A new antibiotic combination for frozen bovine semen. 2. Evaluation of seminal quality

Amikacin, clindamycin, gentamicin, Linco-Spectin, minocin and tylosin were added individually and in combinations at various concentrations to bovine neat semen and to egg-yolk citrate, egg yolk-tris or heated whole milk extenders (nonglycerol fractions) prior to final processing for freezing to -196 degrees C. After thawing samples, seminal quality was measured by progressive motility and acrosomal integrity evaluations. Studies were performed in parallel with microbiological efficacy studies of Shin et al. (7). The antibiotic combination including gentamicin, tylosin and Linco-Spectin at 500 ug/ml, 100 ug/ml and 300/600 ug/ml, respectively, was not detrimental to seminal quality and, in the parallel studies, was most efficacious in controlling microorganisms potentially present in bovine semen.     

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.     

Characterization of three arylsulfatases in semen: seminolipid sulfohydrolase activity is present in seminal plasma.

Sperm cells and seminal plasma of various mammals contain high levels of arylsulfatase. In the present study, we investigated the composition of soluble AS in these compartments of boar semen by analysing sperm cells and seminal plasma using anion-exchange chromatography. Seminal plasma contained both arylsulfatase B (2.4 units per ml), an enzyme which desulfates sulfoglycosaminoglycans and probably sulfoglycoproteins, and arylsulfatase A (10.2 units per ml), an enzyme which desulfates sulfogalactolipids. Sperm cells contained only arylsulfatase A, which differed biochemically from the extracellular arylsulfatase A of seminal plasma (2.6 units per ml). Both types of arylsulfatase A desulfate seminolipid, the natural sulfolipid substrate in sperm, as well as two brain sulfatides. The possible physiological consequences of the presence of extracellular arylsulfatases in seminal plasma for spermatozoa are discussed.     

Use of a semen extender containing antibiotic to improve the fertility of a stallion with seminal vesiculitis due to Pseudomonas aeruginosa

A breeding trial was conducted to determine if a semen extender containing polymixin-B sulfate would improve the fertility of a stallion with seminal vesiculitis due to Pseudomonas aeruginosa . Twenty-three mares were bred to the stallion by one of three methods: artificial insemination with raw semen (Group 1, n = 10), artificial insemination with semen mixed 1:1 with a nonfat dry skim milk/glucose extender containing 1000 units/ml polymixin-B sulfate (Group 2, n = 9), or natural service immediately following infusion of the uterus with 100 ml of the same extender (Group 3, n = 4). Artificial breedings contained a minimum insemination dose of 500 x 10(6) progressively motile spermatozoa. All mares were bred every other day while in estrus. Pregnancy status was determined by transrectal ultrasound examination 15 d after the last breeding. First-cycle pregnancy rate for Group 2 mares (78%) was greater (P < 0.01) than for Group 1 mares (10%). There was a tendency (P = 0.10) for the pregnancy rate of Group 3 mares (50%) to be greater than Group 1 mares. The use of a semen extender containing polymixin-B sulfate improved the fertility of this stallion.     

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%).     

Fertility of frozen-thawed stallion semen extended in lactose-EDTA-egg yolk extender and packaged in 1.0-ml straws

The fertility of frozen-thawed and fresh semen from each of three stallions was compared in an experiment with a randomized block design using 128 mares. Semen was collected every third day, extended in lactose-EDTA-egg yolk extender at a concentration of 500 × 10⁶ progressively motile sperm per 1.0 ml, and frozen in individual-dose, 1.0-ml straws (1.9 mm × 267 mm). The same stallions were collected daily for inseminations with fresh semen. For each insemination dose with fresh semen, 300 × 10⁶ progressively motile sperm were added to 10 ml of heated skim milk extender. Mares were inseminated daily from the second day of estrus through the end of estrus. Of 52 ejaculates processed and frozen, 38% were discarded because < 35% of the sperm were progressively motile after thawing. Based on rectal palpations on day 50 post-ovulation, pregnancy rates for inseminations during one estrus to semen from the three stallions were 17, 33 and 35% for frozen-thawed semen and 60, 62 and 64% for fresh semen. Pregnancy rates with frozen semen from two of the three stallions were 54% of the rates attained with fresh semen.     

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.     

Effect of insemination dose on pregnancy rate in mares

Different insemination doses have been used for artificial insemination(AI) in horses. Since the insemination dose can affect the pregnancy rate, it is important to ensure that an adequate dose be used regardless of the type of inseminationprotocol used. The aim of this study was to find out if it is possible to decrease the insemination dose from 500 x 10(6) progressively motile spermatozoa to 300 x 10(6) progressively motile spermatozoa and still maintain an acceptable pregnancy rate when using extended fresh semen. Thirteen stallions of known fertility and a well-defined group of 64 mares were used in the study. The mares were randomly assigned to 1 of 2 insemination groups. Examination for pregnancy was performed by ultrasonography per rectum approximately 16 d after the last insemination. When using an insemination dose of 300 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 75%. With an insemination dose of 500 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 64%. There was no significant difference in the pregnancy rate between the 2 insemination doses (P = 0.341). We conclude that when using fresh extended semen it is unlikely that an insemination dose of 300 x 10(6) progressively motile spermatozoa would yield a lower pregnancy rate than a dose of 500 x 10(6) progressively motile spermatozoa if stallions with good quality semen are selected.     

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.     

Sperm-induced leukocytosis in the equine uterus

The objective of this study was to investigate the inflammatory reaction induced in the equine uterus by insemination with fresh and frozen semen. Eleven groups (6 to 8 mares per group) were studied during 2 breeding seasons. The mares were inseminated using raw semen, frozen semen, extended fresh and frozen semen, concentrated fresh semen, seminal plasma and seminal extenders only. One group was bred naturally. Six hours after insemination, the uteri were flushed with 50 ml of phosphate-buffered saline (PBS). Seventeen out of 104 samples (16%) exhibited slight bacterial growth. Neutrophil concentrations were significantly (P < 0.05) higher in all treated mares than in the controls. Mares infused with PBS, seminal extenders or the supernatant from centrifuged frozen-thawed semen exhibited only a mild neutrophil response. Insemination with frozen semen resulted in higher neutrophil concentrations than insemination with extended fresh semen (means of 59 vs 5 million neutrophils/ml; P < 0.05). Highest neutrophil counts were found after insemination with frozen semen or concentrated fresh semen. Bacterial contamination of uteri was insignificant 6 hours after breeding. Neutrophilia seems to be induced by spermatozoa rather than bacteria. The intensity of the neutrophil reaction seems to depend on concentration and/or volume of inseminate.     

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = −0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = −0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.     

Intrafollicular insemination for the treatment of infertility in the dairy cow

Intrafollicular insemination (IFI) is based on direct introduction of a sperm suspension into a preovulatory follicle. To our knowledge, the first cases of IFI resulting in pregnancy of the cow are reported here. The experiment was performed on a dairy herd with low fertility during the warm season of the year. Following the sequence of estrus detection, one in three inseminations was intrafollicular (n = 17), whereas deposition of semen was performed into the uterine body (IUI) in the remaining cows (n = 33). Approximately 0.06 ml of a seminal dose containing five million spermatozoa (one quarter of a commercial seminal dose in a 0.25 ml French straw) was injected trans-vaginally into the preovulatory follicle in the IFI group. Four (23.5%) and 3 (9%) cows of the IFI and IUI groups, respectively, became pregnant. On days 8-11 post-insemination, ovulation failure was registered in 3 (18%) and in 4 (12%) cows of the IFI and IUI groups, respectively. Our results suggest that IFI could be used as an alternative procedure to the usual deposition of semen into the uterus in cows of low fertility.     

Effect of dialysis or centrifugation on post-thaw motility and fertility of Santa Gertrudis bull semen collected by electroejaculation

Electroejaculated semen from Santa Gertrudis bulls was used to study the effect of centrifugation (600 x g for 5 min) or dialysis [molecular weight cutoff <14,000 Daltons (Da)] on post-thaw motility and on fertility in beef cattle. Analysis of post-thaw motility showed that the main effects (bulls and semen treatment) were significant (P<0.05). Dialysis significantly improved post-thaw motility in four of seven bulls. Initial volume of seminal plasma in the ejaculate was negatively correlated to post-thaw motility (r = -0.73). No significant improvement in post-thaw motility was observed for bulls with high volumes of ejaculate. In a fertility trial, calving rates of heifers synchronized with PGF(2)alpha and inseminated at 72 and 96 h after the second PGF(2)alpha injection with dialyzed or commercial semen were not statistically different (P>0.05; 54.4% vs 55.4%). These results show that dialysis could be used to improve post-thaw motility of electroejaculated bull semen without altering its fertilizing capacity. However, a high initial volume of seminal plasma seems to have a deleterious effect on sperm freezability that cannot be reversed by dialysis.     

Impact of seminal and serum zinc on semen quality and hormonal status: A population-based cohort study of Russian young men

BackgroundTrace elements are important factors in human reproductive health. Among them, special attention is paid to zinc, which is an essential trace element and is necessary for the normal functioning of the male reproductive system and the process of spermatogenesis. The aim of the study was to investigate the association between seminal and serum zinc concentrations and semen quality and reproductive hormone levels in population of Russian young men.MethodsThe study population consisted of 626 young Russian men (median age 22.5 years), recruited from the general population, regardless of their fertility status. Each participant provided semen and blood sample, information about his lifestyle and ethnicity. Semen quality (sperm concentration, motility and morphology), reproductive hormone levels (testosterone, estradiol, LH, FSH and inhibin B), and serum and seminal zinc concentrations were evaluated. The semen samples were analyzed according to the WHO laboratory manual (WHO, 2010). Serum hormones were measured by enzyme immunoassay, zinc concentrations were determined using spectrophotometry and direct colorimetry without deproteinization.ResultsZinc was present in the seminal plasma in a significantly higher concentration than in the blood serum (median serum Zn concentration was 23.6 μmol/L vs seminal Zn concentration 1571.8 μmol/L). The seminal zinc concentration was positively related to the total sperm count, sperm concentration, progressive motility and normal morphology (Spearman’s test: 0.221; 0.286; 0.269; 0.183, respectively, p < 0.001), while the serum Zn concentration was negatively related to serum testosterone and estradiol levels (r = −0.249 and r = −0.096, respectively, p < 0.001−0.05). It was found that the seminal Zn content in men with normal semen quality was higher compared to men with lowered semen quality (means: 6.37 and 5.03 μmol/ejaculate, respectively, p < 0.001). Similarly, the semen volume, total sperm count, sperm concentration, progressive motility, normal morphology and the serum testosterone level in men with the seminal Zn deficiency were lower than in men with the normal seminal Zn content.ConclusionBased on the results of our population-based study, seminal Zn levels were closely associated with semen parameters in young men, so Zn deficiency may be an important risk factor for lowered semen quality. Seminal Zn determinations should be considered as a useful tool in addition to other parameters in assessing male fertility.     

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.     

Effect of Simmental bull seminal plasma protein in egg yolk-citrate extender on Kacang buck semen fertility

The genetic resources of Indonesia's indigenous Kacang goat require preservation. Artificial insemination is expected to accelerate population increases and preserve genetic resources simultaneously. The present study was the maiden attempt for cryopreservation of Kacang buck sperm. The objectives of this study were to determine whether the supplementation of superior Simmental bull seminal plasma protein in egg yolk-citrate extender could improve the quality of post-thawed Kacang buck sperm, increase conceptions rates, and improve kidding rates. Buck semen was diluted without supplementation (T0) and with supplementation of 2.5 mg (T1) and 5 mg (T2) of Simmental bull seminal plasma protein per mL egg yolk-citrate extender. Extended semen was packed in 0.25 mL straw as cryopreserved frozen semen. Post-thawed semen samples were evaluated for viability, motility, intact plasma membranes, malondialdehyde level, and DNA fragmentation. Estrus was synchronized for sixty Kacang does, which were divided randomly into three groups and inseminated using post-thawed semen. The progesterone serum concentration of the does was measured 7 and 22 days post-insemination to detect ovulation and conception. Pregnancy was confirmed using abdominal palpation at 43 days post-insemination and by observing birth. The T1 group showed the highest (P < 0.05) post-thawed viability, motility, and intact plasma membrane. Conception, pregnancy and kidding rates were also higher in T1 than other treatment groups. In conclusion, the 2.5 mg Simmental bull seminal plasma protein supplementation per mL egg yolk-citrate extender provided the best seminal quality and fertility of post-thawed Kacang buck semen.     

Acrylonitrile-induced sister-chromatid exchanges and DNA single-strand breaks in adult human bronchial epithelial cells

The ability of acrylonitrile to induce cytotoxicity, sister-chromatid exchanges and DNA single-strand breaks was studied in cultured human bronchial epithelial cells. The toxic effect as determined by cloning efficiency was observed at a dose of 600 micrograms/ml but not at doses of both 150 and 300 micrograms/ml. The frequency of sister-chromatid exchange in untreated cells was 3.7 +/- 1.3 per cell. In contrast, cells treated with acrylonitrile at 150 and 300 micrograms/ml exhibited 6.6 +/- 1.3 and 10.7 +/- 1.7 sister-chromatid exchanges per metaphase, respectively. DNA single-strand breaks were induced by acrylonitrile at dose levels of 200 and 500 micrograms/ml. The genotoxic effects on human bronchial epithelial cells that were directly exposed to acrylonitrile are of interest in relation to evidence for the higher lung cancer incidence of acrylonitrile workers in epidemiological studies.     

Evidence for decreasing quality of semen during past 50 years.

OBJECTIVE--To investigate whether semen quality has changed during the past 50 years. DESIGN--Review of publications on semen quality in men without a history of infertility selected by means of Cumulated Index Medicus and Current List (1930-1965) and MEDLINE Silver Platter database (1966-August 1991). SUBJECTS--14,947 men included in a total of 61 papers published between 1938 and 1991. MAIN OUTCOME MEASURES--Mean sperm density and mean seminal volume. RESULTS--Linear regression of data weighted by number of men in each study showed a significant decrease in mean sperm count from 113 x 10(6)/ml in 1940 to 66 x 10(6)/ml in 1990 (p < 0.0001) and in seminal volume from 3.40 ml to 2.75 ml (p = 0.027), indicating an even more pronounced decrease in sperm production than expressed by the decline in sperm density. CONCLUSIONS--There has been a genuine decline in semen quality over the past 50 years. As male fertility is to some extent correlated with sperm count the results may reflect an overall reduction in male fertility. The biological significance of these changes is emphasised by a concomitant increase in the incidence of genitourinary abnormalities such as testicular cancer and possibly also cryptorchidism and hypospadias, suggesting a growing impact of factors with serious effects on male gonadal function.     

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.