Electroejaculation Increases Low Molecular Weight Proteins in Seminal Plasma Modifying Sperm Quality in Corriedale Rams

This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and 2‐D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation.     

Seasonal Variations in Seminal Plasma Proteins of Buffalo

The study was designed to evaluate the influence of season on semen characteristics and seminal plasma protein profile of buffalo bull semen. Thirty‐six ejaculates were collected in three seasons (winter, summer and rainy) from six adult Bhadawari bulls, and semen characteristics were evaluated immediately after collection. The seminal plasma was harvested by centrifugation and protein profiling, and percentage protein fractions were analysed by SDS‐PAGE. The significant effect of season was observed on ejaculate volume, sperm concentration, progressive motility, percentage live spermatozoa, hypo‐osmotic swelling test (HOST) and acrosomal integrity. The electrophoretogram of seminal plasma proteins revealed 20 protein bands in winter, 23 bands in rainy and 25 bands in summer seasons, illustrating the significant effect of seasons on seminal plasma proteins. Among these protein bands, 18 bands were observed common in semen samples of all three seasons while protein bands of 46, 55, 58, 144 and 160 kDa were found in rainy and summer seasons. The protein bands of 48 and 60 kDa were observed only in winter season, whereas 184 and 200 kDa were reported in summer season only. The protein fractions (protein%) of common protein bands observed in three seasons revealed a significant effect of season on protein bands of 24.5, 66, 70, 72, 84 and 86 kDa. From the study, it was pertinent that bull seminal plasma contains specific proteins in particular season, which may be associated with some of the semen characteristics, and these proteins could be used as markers of the semen quality of buffalo bulls.     

Reproductive Development of Santa Inês Rams During the First Year of Life: Body and Testis Growth,Testosterone Concentrations,Sperm Parameters,Age at Puberty and Seminal Plasma Proteins

We have investigated the reproductive development of the tropically adapted Santa Inês ram, the most common hair sheep in Brazil. From 8 to 48 weeks of age, 16 animals were evaluated for body and testis growth, semen parameters, testosterone concentrations and seminal plasma proteins, using two‐dimensional SDS‐PAGE. Animals were weaned at 30 days and kept in feedlots thereafter, receiving hay, concentrate (18% of crude protein) and mineral supplement. Body weight increased from 12.3 ± 0.7 to 54.3 ± 1.6 kg between 8 and 48 weeks (p < 0.05), but changes in thoracic perimeter and scrotal circumference were non‐significant after 36 weeks (p > 0.05). The percentage of motile sperm increased slowly until 23 weeks and more rapidly after that age, but significant changes in progressive motility occurred after 25 weeks. Presence of abnormal sperm related inversely to age. Most significant changes in sperm concentration occurred between 38 and 44 weeks (0.38 ± 0.05 to 1.14 ± 0.24 × 10⁹ cells/ml, p < 0.05) and testosterone reached its highest concentrations at 42 weeks, decreasing afterwards. Rams reached puberty at 28.2 ± 0.8 weeks. The number of protein spots on seminal plasma gels was similar from 15 to 18 weeks (45 and 47 spots; p > 0.05), increased until 24 weeks (141 spots) and 28 weeks (170 spots; p < 0.05) and remained without significant (p > 0.05) changes from 28 to 48 weeks (186 ± 10 spots). Furthermore, the intensity of selected spots on 2D maps increased (p < 0.05) between 15 and 28 weeks, which preceded or coincided with the main developmental changes in sperm motility and percentage of defective sperm in the ejaculates. These results will support future studies designed to characterize specific seminal plasma proteins whose expression relate to the development of testis, epididymis and accessory sex glands.     

Proteomic Characterization of Zinc‐Binding Proteins of Canine Seminal Plasma

The zinc‐binding proteins (ZnBPs) of the seminal plasma are implicated in different processes related to sperm–egg fusion. The aim of this study was to characterize the ZnBPs of canine seminal plasma using two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and mass spectrometry. The ZnBPs were isolated from the ejaculates of five dogs by affinity chromatography and subjected to 2D‐PAGE analysis. The acquired spots, detected across the gels, were analysed by mass spectrometry. Using 2D‐PAGE analysis, it was shown that canine seminal plasma comprised about 46–57 zinc‐binding polypeptides, with molecular mass ranging from 9.3 to 138.7 kDa and pI at pH 5.2–10.0. It was found that zinc‐binding polypeptides of low molecular masses (9.3–19.0 kDa and pI at pH 6.1–10.0) were predominant in the seminal plasma, and seven polypeptides, with molecular masses ranging from 11.7 to 15.4 kDa and pI at pH 6.8–8.7, were characterized by high optical density values. In addition, analysis with mass spectrometry (LC‐MS‐MS/MS) revealed that the identified seven polypeptides are canine prostate‐specific esterase (CPSE), which is the main proteolytic enzyme of the seminal plasma. The findings of this study indicate an important regulatory role of seminal plasma zinc ions in the functional activity of CPSE, which is of great significance for maintaining the normal function of canine prostate and the spermatozoa functions.     

Seminal Plasma Composition from Ejaculates Collected by Artificial Vagina and Electroejaculation in Guirra Ram

This study was conducted to evaluate changes in ram seminal plasma composition from ejaculates obtained using artificial vagina (AV) and electroejaculation (EE). To address this question, we assessed the effect of semen collection method on volume, sperm concentration, sodium concentration, potassium concentration, sodium/potassium ratio, total protein content and protein profile using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide gel electrophoresis. The main findings from this study were: (i) similar volume was obtained, while sperm concentration was significantly lower for EE method; (ii) potassium and sodium/potassium concentration ratio were not influenced by recovery method, while sodium concentration increased significantly when semen was recovered using EE; (iii) approximately 80% of the total relative seminal plasma protein is represented by four protein fractions of molecular weights around 15, 21, 24 and 50 kDa and there were not differences and (iv) focussing the two-dimensional SDS-PAGE gel on the 10–25 kDa rank, the image analysis software detected around 22 spots with isoelectric points ranging from 5.1 to 6.1. Two protein spots (15 kDa and 5.5 and 22 kDa and 5.2 for molecular weight and isoelectric point respectively) increased significantly when semen was recovered using EE. One spot protein with molecular weight around 25 kDa and isoelectric point of 5.2 were only found in the seminal plasma from the semen recovery by AV. As it was demonstrated, ejaculates obtained with EE modify the sodium concentration, alter two proteins concentration and induced the loss of one protein in seminal plasma.     

Can the Genetic Origin Affect Rabbit Seminal Plasma Protein Profile along the Year?

The study was designed to evaluate the influence of genetic origin on rabbit seminal plasma protein profile variation along the year. Seminal plasma of rabbits from line A (maternal line) and R (paternal line) collected during a natural year was subjected to polyacrylamide gel electrophoresis (SDS‐PAGE). The electrophoretic profile of rabbit seminal plasma resulted in multiple protein bands of different intensity ranging from 9 to 240 kDa. Results showed that seven protein bands were significantly different between genetic lines, and among these, three protein bands were significantly different between seasons. The differentially expressed proteins were identified by MALDI‐TOF/TOF or LC‐MS/MS analysis and were the following ones: FAM115E‐like (220, 113 and 59 kDa), ectonucleoside triphosphate diphosphohydrolase 3 isoform X2 (72 kDa), annexin A5 (32 kDa), lipocalin allergen Ory c 4 precursor (19 kDa), and haemoglobin subunit zetalike (13 kDa) between genetic lines and FAM115E‐like (113 kDa), haemoglobin subunit zetalike (13 kDa) and β‐nerve growth factor (12 kDa) between seasons. These results indicate that proteins from rabbit seminal plasma are under both seasonal control and genetic control. Furthermore, the differential presence of these proteins could be one of the causes explaining the differences observed in fertility and seminal parameters between these two lines in earlier studies.     

Interaction of Potential Porcine Sperm Ligands with the Oocyte Plasma Membrane

We previously identified 62, 39, 27 and 7 kDa porcine sperm plasma membrane proteins that demonstrated a predominant affinity for the porcine oocyte plasma membrane by Western ligand blotting. The current experiments were designed to further investigate the potential roles of these molecules in sperm–oocyte plasma membrane interaction. Abilities of these proteins to bind to the oocyte plasma membrane and to inhibit sperm–oocyte interaction were evaluated. Plasma membrane was isolated primarily from the head of ejaculated porcine sperm by nitrogen cavitation and density gradient centrifugation. Fractions containing the 62, 39, 27 and 7 kDa proteins were electroeluted from one dimensional SDS polyacrylamide gels, dialysed and proteins biotinylated. Following incubation with zona‐free porcine oocytes, bound protein was visualized with 20 μg TRITC‐avidin/ml using confocal microscopy. Fractions of the dialysed, electroeluted proteins were added to porcine in vitro fertilization assays. The 62, 39, 27 and 7 kDa proteins all demonstrated binding to the oocyte plasma membrane in contrast to a biotinylated control protein. Addition of unlabelled sperm plasma membrane proteins to the biotinylated protein visibly reduced binding. Addition of each of these protein fractions to in vitro fertilization assays reduced sperm interaction with the porcine oocyte plasma membrane in a concentration‐dependent manner. Binding of these sperm plasma membrane proteins to the oocyte plasma membrane and inhibition of fertilization are consistent with these proteins being involved in sperm–oocyte plasma membrane interaction.     

Retracted: Effects of multiple collections on spermatozoa quality of Persian sturgeon,Acipenser persicus: motility,density and seminal plasma composition

In this study, we investigated the effects of multiple collections of sperm on the endangered Persian sturgeon, Acipenser persicus, in terms of a number of sperm functional parameters (percentage of motile spermatozoa, total time period of motility and sperm concentration) as well as on the ionic composition, protein concentration and osmolality of seminal plasma. Semen samples were collected from 12 induced male fish in three experimental groups that had been injected intramuscularly with LHRH‐A₂, at dosages of 5 μg/kg body weight, at a number of time regimes: at 12 h, 17 h and 24 h after spawning induction (1); at 24, 29 and 34 h after spawning induction (2); and at 36, 41 and 46 h after spawning induction (3). The percentage of motile spermatozoa and the period of sperm motility decreased significantly (p < 0.05) after the second and third collections. The concentration of spermatozoa decreased after the third collection, but this decline was not significant. No significant effect of multiple collections on protein concentration and ionic content (with exception of the Cl^? ion) of seminal plasma was observed. In all experimental groups, a moderate impact of sequential collection on the osmolality (p < 0.05) of seminal plasma was observed. This study provides new data on the effects of multiple collections on spermatological characteristics in the Persian sturgeon. Our results confirm that sequential stripping after the third collections has a negative effect on a number of functional parameters associated with sperm.     

Evaluation of spermiation indices with multiple sperm collections in endangered sterlet (Acipenser ruthenus)

This study investigated the effects of multiple collections of sperm on endangered sterlet (Acipenser ruthenus) sperm functional parameters [spermatozoa motility and curvilinear velocity (VCL)] as well as on protein concentration and osmolality of seminal plasma. The average sperm volume and mean spermatozoa concentration per male were significantly altered with multiple collections. On the other hand, no significant effect of multiple collections on protein concentration of seminal plasma was observed. In all experimental groups, moderate impact of sequential collection on osmolality (p < 0.05) of seminal plasma was observed. Ninety to 100% of motile spermatozoa were observed at 15 s after activation, with an average VCL of 181.12 ± 19.10 μm/s. After 90 s, average VCL decreased to 130 ± 26 μm/s. Motility was maintained for up to 4 min. The maximum percentage of motile spermatozoa was observed after the third collection of sperm. The spermatozoa VCL increased significantly with subsequent collections. The results of this study provide new data on the effects of multiple collections on quantitative and qualitative parameters of sperm in sterlet. The data confirmed that the sequential stripping has no negative effect on the percentage of motility and spermatozoa velocity. This should be beneficial for the development of sterlet aquaculture programs.     

Osteopontin in Seminal Plasma and Sperm Membrane of Dogs

The objective of this work was to describe the presence of osteopontin (OPN) in canine seminal plasma and sperm membranes. A pool of seminal plasma and sperm membrane extract from 30 dogs was used. Polyacrylamide electrophoresis gels were performed and the bands were transferred to nitrocellulose paper and Western blot was undertaken using an antibody anti-OPN. Two and 12 bands were marked in the seminal plasma (77.2 and 15.6 kDa) and sperm membrane extracts (70.6–26.6 kDa), respectively. However, from 12 marked bands in the sperm membrane extract, only three (46.4, 37.7 and 36.5 kDa) were strongly marked. We conclude that, seminal plasma and sperm membranes from dogs contain different isoforms of OPN; yet, further studies will be necessary to determine their function in this species.     

家蚕病原性微孢子虫的蛋白质化学性质的研究

抽提了微 孢子虫的总 蛋白和选择 性分离纯 化了主要 蛋白组 分。对 总蛋白 进行了 酸性 Ｐ Ａ Ｇ Ｅ、碱性 Ｐ Ａ Ｇ Ｅ、 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 和氨基酸组成分析，并对 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 结果进行了薄层扫描分析。发现每一样品总蛋白在 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 图谱上均分离出３０ 多条蛋白带，均有５ 条主带，但位置不同，各条蛋白带的相对含量不同。氨基酸组成分析结果中发现各种孢子总蛋白所含的氨基酸种类基本相同，均含有１６ 种氨基酸，但各种氨基酸的相对含量不同。对主要蛋白组分进行了 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 氨银染色法纯度鉴定、薄层扫描分析和氨基酸组成分析，发现彼此之间有共同点，但也存在一定的差异。     

Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

Identification of RSVP14 and RSVP20 components by two-dimensional electrophoresis and Western-blotting.

We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4-7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (M(r)) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e, f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases.     

绵羊精浆蛋白质组2-DE图谱的构建及初步分析

本研究比较了丙酮沉淀法制备蛋白质不同上样量对绵羊精浆蛋白质二维电泳图谱的影响。结果显示,精浆总蛋白经SDS-PAGE电泳,得到分子质量在14.4~116 ku的28条蛋白质条带。二维电泳图经PDQuest 8.0分析,上样量为0.8、1.0、1.2 mg时检测出的蛋白质点分别为207±10、281±13和374±16个,分子质量基本分布在20~80 ku、等电点为4~9的区域内,随着上样量的增加,分子质量在20~80 ku的蛋白质点明显增多,但每个分子质量区间的蛋白质点所占的比率较为恒定。研究结果表明,采丙酮沉淀制备精浆蛋白结合合适的上样量能够建立绵羊精浆全蛋白质图谱,为进一步研究绵羊精浆蛋白质组学奠定基础。     

Prostasomes of Canine Seminal Plasma – Zinc‐Binding Ability and Effects on Motility Characteristics and Plasma Membrane Integrity of Spermatozoa

Prostasomes are small lipid membrane‐confined vesicles that are involved in various fertilization‐related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross‐bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc‐affinity chromatography on a Chelating Sepharose Fast Flow – Zn^{2 +} showed that from 93 to 100% of the prostasome proteins bind zinc ions (P⁺Zn). SDS‐PAGE revealed that canine P⁺Zn comprised four protein bands, with low molecular weights (10.2–12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold‐stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.     

Upregulation of CRISP-3 and kallikrein in stallion seminal plasma is associated with poor tolerance of cooled storage

For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expression in SP with either positive or negative effects on sperm survival during storage was studied using two-dimensional differential gel electrophoresis and liquid chromatography–mass spectrometry. Lower sperm chromatin integrity was associated with upregulation of the proteins kallikrein, CRISP-3 and HSP-1, while higher chromatin integrity was associated with upregulation of TIMP-2. In the sperm-rich fractions, kallikrein and CRISP-3 differed significantly between SP samples with differing effects on sperm chromatin integrity. In the sperm-poor fractions, TIMP-2 and HSP-1 differed significantly between the two SP groups. Differences in the seminal plasma proteome are associated with sperm longevity during cooled storage.     

Differences Between High‐ and Low‐Motility Buffalo Sperm Identified by Comparative Proteomics

This study was undertaken to investigate differences in protein expression between high‐ and low‐motility sperm of swamp buffalo. The research used two‐dimensional gel electrophoresis (2DE) coupled to matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (MALDI‐TOF/TOF‐MS) to analyse the different proteins. The results showed 18 different expression protein spots between high‐ and low‐motility buffalo sperm; eight of these proteins were up‐regulated in low‐motility sperm, five were down‐regulated, one deleted and four proteins specifically expressed. Finally, four proteins were successfully identified by MS as belonging to three unique proteins; they are outer dense fibre of sperm tails protein 2 (ODF2), ATP synthase subunit alpha (ATP5A1) and succinyl‐CoA synthetase subunit beta (SUCLG2). In summary, these results help to develop an understanding of the molecular mechanisms associated with low‐motility sperm and provide clues for finding molecular markers associated with sperm motility.     

Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.     

Induction of intradermal skin reactions in the bovine by fractionated proteins of Hypoderma lineatum

Protein species found in crude extracts of Hypoderma lineatum (Villers) 1st-instar gullet-stage larval homogenates were fractionated by chromatography and further identified by conventional polyacrylamide gel electrophoresis (PAGE) and SDS/PAGE techniques. Seven major fractions were resolved by ion exchange chromatography. Conventional PAGE of the crude antigen preparation revealed a minimum of 14 protein species, while SDS/PAGE revealed 3 major protein species. The ability to elicit cutaneous reactivity by isolated proteins was determined by the intradermal skin test. An immediate-type hypersensitivity reaction was elicited in the skin of vaccinated and previously infested animals by the components of the 4 protein fractions tested. Only one protein fraction, fraction 4, elicited an apparent delayed reaction at 48 h in vaccinated and previously infested animals. A non-specific background reaction was elicited in control animals by the fractionated proteins and was most notable between 1- and 4-h post-injection.