the semen that moved across the membrane after incubation at 37 degrees C for 3 hr. The sperm motility as measured by this method correlated well with that measured by direct microscopic examinations. The measurement was more reliable using an 8-microns instead of a 5-microns pore-size membrane. The method was found to work equally well for the sperm motility measurement of the semen with a sperm concentration between 1.5 x 10(8)/ml and 6.0 x 10(8)/ml. The results indicate that this method is a simple, objective, quantitative, and reproducible design for the measurement of boar sperm motility.     

Different signaling pathways in bovine sperm regulate capacitation and hyperactivation

Hyperactivated sperm motility is characterized by high-amplitude and asymmetrical flagellar beating that assists sperm in penetrating the oocyte zona pellucida. Other functional changes in sperm, such as activation of motility and capacitation, involve cross talk between the cAMP/PKA and tyrosine kinase/phosphatase signaling pathways. Our objective was to determine the role of the cAMP/protein kinase A (PKA) signaling pathway in hyperactivation. Western blot analyses of detergent extracts of whole sperm and flagella were performed using antiphosphotyrosine antibody. Bull sperm capacitated by 10 microg/ml heparin and/or 1 mM dibutyryl-cAMP plus 100 microM 3-isobutyl-1-methylxanthine exhibited increased protein tyrosine phosphorylation without becoming hyperactivated. Procaine (5 mM) or caffeine (10 mM) immediately induced hyperactivation in nearly 100% of motile sperm but did not increase protein tyrosine phosphorylation. After 4 h of incubation with caffeine, sperm expressed capacitation-associated protein tyrosine phosphorylation but hyperactivation was significantly reduced. Sperm initially hyperactivated by procaine or caffeine remained hyperactivated for at least 4 h in the presence of Rp-cAMPS (cAMP antagonist) or PKA inhibitors H-89 or H-8. Pretreatment with inhibitors also failed to block induction of hyperactivation; however, the inhibitors did block protein tyrosine phosphorylation when sperm were incubated with capacitating agents, thereby verifying inhibition of the cAMP/PKA pathway. While induction of hyperactivation did not depend on cAMP/PKA, it did require extracellular Ca(2+). These findings indicate that hyperactivation is mediated by a Ca(2+) signaling pathway that is separate or divergent from the pathway associated with acquisition of acrosomal responsiveness and does not involve protein tyrosine phosphorylation downstream of the actions of procaine or caffeine.     

Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).     

Unilateral intrauterine insemination with prostatic fluid-sensitized frozen caudal epididymal sperm in beagle dogs

The effects of the absence or presence of prostatic fluid (PF) during cauda epididymal sperm retrieval were assessed as regards semen quality after freezing semen in egg yolk Tris-fructose citrate solution (EYT-FC). Epididymal spermatozoa from the left testis of each of 10 dogs was retrieved into PF, whereas that from the right testis into EYT-FC only. At sperm recovery, the only difference between the two groups was that the incidence of spermatozoa with cytoplasmic droplets (immature sperm) was lower in the PF group (P < 0.01). In contrast, after freezing-thawing, (mean +/- S.E.) sperm motility (32.0 +/- 1.4 versus 12.5 +/- 2.0%, P < 0.01) and viability (58.2 +/- 3.5 versus 41.8 +/- 5.6%, P < 0.05) were higher in the PF group than in the EYT-FC group, respectively. Furthermore, 25.6 +/- 2.7% spermatozoa in the PF group were still motile after being maintained at 20 degrees C for 6 h. The incidence of immature spermatozoa post-thaw was lower compared to that after recovery in the EYT-FC group (P < 0.01), but was still higher than that in the PF group (P < 0.05). Frozen-thawed spermatozoa (2 x 10(8)) were used for unilateral intrauterine AI. The conception rate of PF-unsensitized sperm was 20% (2/10), but that of PF-sensitized sperm was 80% (8/10; P < 0.01). Therefore, sperm recovered in PF and frozen-thawed were of good quality. Sensitization of epididymal sperm with PF before freezing clearly improved the conception rate to AI of spermatozoa derived from the cauda epididymus.