Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process

Experiments were conducted to investigate the effect of seminal plasma on sperm motility during the cryopreservation process. Ejaculated and epididymal spermatozoa from the ram and the bull were washed by centrifugation and resuspended in either seminal plasma or a modified Tyrode's medium (TALP) prior to dilution in medium suitable for cryopreservation. Resuspension of washed ejaculated ram spermatozoa in seminal plasma resulted in higher percentages of motile spermatozoa than resuspension in TALP after the spermatozoa were cooled to 5 degrees C (52 vs 35%), and after thawing (14 vs 9%), respectively. Resuspension of epididymal ram spermatozoa in seminal plasma had no beneficial effect in maintaining sperm motility after cooling (78 vs 73%); however, seminal plasma was beneficial to epididymal ram spermatozoa after thawing (34 vs 3%), respectively. Resuspension of washed ejaculated bull spermatozoa in either seminal plasma or TALP had no effect on the percentage of motile spermatozoa after cooling to 5 degrees C (73 vs 75%) or after thawing (60 vs 60%), respectively. In addition, seminal plasma had no beneficial effect on the percentage of motile epididymal bull spermatozoa when compared with that of TALP-treated spermatozoa after cooling (75 vs 72%) or after thawing (66 vs 63%), respectively. Seminal plasma from different sires (ram and bull) affected epididymal sperm motility. The ability of sperm cells to withstand damage during cryopreservation, however, appears to reside in the sperm cells themselves, probably due to sperm cell composition.     

Effect of increasing trypsin concentrations on seminal coagulum dissolution and sperm parameters in spider monkeys (Ateles geoffroyi)

Seminal coagulum formation in spider monkeys (Ateles geoffroyi) interferes with the efficient recovery and evaluation of spermatozoa. The main objective was to assess the effect of increasing concentrations of trypsin on dissolution of seminal coagulum and spermatic parameters. Seminal coagulum was incubated at 37 °C without trypsin or in the presence of increasing trypsin concentrations (0.1%, 1.0%, and 5.0%). For each sample, coagulum dissolution time was measured, and sperm concentration, viability, motility, and morphology were evaluated using light microscopy and/or transmission electronic microscopy (TEM). Trypsin concentrations of 1.0% and 5.0% more rapidly liquefied seminal coagulum, averaging 32 and 21 min, respectively, compared with nontrypsinized controls, with maintenance of greater sperm viability (70.8% and 72.5%, respectively). Coagulum treated with 1.0% trypsin and the liquid ejaculate fraction averaged higher sperm motility (40.1% and 55.6%, respectively) than control samples, and both 1.0% and 5.0% trypsin treatment allowed recovery of increased numbers of motile spermatozoa. There was greater sperm fragmentation at the head and midpiece level after treatment with 1.0% and 5.0% trypsin (55.8% and 55.9%); however, the percentage of normal morphology in structurally intact spermatozoa did not differ relative to controls. With transmission electronic microscopy imaging, there were similar percentages of spermatozoa with plasma membrane swelling in the midpiece and acrosomal regions in trypsin-treated samples and controls. In conclusion, trypsin treatment of spider monkey seminal coagulum exerted a concentration-dependent effect on dissolution time and various spermatic parameters. Higher trypsin concentrations caused more rapid liquefaction of coagulum and recovery of greater numbers of motile spermatozoa, but may adversely affect fragmentation of spermatozoa and could compromise sperm function and cryopreservation potential.     

Identification of sperm forward motility-related proteins in human seminal plasma

Seminal plasma, an amorphous material that exists in semen, contains proteins related to sperm forward motility. Employing affinity chromatography with ConA beads and protein ultrafiltration, we isolated and concentrated proteins from heated human seminal plasma. Results of computer-assisted semen analyses (CASA) demonstrated that the forward motility index of bovine spermatozoa from the epididymal caput, incubated with proteins and theophylline, was significantly different from that of spermatozoa incubated with theophylline alone (P < 0.01). The electrophoreses revealed that the protein bands with high molecular weights in the gel of PAGE changed into low molecular weights in the gel of SDS-PAGE. Furthermore, proteins from a separated portion of the PAGE gel were still able to stimulate spermatozoa from the epididymal caput to gain forward motility. Two-dimensional (2D)-gel electrophoresis and mass spectrometry indicated that spots focused on the portion seemed, according to their amino acid sequences, to be like human alpha-1-antitrypsin and zinc-alpha-2-glycoprotein (ZAG) precursors. Western blot analysis showed the presence of these two proteins in seminal plasma. These proteins, related to the forward motility of spermatozoa in human seminal plasma, may play important roles during maturation of spermatozoa, from the epididymis through fertilization in the female reproductive tract.     

The maintenance of motility and the surface properties of epididymal spermatozoa from bull, rabbit and ram in homologous seminal and epididymal plasma.

Epididymal spermatozoa from bull, rabbit and ram were incubated in homologous epididymal plasma or seminal plasma in a buffered saline-based medium with or without serum albumin. The spermatozoa were either diluted directly into the medium or were washed first. No effect of washing was observed on the subsequent reaction of the cells to the different media. A considerable proportion of the populations of epididymal spermatozoa survived (i.e. continued to exhibit motility) for up to 22 h at 30 degrees C in the simple saline-based medium. Initially epididymal plasma had a slight stimulatory effect on sperm motility in ram and bull but it had no effect on sperm survival in any of the 3 species. Seminal plasma stimulated motility markedly in ram initially, but in all 3 species seminal plasma was detrimental to survival: in ram even a 15-min exposure to the fluid reduced survival. Serum albumin also stimulated motility; it delayed, but did not prevent, the detrimental effect of seminal plasma, although it had no effect itself on survival. The effects of epididymal plasma, seminal plasma and serum albumin on surface properties of epididymal spermatozoa, i.e. agglutination, sticking-to-glass and eosinophilia, were also noted. These varied between species and there was no correlation between these effects and the effects on motility and survival.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Exogenous DNA uptake by South American catfish (Rhamdia quelen) spermatozoa after seminal plasma removal

Sperm from different species shows biological differences, determining the success or failure of the sperm-mediated gene transfer (SMGT) technique. There is evidence that exogenous DNA uptake by the spermatozoa is a species-specific and highly regulated phenomenon. Problems involving SMGT procedures might be related to activation of defenses in spermatozoa and in seminal plasma such as DNase enzymes. The objective in the present study was to transfect South American catfish spermatozoa after seminal plasma removal. Seminal plasma had a strong DNase activity that is reduced after sperm washes in isosmotic solution, in which Western blot analysis demonstrated a reduction in the DNase content after washes and Southern blot evaluations show the presence of plasmid after sperm washes. The seminal plasma DNase digests exogenous DNA in a few minutes and has an optimal activity at 43°C. Also, EDTA at 30 mM concentration inhibits the DNase activity. Using PCR the pEGFP vector was internalized by sperm cells even at lesser concentrations (5-40 ng/10(6) spermatozoa) without motility loss after seminal plasma removal. Conversely, using greater pEGFP concentrations (100 ng/10(6) spermatozoa), there were no motile cells, suggesting toxicity of exogenous DNA for sperm cells. These results are interpreted to provide information that can improve the protocol for generation of transgenic South American catfish.     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

Evidence for specific binding of uncapacitated boar spermatozoa to porcine zonae pellucidae in vitro

Washed ejaculated boar sperm and sperm from the cauda epididymis bind to the zona pellucida of fixed porcine eggs in large numbers. Sperm incubated in the presence of dextran sulfate (8 K daltons or 500 K daltons) or fucoidan and then washed no longer bind to eggs. Other acid carbohydrates (heparin, chondroitin sulfates, inositol hexasulfate, carboxymethylcellulose) fail to block sperm-egg binding even when added directly to sperm-egg suspensions. Seminal plasma and the seminal vesicle secretion contain basic proteins which bind tightly to sperm and bind reversibly to eggs preventing sperm from binding to eggs. When dextran sulfate or fucoidan are mixed with the vesicular secretion, from which seminal plasma basic proteins originate (Hunt et al., '83), the secretion loses the capacity to prevent sperm from binding to eggs; this suggests that seminal vesicle proteins can bind to the same site on zonae as do sperm and thus seminal plasma may modify sperm-egg interactions. Corpus and cauda epididymal sperm also bind in large numbers to the zona pellucida of isolated eggs but high concentrations of caput sperm, which exhibit high motility in the presence of caffeine, bind only in few numbers. Thus a component that enhances sperm-zona binding is apparently formed on the plasma membranes of uncapacitated sperm during passage through the epididymis. This finding, and an earlier observation that antibodies raised against uncapacitated sperm plasma membranes block sperm-egg binding in vivo (Peterson et al., '83) suggest that this component may be involved in sperm zona interaction in vivo.     

Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.     

Cryopreservation of epididymal sperm in tree shrews (Tupaia belangeri)

Cryopreservation of sperm from tree shrews, which are considered primitive primates, would enhance genetic management and breeding programs. Epididymal sperm were surgically harvested from male tree shrews, cryopreserved in two Tes-Tris-based cryodiluents, and used in four experiments. In Experiment 1, there were no significant differences in motility and acrosome integrity among five concentrations of egg yolk in TTE after cooling to 4 °C. However, sperm frozen in TTE containing 20% egg yolk at −172 °C/min had better (P < 0.05) post-thaw motility and acrosome integrity. In Experiment 2, sperm held for 10 min prior to storage in liquid nitrogen had greater motility than those held for 5 or 15 min (P < 0.05), but acrosome integrity was not different (P > 0.05) among treatments. In Experiment 3, sperm frozen in TTE diluent had higher (P < 0.05) motility and acrosome integrity than those in TEST diluent. In Experiment 4, there were no differences (P > 0.05) in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes, following AI with fresh versus frozen sperm. In conclusion, tree shrew epididymal sperm were successfully cryopreserved, as assessed by post-thaw motility, acrosome integrity, and fertilizing ability.     

Characterization of seminal plasma proteins stabilizing the sperm viability in rainbow trout (Oncorhynchus mykiss)

In seminal plasma of the rainbow trout 12 proteins were detected by SDS-PAGE, ranging in their molecular weight from 135 to 16 kDa. Only those proteins with a molecular weight of 65, 54, 47 and 16 kDa occurred in all investigated seminal plasma samples. The 65 and the 54 kDa protein were found in highest quantities (34-45% of the total quantified protein content) followed by the 47 and the 16 kDa protein (6-7% of the total quantified protein content). The 65 and the 48 kDa protein were glycoproteins as they stained positively with Periodic-Acid-Schiff reagent (PAS) specific for carbohydrates as well as with Coomassie Blue. The 90 and 19 kDa protein were found in 82-91% of the investigated samples, all other proteins in lower frequencies of 36-73%. Seminal plasma contained no lipoprotein as staining with Sudan black B was negative. To find out which proteins positively affected the sperm viability (defined as sperm motility which could be activated) spermatozoa were incubated in sperm motility inhibiting saline solution containing different seminal plasma protein fractions. Sperm motility which could be activated after an incubation period of 48 h was highest in those fractions which shared the 54, 47, and the 16 kDa protein. When spermatozoa were incubated in untreated seminal plasma sperm viability was still higher than in the seminal plasma protein fractions indicating that other components of the seminal plasma positively affected sperm viability, too. The possible influence of seminal plasma proteins on sperm physiology is discussed.     

Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation

Effects of seminal plasma on post-thaw motility and membrane integrity of cryopreserved horse spermatozoa were investigated. Carboxyfluorescein diacetate staining was used for the assessment of sperm membrane integrity. Adding 30% of seminal plasma from stallions with high post-thaw sperm motility to ejaculates from stallions with low post-thaw sperm motility increased progressive motility from 24.0 +/- 1.6 to 34.5 +/- 1.9% (P < 0.05) and membrane integrity from 27.0 +/- 2.1 to 34.3 +/- 2.3% membrane-intact spermatozoa (P < 0.05). Conversely, the addition of seminal plasma from stallions with low post-thaw sperm motility to ejaculates from stallions with high post-thaw motility decreased progressive motility from 36.0 +/- 1.6 to 30.0 +/- 2.7% (P < 0.05) but did not induce changes in membrane integrity. Seminal plasma from stallions with opposite post-thaw motility therefore clearly influenced the resistance of spermatozoa to the freezing and thawing process. We conclude that the individual composition of seminal plasma affects the suitability of stallions for semen cryopreservation.     

Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin

Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.     

Testicular growth, hormone concentrations, seminal characteristics and sexual behaviour in stallions

Puberty was studied using 15 colts of Quarter Horse phenotype. Total scrotal width was measured every 8 weeks from 48 to 96 weeks. Blood samples were taken from 8 colts at 8, 16 and 24 weeks and then every 4 weeks until 100 weeks to measure changes in LH, FSH and testosterone concentrations. Seminal collections were attempted monthly from 48 to 64 weeks and every 2 weeks thereafter until puberty resumed every 3rd day from 96 weeks for 15 ejaculates. For all collections, times to erection, mount and ejaculation and seminal characteristics were recorded. Age at puberty was defined as the first ejaculate containing 50 x 10(6) spermatozoa, with greater than or equal to 10% motile. Colts were castrated at 2 years to enable determination of daily sperm production (DSP), epididymal sperm reserves and normality of spermatogenesis. Total scrotal width increased linearly from 48 to 96 weeks. Age at puberty averaged 83 weeks (56-97 weeks). Changes in serum concentrations of LH and FSH were parallel, rising at 36-40 weeks, declining after 40 weeks and rising again at 68-80 weeks. Testosterone was low until 68 weeks after which concentrations rose slowly to 80 weeks and increased rapidly to a plateau at 92 weeks. Sexual behaviour and seminal characteristics differed (P less than 0.05) between puberty and 2 years, except for time to erection, time to mount, and percentage of motile spermatozoa. DSP at 2 years averaged 1.7 x 10(9) and daily sperm output (DSO) averaged 1.1 x 10(9). The correlation between DSP and DSO was 0.83 (P less than 0.01). There were 9.57 x 10(9) spermatozoa/epididymis of which 67% were in the cauda.     

Impact of feeding ethanolic extract of root bark of Cananga odorata (Lam) on reproductive functions in male rats

The 50% ethanolic extract of the root bark of C. odorata administered orally at the dose of 1g/kg body weight/day for 60 days resulted in decreased epididymal sperm motility and sperm count in male albino rats. Morphological abnormalities were also observed in the sperms. The testicular glycogen, the activities of 3beta hydroxy steroid dehydrogenase, glucose 6-phosphate dehydrogenase, malic enzyme, sorbitol dehydrogenase in seminal vesicle, fructose in seminal plasma and serum testosterone were significantly decreased in treated group. While testicular cholesterol level, the concentration of the fecal bile acids, urinary excretion of 17 ketosteroids, the activities of 17beta hydroxy steroid dehydrogenase, epididymal lactate dehydrogenase and that of testicular HMG CoA reductase were increased in treated group when compared to control. The results suggest that the ethanolic extract of C. odorata possesses the spermatotoxic effects in male albino rats.     

Effects of hexavalent chromium on reproductive functions of male adult rats

Hexavalent chromium is an environmental contaminant which may be associated with reproductive abnormalities in male rats. In the present study, we examined the effect of hexavalent chromium on male reproductive function of rats. Male Wistar rats received a daily intraperitoneal injection of potassium dichromate (1 or 2 mg/kg body weight) for fifteen consecutive days. A decrease in testis weight and an increase in seminal vesicles and prostate weights were demonstrated after chromium treatment. Moreover, a dose-dependent increase in blood and testis chromium levels as well as an increase in FSH and a decrease in LH and testosterone serum levels were detected in treated rats. Histological analysis revealed pronounced morphological alterations with enlarged intracellular spaces, tissue loosening and dramatic loss of gametes in the lumen of the seminiferous tubules of treated rats. In addition, a decreased sperm motility and number of epididymal spermatozoa together with an increased sperm abnormality rate was found in chromium-treated rats in comparison to controls. In rats receiving the higher chromium dose, histological images presented considerably increased areas filled with seminal vesicle and prostate secretions. The mucosal crypts of seminal vesicles and the typical invaginations of prostate were altered. The results suggest that subacute treatment of potassium dichromate promotes reproductive system toxicity and affects testicular function of adult male rats.     

Seminal plasma non-heparin binding proteins (NHBP) reduce the cryoinjury to buffalo cauda epididymal spermatozoa induced by heparin binding proteins (HBP)

Previous cryopreservation studies with buffalo cauda epididymal spermatozoa have reported a deleterious effect of seminal plasma heparin binding protein (HBP). The amount of HBP used in these studies was meager compared to the normal level of HBP in the buffalo ejaculate, still the damage induced upon the spermatozoa was substantial when compared to that incurred to the spermatozoa during routine freezing of ejaculated semen. Thus there might be some factor(s) in the seminal plasma, which reduce the deleterious effect of HBP on spermatozoa during cryopreservation of ejaculated semen. This study was conducted to investigate for the presence of any such factor in buffalo seminal plasma. Seminal plasma proteins were separated on their heparin binding properties as heparin binding (HBP) and non-heparin binding (NHBP). The separated proteins were added to the extender of buffalo cauda epididymal semen for cryopreservation either alone or in combination. The spermatozoa were assessed for progressive motility, viability, acrosomal integrity and response to hypo-osmotic solution test (HOST) at prefreeze and post-thaw stages of cryopreservation. NHBP was found to provide some degree of protection to buffalo spermatozoa against cryopreservation stress as well as the deleterious effect of HBP during cryopreservation.     

角马蜂贮精囊及精子的形态学观察

昆虫贮精囊和精子的形态多样性是重要的分类和系统发育分析特征之一, 然而在马蜂亚科乃至整个胡蜂科中却鲜有涉及。本文首次解剖了角马蜂Polistes chinensis antennalis Pérez的雄性生殖系统, 着重对其贮精囊的超微结构进行描述, 并简要报道了精子的外部形态。角马蜂的贮精囊由输精管亚前端膨大而成, 有一层发达的柱状上皮细胞贴在基底膜内壁： 细胞核位于柱状细胞基部, 上皮细胞端半部线粒体密集, 顶膜特化成微绒毛。角马蜂精子头长21.4 μm, 体长94 μm, 是已报道胡蜂科精子中长度最短、 相对头长最长的种类。研究结果为胡蜂科昆虫系统发育以及繁殖生理提供理论依据。     

The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa

In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n=3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6h. Catalase had no effect (P>0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced (P<0.05) sperm quality after post-thaw incubation for 0 h (motility), 3h (motility, average path velocity, viability and acrosome integrity) and 6h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect (P<0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline (P<0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements (P<0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa.