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.     

Biochemical observations on beagle dog semen.

Semen samples were collected at weekly intervals for six weeks from eight sexually mature beagles previously shown to produce normal ejaculates. Seminal plasma and sperm fractions were separated by centrifugation and the sodium, potassium, alanine and aspartate aminotransferases, acid and alkaline phosphatase concentrations in the two fractions determined. Regression analysis of the mean weekly values obtained from physical and biochemical examination of the ejaculates showed that sodium ion concentration was highest in seminal plasma. The highest levels of aminotransferases were found in sperm fractions. Those enzymes may be indices of abnormal or damaged spermatozoa. Acid and alkaline phosphatase activity was 100 times greater in seminal plasma than in sperm fractions. Phosphatase concentrations are likely to be dependent on prostate activity. Measurement of acid phosphatase in canine semen therefore may be a useful index of prostate function. The motility of the semen samples was independent of the potassium concentration in seminal plasma. However, there was some evidence of a correlation between sperm motility and the enzyme and sodium content of seminal plasma.     

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.     

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.     

A comparative study on the chemical composition of plasma from the cauda epididymidis, semen fractions, and whole semen in boars

A comparative study was carried out on the chemical composition of plasma from the cauda epididymidis, semen fractions, and whole semen of boars. A total of 22 boars were used in this study. The boars, which ranged in age from 8 to 14 months, were of Swedish Landrace and Swedish Yorkshire breed. All boars used presented a normal semen picture. A dummy sow and an artificial vagina were employed for semen collection. The semen was collected as whole semen and as semen fractions in 10 nil volumes. The contents of the cauda epididymidis was removed post mortem.The following parameters were investigated: sperm concentration, dry weight of spermatozoa and of seminal plasma, osmotic pressure, sodium, potassium, chloride, inorganic phosphorus, calcium, magnesium, total protein, GOT, GPT, and alkaline phosphatase in seminal plasma. Paper electrophoresis was carried out on seminal plasma. Tlxe results of the analysis are summarized in Tables 1–6.The sperm concentration was approximately 3.2 mill./mm3 in the cauda epididymidis, 1 mill./mm³ in the sperm-richest fraction (II) and 0.25 mill./mm3 in whole semen. The dry weight (expressed in per cent dry matter) of spermatozoa was highest in the cauda epididymidis (25.47 %), showing a tendency to decreasing in semen fractions I—IV and was lowest in whole semen (15.29 %). The per cent dry weight in plasma was higher in the cauda epididymidis (4.56 %) than in semen fraction I (2.20 %). In semen fractions I—IV the per cent dry weight rose from 2.20 (U to 4.51 % and reached the level of approximately 3.80 % in the sperm-free fractions V—VII. The osmotic pressure was significantly higher in the cauda epidi-dymal plasma than in the whole seminal plasma or the seminal plasma fractions. The same phenomenon was observed in a boar where the cauda epididymal content was collected in vivo from a patent established fistula. There appears to be a connection between the per cent dry weight of spermatozoa and the osmotic pressure, which means that the per cent dry weight of the cauda epididymal spermatozoa decreases when mixed with the accessory gland secretions, which have a lower osmotic pressure. The fall in per cent dry weights is thought to be caused by an intake of water.The amount of sodium, chloride and magnesium was higher in ejaculated seminal plasma than in cauda epididymal plasma. The reverse was true for inorganic phosphorus and potassium. Moreover the sperm-free fractions contained more sodium, chlorides and magnesium than the sperm-containing fractions, while the concentration of potassium and inorganic phosphorus was comparatively higher in the sperm-containing fractions. A connection is apparent between sperm concentration and the potassium, inorganic phosphorus and magnesium levels. Statistical analysis of the values of chloride and magnesium revealed significant differences between individual boars for most of the semen fractions.The concentration of plasma proteins in the cauda epididymidis was approximately the same as in whole semen and in the semen fractions except for fraction I, which contained a relatively low concentration. As regards total protein there were significant differences between individual boars in most of the semen fractions as well.The paper electrophoretic pattern of epididymal plasma was different from that of semen plasma. Thus there were three or four distinct components in the cauda epididymidis numbered 1, 2, 3, and 4, and three distinct components in whole seminal plasma numbered 3, 4, and 5, while the sperm-richest semen fractions contained four components (2, 3, 4, and 5) and the others three components, namely 3, 4, and 5.The level of GOT was high in the cautlu cpiflidymill contents (99.1 i. u./ml) compared with that for whole seminal plasma (99.1 i.u/ml). In semen fractions there was a clear positive correlation between the level of GOT and the sperm concentration. The GPT concentration wis as a whole low and. in contrast to GOT. somewhat higher in the sperm-free fractions than in the sperm-containing fractions. The concentration of alkaline phosphatase was very high in cauda epididymal plasma (31,463 i. u./ml) as well as in the sperm-rich fractions (e.g. 7,096 i. u./ml in fraction II). Preliminary investigation has moreover revealed a very low alkaline phosphatase concentration in seminal plasma of vasectomized boars, which condition suggests thai the main origin for alkaline phosphatase in boars is the testis and epididymis.     

Level of Glycolyzable Substrates in Stallion Semen: Effect of Ejaculation Frequency on Sperm Survival after Cool Storage during the Nonbreeding Season

Although seminal characteristics are routinely evaluated in the stallion, the effect of collection schedules and seminal plasma on semen quality during cool storage is not well understood, specifically during the nonbreeding season when cryopreservation of stallion semen is preferentially performed. To address these issues, behavioral characteristics, seminal parameters, and biochemical markers (d-glucose, fructose, and citric acid) were measured in ejaculates (n = 60) obtained during the nonbreeding season. Semen was collected from three stallions, twice a day (1-hour gap between successive collections) and two times in a week. Differences between the means of first and second ejaculates were observed for erection latency (P < .001), which was higher in second ejaculates and determined a higher total breeding time (P < .1). Variations introduced by the stallion were significant for number of mounts (P < .05, in first ejaculates), erection latency (P < .001, in second ejaculates), and total breeding time (P < .001, in second ejaculates). First and second ejaculates differed significantly for sperm motility and sperm concentration (P < .001, higher in first ejaculates) and pH (P < .01, higher in second ejaculates). d-glucose was present in seminal plasma at a much higher concentration than fructose (P < .001) in both ejaculates. There were no significant stallion-associated differences in sperm vitality and pH in the first and second ejaculates as well as in sperm concentration for the second ejaculates. The effect of seminal plasma on equine sperm survival during cooled storage was analyzed by monitoring sperm motility and cell morphology after conservation in an extender medium with and without seminal plasma. When statistically considering seminal plasma and conservation time simultaneously, it was found that these variables affected acrosomal status and midpiece morphology.     

CASA Assessment of Kinematic Parameters of Ram Spermatozoa and their Relationship to Migration Efficiency in Ruminant Cervical Mucus

Sperm motility is an indicator of male fertility because of its importance for sperm migration through the female genital tract and for gamete interaction at fertilization. This study analyses the relationship between computer assisted semen analysis (CASA) motility patterns and sperm migration of rams in ruminant cervical mucus. In experiment 1, spermatozoa extended with sperm analysis medium (SAM) and seminal plasma were compared in terms of motility. In experiment 2, 56 semen samples were collected either with artificial vagina (AV) or electroejaculator to be compared in terms of motility performance. In experiment 3, 104 ejaculates collected by AV from 26 males were analysed via the CASA system to characterize their motility patterns. In experiment 4, ejaculates from pairs of rams (20 rams in total) were simultaneously assessed for mucus migration (ovine, caprine, bovine) and motility patterns to evaluate the correlations between both parameters. Semen collected by AV and extended in SAM allows the most reliable assessment for sperm motility. Ram spermatozoa move fast and follow a linear trajectory compared with other ruminants. Continuous line velocity (VCL) and average path velocity (VAP) are the only sperm kinematic parameters that presented significant positive correlations with the ability to migrate in sheep cervical mucus (p < 0.05). Continuous line velocity, VAP, straight line velocity and linearity are highly significantly related with migration efficiency in goat cervical mucus (p < 0.01) and only lateral head displacement is negatively related to sperm migration in bovine cervical mucus (p < 0.05). These results suggest that specific kinematic parameters confer the ability of spermatozoa to colonize and migrate through epithelial mucus with different rheological properties.     

Concentration,Activity and Biochemical Characterization of Myeloperoxidase in Fresh and Post‐Thaw Equine Semen and their Implication on Freezability

Myeloperoxidase (MPO) is a pro‐oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm‐rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme‐linked immunosorbent assay and specific immunological extraction followed by enzymatic detection. MPO subunits present in semen were characterized by Western blot. Purified active MPO was added in saline solution and freezing extender to control its activity during freezing procedure. Differences between medians were determined using Kruskal–Wallis test, and correlations were determined using Spearman's test for nonparametric data. Active MPO concentration was low in seminal plasma and thawed semen, but high in pellet (p = 0.0058), as the opposite relation was observed for total MPO concentration (p < 0.0001). In seminal plasma and post‐thaw semen, inactive 86‐kDa MPO precursor was mainly observed. Purified MPO activity was decreased in the extender (p = 0.0286). MPO activity in pellet was highly correlated with thawed progressive motility (r = ?0.5576, p = 0.0086). Inactive MPO precursor and unknown low molecular weight inactive MPO precursor subunits explain low MPO activity in semen. Major MPO activity was observed in pellet, and post‐thaw loss of activity is partially explained by MPO inactivation in extender. Thawed semen motility was negatively correlated with MPO activity in pellet, becoming a potential freezability predictor.     

Effect of seminal plasma concentration and various extenders on postthaw motility and glass wool-Sephadex filtration of cryopreserved stallion semen

OBJECTIVE: To compare the effect of semen extender and seminal plasma on postthaw motility and filtration through a glass wool-Sephadex (GWS) filter for frozen stallion semen. SAMPLE POPULATION: 7 stallions from which we collected > or = 3 ejaculates/stallion. PROCEDURES: 4 experiments were conducted to evaluate postthaw quality of frozen stallion semen. Kenney extender was compared with glucose-EDTA extender by use of various dilution rates that resulted in differing concentrations of seminal plasma. Stallions known to produce semen with poor postthaw quality were used to investigate whether a particular extender or dilution rate could improve ability of such semen to survive freeze-thaw procedures. RESULTS: Use of Kenney extender as the centrifugation extender significantly improved postthaw motility and GWS filtration, compared with glucose-EDTA. Extending semen at a dilution of 1:3 was significantly better than 1:1 for both motility and GWS filtration. In addition, including seminal plasma at a concentration of 5% in the cryopreserved semen resulted in significantly higher yield of spermatozoa after GWS filtration, compared with complete removal of SP or use of seminal plasma at 25%. Lastly, semen with poor postthaw quality had significantly improved postthaw quality in regard to motility and GWS filtration when semen was frozen with seminal plasma at a concentration of 5%, compared with semen frozen with seminal plasma at a concentration of 25%. CONCLUSIONS AND CLINICAL RELEVANCE: Use of Kenney extender at a high dilution (> or = 1:3) immediately after collection of semen can improve postthaw quality of frozen stallion semen.     

Effect of electroejaculation on progesterone and cortisol excretion in bovine semen

Artificial vagina (AV) and electroejaculation (EE) are the 2 methods used to obtain semen from bulls. The purpose in the present study was to evaluate these 2 methods of collection when 2 markers, cortisol and progesterone, were injected IV. During period 1 (control measurement), semen was obtained by EE at 0, 20, 60, 120, and 180 minutes. In period 2, bulls were injected (3 days later) with a mixture of cortisol (113 mg) and progesterone (100 mg), and then samples were obtained by EE. In period 3, cortisol and progesterone were injected (3 weeks later), and samples were obtained by AV. Seminal plasma concentrations of cortisol and progesterone were maximal at 20 minutes in EE and AV collections. Seminal plasma concentrations of progesterone and cortisol were roughly 50% less in EE than in AV collection. However, the total excretion of progesterone and cortisol per collection was similar in both techniques. Excretion of cortisol was 14 to 33 times greater than that of progesterone. It was concluded that concentrations of markers in the EE were significantly less than those in AV collection. For this reason, total excretion and concentration of marker in semen should be accounted for when conducting excretion studies.     

Relationship of cysteine‐rich secretory protein‐3 gene and protein with semen quality in stallions

Cysteine‐rich secretory protein‐3 (CRISP‐3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP‐3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP‐3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP‐3 was identified and haplotypes were derived. Semen quality was assessed through the total motility (MOT), sperm vitality (SV), normal morphology (NM), functional integrity of membrane (MI) and a seminal quality index (SQi). CRISP‐3 protein content of seminal plasma (SP) was determined by ELISA. The effect of the genotype, the haplotype and the concentration of the CRISP‐3 protein on the seminal quality were evaluated through generalized linear models and linear regression analyses. Homozygous genotypes for SNP1, SNP2 and SNP3 and the heterozygous genotype for SNP4 showed a positive effect on seminal quality. Different haplotypes with positive effect on MOT, SV, NM, MI and SQi were identified. The allelic substitution analysis resulted in positive regression coefficients for MOT (SNP2) and MI (SNP2 and SNP3). A high level of CRISP‐3 resulted in a higher MOT and SQi. It is concluded that the quality of stallion semen is influenced by the genotype of CRISP‐3 and the concentration of CRISP‐3 protein in SP.     

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.     

不同季节肉用种公牛精液品质与生理常值、血清及精清生化指标相关关系研究

以利木赞和夏洛来种公牛为试验牛，研究不同季节对其精液品质、冻精产量、生理常值、血清及精清生化指标的影响。结果表明：（1）与其它季节相比，夏季肉用种公牛的原精活力、冻精活力、活精于百分数、顶体完整率均显著降低，而精于畸形率显著升高（P〈0．05）。精子密度以秋季最低。（2）冻精产量夏季处于最低水平，平均每月仅为7388支。（3）夏季肉用种公牛的血清LH、睾酮、T3、皮质醇的含量分别为48．12、4．15、1．11、4．89ng／L，显著低于春、秋、冬季（P〈0．01）。（4）夏季肉用种公牛血清钠、钾、钙、镁均处于最低水平，显著低于其它季节（P〈0．01）。（5）夏季肉用种公牛精清睾酮水平明显下降，显著低于春季、秋季和冬季。（6）精清钠、钾、钙夏季含量最低，分别为83．12mmol／L、18，77mmol／L和4．63mmol／L；精清镁含量由春季至秋季呈显著下降趋势；精清磷含量夏季最高，为4．65mmol／L，显著高于春季、秋季和冬季。（7）肉用种公牛精清ALP夏季含量为1784．3U／L，显著高于春、秋两季，但低于冬季。（8）精清睾酮与精液品质各指标存在显著相关；血清生化指标与精清生化指标存在显著相关；直肠温度、呼吸频率与精液品质及血清生化指标存在显著相关性，与精清生化指标相关性不大。     

Addition of Seminal Plasma to Post‐thawing Equine Semen: What is the Effect on Sperm Cell Viability?

Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autologous seminal plasma after semen thawing (ASP). The addition of 20% of seminal plasma led to an increase in the cell population that simultaneously show plasma and acrosomal membrane integrity (p < 0.05). The addition of seminal plasma did not alter the total motility, the amount of cells with mitochondrial membrane potential or the sperm velocities (average path velocity, straight-line velocity and curvilinear velocity). However, the beat/cross-frequency, straightness and linearity were reduced in ASP and HSP groups (p < 0.05). Unexpectedly, the addition of homologous seminal plasma reduced the proportion of cells with progressive motility (p < 0.05) and the addition of autologous seminal plasma reduced the amplitude of the lateral head displacement (p < 0.05). Based on the increase in the cell populations that had the plasma and acrosomal membrane integrity simultaneously identified in this study, we proposed that the addition of seminal plasma (autologous or homologous) into post-thawed semen before insemination could increase semen fertility.     

Methods of collection,extender type,and freezability of semen collected from creole bulls raised in the tropical highlands of Ecuador

This study was conducted to determine the best combination between two collection method and two extenders in the cryopreservation of semen from creole bulls adapted to highlands of the Ecuadorian Andes. Sixty ejaculates from three adult Creole bulls were evaluated after collection by artificial vagina (AV) and electroejaculation (EE). Semen samples were split into two aliquots and diluted with a soy lecithin extender (Andromed®; A) or an egg yolk-containing extender (Triladyl®; T) and packed in straws of 0.25 ml with 20 × 10⁶ sperms. Optical microscopy and computer-assisted semen analysis system (CASA) were used to evaluate semen quality characteristics. The effects of collection methods and extender type as well as its interaction were evaluated by a factorial ANOVA and Bonferroni’s test. Semen samples collected with EE and frozen with T (EE-T) and A (EE-A) had greater proportion of spermatozoa with optical assessed individual progressive motility (IPM), plasmatic membrane intact (HosT), and lower tail abnormalities than those obtained with AV and frozen with the same extenders (AV-T and AV-A); however, differences were significant only between EE-A and AV-T. CASA assessment indicated that the total mobility (TM) was greater (P < 0.05) in semen samples diluted with T, although these samples had a greater proportion (P < 0.05) of sperms with local motility (LM) and fewer immobile sperms (IS), than those extended with A. Generally, semen samples obtained with EE or AV and diluted with T seems to be the best option to ciopreserve gametes of Creole bulls raised in highlands of Ecuadorian Andes.

     

高温对荷斯坦种公牛精液品质及精清生化指标的影响研究

以荷斯坦种公牛为试验牛研究了高温对其精液品质和精清生化指标的影响。结果表明:(1)高温可造成种公牛精液品质显著下降。使原精活力、精子密度、活精子百分数和顶体完整率分别比春季下降3·13%(P<0·05)、34·8%(P<0·01)、15·0%(P<0·01)和17·8%(P<0·01),精子畸形率比春季上升25·5%(P<0·01)。(2)夏季荷斯坦种公牛精清睾酮含量仅为4·31pg/mL,极显著低于非高温季节(P<0·01)。(3)高温环境使得荷斯坦种公牛精清钾、钙、镁处于最低水平(P<0·01),精清碱性磷酸酶(ALP)、酸性磷酸酶(ACP)处于最高水平(P<0·01),使精清钠由春季至夏季呈显著下降趋势。     

Alkaline and Acid Phosphatase Activity, pH and Osmotic Pressure of Boar Semen

Alkaline phosphatase activity was recorded in forty ejaculates of the sperm rich fraction of boar semen as 9,790 ± 5,250 Klein-Babson-Read units per 100 ml. of seminal plasma. Acid phosphatase activity in the same ejaculates was 681 ± 304 Babson-Read units per 100 ml. of seminal plasma. No alkaline phosphatase activity was detected in the seminal plasma of vasectomized boars.

The pH of the sperm rich fractions was 7.69 ± 0.33 and the osmotic pressure was 313.56 ± 7.98 milliosmols.

     

Estimation of endogenous levels of osteopontin,total antioxidant capacity and malondialdehyde in seminal plasma: Application for fertility assessment in buffalo (Bubalus bubalis) bulls

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high‐fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high‐fertile bulls was found to be higher (p < .05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p < .05) in high‐fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r = .678, p = 0.013) and TAC (r = .648, p = .042) were found to be positively correlated with bull fertility and the level of MDA (r = ?.718, p = .019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds.     

Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

The effect of collection method and housing system on semen production and fertility of Alabio drakes

Three methods of semen collection from Alabio drakes were compared: the use of an artificial vagina (AV), electro ejaculation (EE) and manual massage (MM). For the latter two methods, drakes were housed in individual cages or in groups in floor pens, while for the AV method all drakes were kept in individual cages. Training success rates of drakes for the three collection methods were similar. Drakes housed in cages produced significantly more semen with a higher sperm concentration, which maintained fertility longer than semen from floor-penned drakes. The average semen volume per ejaculate, sperm concentration and total number of sperm per ejaculate were highest for the AV method while the other two methods did not differ. The average duration of fertility was significantly longer for the AV method (8.8 d) than the EE (6.5 d) or MM methods (5.1 d). The proportion of fertile eggs for 7 d following the insemination of 2 X 10(8) spermatozoa was also highest for the AV method. Although greater skill was required from the operator, the AV method is recommended for general use because of its significantly higher semen yield and longer duration of fertility.