A new antibiotic combination for frozen bovine semen, 3. Evaluation of fertility

Field fertility (nonreturn rate) studies were performed independently by three artificial insemination organizations to evaluate bovine semen processed for freezing using the antibiotics gentamicin, tylosin and Linco-Spectin at concentrations of 500 ug, 100 ug, and 300/600 ug, respectively, per milliliter of neat semen and per milliliter of nonglycerol portion of the extender. The antibiotic combination including penicillin, dihydrostreptomycin, polymyxin B sulfate, with/without Linco-Spectin (500 units/ml, 2000 ug/ml, 1000 units/ml and 300/600 ug/ml, respectively) was used as the control treatment. Results indicated no significant effect on seminal quality as measured by field fertility under the conditions of these experiments using heated whole milk or egg yolk-sodium citrate seminal extenders. Use of the new antibiotic combination has been adopted by Certified Semen Services.     

A new antibiotic combination for frozen bovine semen 1. Control of mycoplasmas, ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus somnus

Systematic evaluations of new combinations of antibiotics for the control of bovine mycoplasmas, ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus somnus in a bovine frozen semen process were made. These organisms were standardized to 10(5) to 10(6) colony forming unit (CFU) and inoculated into each ml of raw semen. Antibiotics in a final volume of 0.02 ml were added to each ml of the raw semen and were contained at the same concentration in the nonglycerol portion of the extenders (whole milk, 20% egg yolk citrate, 20% egg yolk tris, Plus-X, and 28% egg yolk tris). The combination of gentamicin (500 ug/ml) tylosin (100 ug/ml) and Linco-Spectin (300/600 ug/ml) was more effective for the control of mycoplasmas and ureaplasmas and equally effective for the control of C. fetus subsp. venerealis and Haemophilus somnus than the standard combination of penicillin, dihydrostreptomycin and polymyxin B sulfate.     

Effect of antibiotics on motion characteristics of cooled stallion spermatozoa

The control of bacteria in semen of stallions has been most effective with the use of seminal extenders containing suitable concentrations of antibiotics. However, the detrimental effect of antibiotics on sperm motility may be greater in stored, cooled semen due to the prolonged exposure to the antibiotic. Therefore, a study was conducted to determine the effect of various antibiotics on sperm motion characteristics following short term exposure and during cooled storage of semen. Reagent grade amikacin sulfate, ticarcillin disodium, gentamicin sulfate and polymixin B sulfate were added to a nonfat, dried, skim milk - glucose seminal extender at concentrations of 1000 or 2000 mug or IU/ml. Aliquots of raw semen were diluted with extender-antibiotic combinations to a concentration of 25 x 10(6) spermatozoa/ml. An aliquot was also diluted with extender without antibiotic. Aliquots were incubated at 23 degrees C for 1 h. In addition, portions of the aliquots were cooled from 23 to 5 degrees C and stored for 48 h. During 1 h of incubation of extended semen at 23 degrees C, there was a significant (P<0.05) reduction in the percentage of progressively motile spermatozoa for samples containing gentamicin sulfate. After 24 h of storage at 5 degrees C, 2000 mug/ml of gentamicin and levels equal to and greater than 1000 IU/ml of polymixin B in seminal extender resulted in significant (P<0.05) reductions in the percentages of motile and progressively motile spermatozoa. After 48 h of cooled storage, a level of 1000 mug/ml of gentamicin sulfate. resulted in significant (P<0.05) reductions in the percentages of motile and progressively motile spermatozoa. Levels equal to or greater than 1000 IU/ml of polymixin B sulfate also resulted in a significant (P<0.05) reduction in mean curvilinear velocity. Levels up to 2000 mug/ml of amikacin sulfate and ticarcillin disodium had no significant effect on sperm motion characteristics during short-term incubation at 23 degrees C or storage for 24 h at 5 degrees C. Overall, the addition of antibiotics to extender did not significantly (P>0.05) improve motion characteristics of spermatozoa over control samples. However, levels of gentamicin sulfate greater than 1000 mug/ml and of polymixin B sulfate equal to or greater than 1000 IU/ml should be avoided in seminal extenders used for cooled semen.     

Failure of antibiotics gentamycin, tylosin, lincomycin and spectinomycin to eliminate Mycoplasma bovis in artificially infected frozen bovine semen

To study the effect of antibiotics upon Mycoplasma bovis in fresh bovine semen just before freezing, specimens of bovine semen were artificially infected with 1 of 9 different strains of M. bovis. Inocula of each strain were prepared to contain 10(5) to 10(6)/mL colony-forming units of M. bovis at 3 different stages of the growth phase. The infected semen was diluted with a Tris extender by a 3-step procedure using an antibiotic mixture of gentamicin, tylosin, lincomycin and spectinomycin (GTLS). This semen-antibiotic mixture was placed into French straws that were stored at -196 degrees C. The control semen specimens contained no antibiotics Mycoplasmas were counted after 8 d of storage in 3 decimal dilutions of the frozen semen. No evident effect was noticed upon the 9 tested strains of mycoplasmas in the semen frozen with the antibiotics, compared with that of the untreated control samples. It was further shown that this lack of effect was irrespective of the stage of the growth phase of the mycoplasmas. It was concluded that the antibiotic mixture (GTLS) in semen specimens is not capable of total elimination of mycoplasmas in frozen bovine semen.     

Thermotemporal dynamics of contaminant bacteria and antimicrobials in extended porcine semen

Bacterial contamination of extended porcine semen has been associated with deleterious effects on both semen quality and sow fertility. Retrospective, prospective and in vitro studies were performed to delineate the prevalence and behavior of certain bacterial contaminants in extended semen, and antimicrobial pharmacodynamics in various semen diluents. Retrospective review of extended semen samples submitted from North American boar studs for microbiological screening at the University of Pennsylvania Reference Andrology Laboratory in 2005 and 2006 yielded bacteriospermia prevalence rates of 17% (144/832) and 26% (256/984), respectively. In a prospective study of regional boar studs, of 91 extended semen samples tested over 1-y, 29% were positive for bacteriospermia. Retrospective and prospective studies both showed that the preponderance of contaminant positive samples occurred during the fall months (P<0.05). To better understand behavior of select contaminant bacteria, generation intervals were determined for Serratia marcescens (SM) and Achromobacter xylosoxidans (AX) at 16, 22 and 37 degrees C. Generation times were temperature-dependent, with intervals decreasing two- to four-fold as incubation temperature increased. Growth patterns for SM, AX and Burkholderia cepacia were evaluated in various semen diluents. The different diluents exhibited constant or episodic patterns of growth within and among bacteria throughout the 5-d test period. Kill-time kinetics at 37 degrees C of several genera of bacteria in four semen diluents containing amoxicillin, gentamicin, tylosin, and lincomycin/spectinomycin (single drug or combination) ranged from 75 to over 360min, and was highly dependent (P<0.05) upon both type of bacteria and semen diluent.     

Tissue kallikrein activity in seminal plasma of bovine ejaculates with different quality

Tissue kallikrein activity was monitored in seminal plasma from 3 groups of bovine ejaculates: those with normal total sperm motility (78.43%), with reduced sperm motility (49.29%), and with reduced sperm count (0.68 x 10(9) cells/ml). The tissue kallikrein activity was measured spectrophotometrically by using the specific chromogenic substrate S-2266. It was found that the semen samples with normal sperm motility manifested 1.083 microkat/L, on an average, or 29% higher than the activity recorded in ejaculates with reduced sperm motility (P < 0.05). After storage of a group of ejaculates of normal quality for 5 h at room temperature, sperm motility dropped by approximately 80%, expressed as a percentage of the initial motility, while the tissue kallikrein activity in the respective seminal samples decreased by 23%. No significant differences were found in kallikrein activity between ejaculates with normal and reduced sperm counts. It is concluded that a relationship exists between the level of tissue kallikrein activity in the seminal plasma of bovine ejaculates and sperm motility.     

The concentration of pepsinogen C in human semen and the physiological activation of zymogen in the vagina

The relationship between male infertility and the pepsinogen C content in semen has been investigated. The activation of the seminal pepsinogen C in the vagina has been studied under physiological conditions. Samples of semen from 48 vasectomized males and from 46 males of infertile couples were analyzed for pepsinogen C by radioimmunoassay. No correlation was found between the level of pepsinogen C and seminal characteristics, including sperm concentration, motility, and morphologic features. The mean concentration of pepsinogen C was 42.2 micrograms/ml; the first, second, and third quartile were 18.4, 29.6, and 57.6 micrograms/ml, respectively. No significant difference in the level of pepsinogen C was observed between semen of normal quality, semen of reduced quality, and semen with aspermia. Activation of pepsinogen C occurred within 3 h when semen was incubated at pH below 5.0 at 37 degrees C. Intravaginal activation was investigated in six experiments in which semen from two males was instilled in three females. In four experiments with two couples, post-coital activation was investigated. Pepsin C activity in vaginal fluid was detected an average of 3 h (range 2-5 h) and 5 h (4-7 h) after instillation or ejaculation, respectively. Vaginal pH had then been below 4.5 for approximately 1 h. Pepsin C activity was present in the vagina for more than 24 h thereafter. It is most likely that seminal pepsin C is without influence on the fertilizing spermatozoon. However, pepsin C may exert a local effect in the vagina by degrading seminal proteins, thus preventing an immunogenic response in females.     

Isolation of motile spermatozoa by density gradient centrifugation in Percoll®

A procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.     

Morphological change of the acrosome on motile bovine spermatozoa due to storage at 4 degrees C

Swelling of the apical ridge and anterior acrosome of motile bovine spermatozoa was observed during in-vitro storage using differential interference-contrast optics. This morphological alteration is different from that described as the false acrosome reaction on immotile spermatozoa, apparent in ageing semen samples and which has been associated with cell death. In this study, transmission electron microscopy revealed that the apical ridge acrosomal matrix was extended into complex folds and/or projections. Acrosomal and plasma membrane integrity was retained. Storing spermatozoa (1500 X 10(6)/ml) in seminal plasma at 4 degrees C for 1 day was most conducive to the swelling of the apical ridge. Replacing seminal plasma with egg yolk-citrate inhibited swelling. However, incubating semen at 37 degrees C in egg yolk-Tris-fructose extender (25 X 10(6) spermatozoa/ml) after storage in egg yolk-citrate at 4 degrees C for greater than or equal to 3 days restored the swelling characteristic.     

Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 degrees C

Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0 ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (SigmaDiagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36 +/- 0.02 Sigma units/ml, (mean + S.E.M.; n = 16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300 x g for 15 min) and resuspended in NFDSM to a final concentration of 25 x 10(6) spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100 degrees C for 5 min) while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5 degrees C, and stored at 5 degrees C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72 h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100 units/ml but not 10 units/ml of pPL decreased (P < 0.01) total and progressive motility of stored sperm. Heat-inactivated pPL (Treatment 5) did not significantly decrease motility of spermatozoa during storage. Because the lipase activity assayed (Sigma units) and the lipase activity added to cooled semen (pPL units) were not equivalent, pPL was assayed in the Sigma Diagnostic Lipase assay. The relationship between Sigma Units (Y) and pPL units (X) appeared to be a log-linear relationship with log(Y) = -0.912 + 0.007X; R2 = 0.90. Mean lipase activity assayed in stallion seminal plasma was equivalent to approximately 64 pPL units/ml. These data suggest that endogenous lipase activity in stallion seminal plasma may be a factor in the adverse effects of seminal plasma on cooled spermatozoa in some stallions.     

Effects of oxytocin on semen release response in African catfish (Clarias gariepinus)

In silurid fishes, semen collection is practically impossible, even after hormonal stimulation. Instead, males are killed and testes macerated to obtain sperm. To understand the endocrine control of semen release in catfishes, we investigated the role of smooth muscle contractors in semen release and semen quality of African catfish (Clarias gariepinus). In in vitro experiments, testis slices were incubated with oxytocin (1 and 10 IU), isotocin (2 and 20 ug), vasopressin (0.2 and 2 ug), epinephrine (1 and 10 ug), PGF2alpha (1 and 10 ug), purified Clarias LH (300 ng) and partly purified Clarias pituitary extract (containing 300 ng LH). Only oxytocin increased sperm concentration of the medium (assessed by optical density measurements) compared to control incubations. Oxytocin was then tested in vivo in two groups of fish: normal males, and males that had been treated with 17alpha-methyltestosterone during larval stages to inhibit seminal vesicle development (MT males). Both groups of fish received two doses of carp pituitary suspension (8 and 10 mg/kg, respectively i.m.) with or without subsequent oxytocin treatment (5 IU/kg i.v.; cPS-OT treatment and cPS treatment, respectively). There was no effect of oxytocin on the number of strippable males. Of cPS and cPS-OT treated fish, 87% MT males and 60% normal males were strippable. The stripped semen volume was low in both groups but MT males produced higher (P < 0.001) hatching rates (63.1%) than did normal males (2.1%).     

Protein, cholesterol, acid phosphatase and aspartate aminotransaminase in the seminal plasma of turkeys (Meleagris gallopavo) producing normal white or abnormal yellow semen

Turkeys which produce yellow semen have abnormal ductuli efferentes' epithelial morphology, with blebbing of cytoplasmic material into the ductal lumen. This could possibly increase the activity or concentration of seminal plasma components. In the present study, seminal plasma from 270 Large White breeder turkeys was evaluated for protein and cholesterol concentrations and the activities of acid phosphatase and asparate aminotransaminase. In a separate experiment, protein concentrations of turkey seminal plasma were estimated by biuret or Bradford methods. Bradford estimates were 46.6% less than those obtained with the biuret assay, using bovine serum albumin as the standard. Estimates of seminal plasma protein concentration in the main study were obtained using the Bradford method, and should be adjusted accordingly when compared with other studies using the biuret technique. Abnormal yellow seminal plasma, compared to normal white seminal plasma, had elevated levels of total protein and cholesterol and increased activities of acid phosphatase and aspartate aminotransaminase. Overall means were: 14.3 mg/ml, 38.9 mg/dl, 232.6 IU/ml, 81.0 IU/ml, respectively. Correlation coefficients for cholesterol concentration, acid phosphatase and aminotransaminase activity with protein concentration were +0.65, 0.70 and 0.50 (P less than 0.0001), respectively. Specific activities of both enzymes showed a significant reduction as seminal plasma protein increased, indicating a disproportionate increase in proteins other than these enzymes in yellow seminal plasma.     

Treatment of Gram-Negative Bacillary Meningitis With Intrathecal Gentamicin

In order to evaluate the effect of intrathecal gentamicin on gram-negative bacillary meningitis, twenty-eight patients were treated with intralumbar or intraventricular gentamicin in combination with systemic gentamicin and with other antibiotics. Sterile cerebrospinal fluid was achieved in 21/22 (95%) episodes of documented gram-negative bacillary meningitis in patients who received more than one day of therapy. Seventy-seven percent of these patients survived their infection. The mean cerebrospinal fluid gentamicin level measured 24 hours after intrathecal administration was 5.9 ug/ml following intralumbar administration and 11.1 ug/ml following intraventricular administration. Toxic side effects due to intrathecal administration of gentamicin were not noted. These findings suggest that both intralumbar and intraventricular administration of gentamicin are safe and efficacious in the treatment of gram-negative bacillary meningitis.     

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.     

Effects of bull and heparin and sperm concentrations on in vitro fertilization of buffalo (Bubalus bubalis ) oocytes matured in vitro

A study was undertaken to assess the ability of spermatozoa from 6 buffalo bulls, at different levels of heparin and sperm concentrations, to achieve an acceptable level of fertilization in vitro. Frozen-thawed spermatozoa, 3 dosages of heparin (0, 10 and 100 ug/ml) in the presence and absence of penicillamine, hypotaurine and epinephrine (PHE), and 4 sperm concentrations (1 x 10(6), 2 x 10(6), 3 x 10(6) and 4 x 10(6) /ml) were studied using 3202 buffalo oocytes. The mean proportions of fertilized oocytes in the group treated with 10 ug/ml of heparin were significantly higher (P<0.05) with the semen of Bulls A, B and C (44.7 to 64.3%) than in medium devoid of heparin. An increase in the dosage of heparin from 10 ug/ml to 100 ug/ml reduced the overall fertilization rate. However, optimal fertilization (30.9%) at 100 ug/ml heparin was observed for semen from Bull D. Bulls E and F yielded the lowest fertilization rate (9.6 and 14.2%, respectively) at the above mentioned heparin dosage. Analysis of sperm density revealed that a concentration of 2 x 10(6) spermatozoa yielded optimal fertilization rates in vitro. Higher sperm concentrations (3 x 10(6) or 4 x 10(6)) resulted in higher oocyte penetration rates but gave rise to polyspermy.     

Endogenous prostaglandin F2α concentrations in bovine whole semen, seminal plasma, and extended semen

A series of experiments were conducted to quantify PGF_2α in bovine semen, seminal plasma, and extended semen, and to determine if PGF_2α was synthesized or released during extension of bovine semen. Concentrations of PGF_2α were measured in paired samples of whole and extended semen from beef and dairy bulls. Concentrations of PGF_2α did not differ between beef and dairy (mean ± SEM, 273.8 ± 42.8 vs. 210.3 ± 18.5 pg/mL, respectively; P = 0.12), but tended (P = 0.08) to be greater for whole compared with extended semen (255.5 ± 29.8 vs. 194.5 ± 17.0 pg/mL). Whole semen was extended at eight dilution rates (regardless of initial sperm concentration), using a diluent consisting of two fractions. Samples collected after each dilution step resulted in four subsamples. Concentrations of PGF_2α in subsamples decreased (P < 0.001) at higher dilution rates and later steps of extension. Subsequently, whole semen and seminal plasma were extended at three dilution rates. Initial PGF_2α concentration was greater (P < 0.001) for whole semen compared with seminal plasma. During extension, PGF_2α synthesis or release resulted in less disparity, but the amount synthesized or released was greater (P = 0.03) for semen compared with seminal plasma. We concluded that synthesis or release of PGF_2α during extension resulted in concentrations similar to whole semen.     

Detection of bovine papillomavirus type 2 DNA in commercial frozen semen of bulls (Bos taurus)

Papillomaviruses are found in epithelial lesions and are linked to different carcinogenic processes in humans and other animals. Although bovine papillomavirus (BPV) has been characterized as epitheliotropic, the presence of viral DNA has been detected in other sample types, including fresh semen. The aim of this study was to evaluate the presence of BPV DNA in spermatozoa and seminal plasma samples of commercial frozen semen taken from bulls (Bos taurus) and its effects on semen function. PCR assays were conducted with specific primers to detect BPV types 1-6 in 40 semen samples of dairy Gir bulls. The semen quality was assessed by the use of parameters such as motility, vigor, acrosomal integrity and DNA integrity. BPV-2 DNA was detected in all of the sperm cell samples and all the seminal samples; however BPV-1, 3, 4, 5 and 6 could not be detected. The presence of BPV DNA was apparently not a cause of reduced sperm function. This is the first record of BPV-2 DNA the commercial frozen semen taken from dairy Gir cattle by several companies that provide semen. Further studies are needed to assess the viability of the virus and the extent to which it can be spread through semen.     

Fertility test of frozen boar semen.

The fertility results of two experiments are presented. In experiment 1, the semen was frozen in tris-fructose-EDTA or BF3 diluents at 0-25 X 10(9)/ml sperm concentration and extended after thawing with either seminal plasma (SP) or the freezing medium (FM) containing no cryoprotective agent. In the second experiment the semen was glycerolated by two methods, frozen at 1-0 X 10(9)/ml sperm concentration, and extended wtih FM before insemination. Fertility after double insemination within one oestrus with semen frozen in tris-fructose-EDTA or BF3 diluents varied depending on the medium used for extension of thawed semen. The farrowing rates for semen frozen in the former diluent with FM and SP post-thawing media were 4/8 and 1/8 respectively, and for semen frozen BF3 diluent with FM and SP post-thawing extenders 1/8 and 5/8. The mean farrowing for the 32 animals inseminasted was 34-4%. Pregnancies for semen frozen in tris-fructose-EDTA and glycerolated at 30 or 5 degrees C were 5/12 and 4/12 respectively, and for single and double inseminations 6/12 and 3/12 respectively. Of 24 animals inseminated 37-5% farrowed.     

Effects of vitamin E on fowl semen storage at 4 degrees C

Vitamin E was assayed for either in chicken spermatozoa or seminal plasma. Effects of vitamin E on the motility and fertilizing ability of chicken semen stored for 24 hours at 4 degrees C were also studied. A mean of 0.25 mug vitamin E 10 (9) cells was found in spermatozoa and 0.074 mug in seminal plasma. When the medium for in vitro storage of semen was supplemented with vitamin E the motility of spermatozoa was not affected. However, vitamin E improved the fertilizing ability of semen stored for 24 hours at 4 degrees C, especially at the dose of 8 mug/ml of semen diluent.     

SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the protein profiles of seminal plasma in buffalo bulls and to examine their correlation with semen characteristics. Semen of 10 buffalo bulls were collected by a bovine artificial vagina. Semen characteristics (motility, morphology, viability and concentration) were recorded. A part of the semen sample (1 ml) was diluted by tris-egg yolk-glycerol extender, packed in French straws and was frozen in liquid nitrogen. The straws were later thawed and semen characteristics were compared with those of the fresh semen. Seminal plasma was harvested by centrifugation; treated with cold ethanol and then, underwent SDS-polyacrylamide gel electrophoresis (PAGE). Twenty five protein bands were identified on the gel, of which those of <35.5 kDa were prominent (72% of the bands). Of these protein fractions, 24.5 kDa was significantly correlated with sperm progressive motility in fresh and viability in frozen-thawed semen while 45 kDa bands were correlated with abnormal morphology in frozen-thawed semen; 55 kDa protein fractions were correlated with sperm viability of fresh semen. Progressive motility, viability and abnormal sperm morphology of frozen-thawed semen were highly correlated with these parameters in the fresh semen. In conclusion, seminal plasma protein fractions in buffalo bulls are similar to those reported in other animal species and have some correlations with semen characteristics before and after freezing.