Ml Of Semen Research Articles

Semen collection, cryopreservation and artificial insemination in the dromedary camel

Collection of semen with a bovine artificial vagina (AV) was attempted with each of 14 camels over a period of 2 years. Semen samples were evaluated, extended and cryopreserved. Frozen thawed semen, diluted cooled semen or whole semen was used to inseminate some female camels which were induced to ovulate with hCG. Males ejaculated semen into the AV in 74.6% collection attempts. The male copulated for at least 200 s in 62.9% attempts. The remaining copulations were of shorter duration. Similarly, 49.3% ejaculates were at least 3 ml of semen. Libido and donation of semen improved from December onwards and reached a peak after mid January with peak performance persisting until April. It declined during May. The majority of camels had lost libido and refuse to donate semen by the end of May. Camel semen is in gel form. While 35.9% of 203 semen samples exhibited no individual sperm motility, 28.5% exhibited low to fair grade individual sperm motility and only 35.4% exhibited >50% sperm motility. Differences existed between animals ( P<0.01) and months ( P<0.05) of collection, while effect of copulation time was not significant. Mass motility was not observed in camel semen. Individual sperm motility develops after liquefaction of semen. Addition of caffeine but not chymotrypsin improved the individual motility. The mean live percent sperm count and normal acrosome were 73.3±1.0 and 92.0±0.5, respectively. Only 51.1% of 45 semen samples with pre-freeze motility of >50% and 25% of 16 semen samples from low pre-freeze motility group with an overall success of 44.2% of 61 semen samples were successfully preserved. Wide variation was observed in the freezability of semen from different males. Attempts to impregnate female camels with liquid semen, frozen thawed semen and whole semen after hCG induced ovulation resulted in 0/10, 1/13 and 4/10 pregnancies.

Manipulation of Fertilization Procedures to Improve Hatchery Walleye Egg Fertility and Survival

Abstract The purpose of this investigation was to determine the effect of egg depth and the timing of semen presentation on the egg survival of walleye Stizostedion vitreum and to alter these procedures to improve egg fertility and subsequent survival and hatch. Tests were conducted evaluating egg depths of 4, 7, and 10 mm and one to three semen additions during the first minute of fertilization. The findings indicated that an egg depth of 4 mm resulted in significantly more fertile eggs (86%) than depths of 7 mm (77%) and 10 mm (65%) and that a 7-mm egg depth will yield significantly more fertile eggs than a 10-mm depth. Tests using the multiple addition of semen (extended 2:1) to 473 mL of eggs at a depth of 4 mm resulted in the highest fertility rate of 83% when 3.0 mL of semen was added initially and then 30 s and 60 s later, compared with a 79% fertility rate for a single, 9.0-mL addition (P < 0.05). Using an egg depth of 12 mm and 9.0 mL of extended semen resulted in a fertility rate of 70%.

PCR Assessment of Chlamydia trachomatis Infection of Semen Specimens Processed for Artificial Insemination

In order to ascertain the microbiological quality of stored semen specimens processed for artificial insemination by a donor (AID), we developed a PCR assay targeting the chlamydial plasmid to detect Chlamydia trachomatis in semen. The lower limit of detection of this assay corresponded to 2.5 to 5 elementary bodies per microl of semen. A total of 669 cryopreserved ejaculates from 97 asymptomatic donors were tested for C. trachomatis infection. Twelve ejaculates, originating from four donors, were found to be positive, indicating a 4% prevalence of C. trachomatis infection among the donor population studied. Cross-contamination between the cryopreserved specimens in the storage container was studied by typing using sequence analysis of PCR-amplified omp1 genes of the strains. Two donors were infected with serovar E, one was infected with serovar F, and one was infected with serovar K. For two donors, the duration of C. trachomatis positivity could be assessed. One donor donated C. trachomatis-positive semen for at least 4 successive months, and the other did so for at least 16 months. The occurrence of C. trachomatis infection in cryopreserved donor semen indicates that ejaculates from donors not tested for a C. trachomatis infection just prior to donation should be tested for infection by a direct test such as the PCR described here. Direct testing of semen specimens will detect not only donors with an active infection but also C. trachomatis-infected ejaculates already stored and will thus improve the microbiological quality of AID, since discrepancies in the presence of C. trachomatis in urine and semen specimens have been reported.

High catalase content of rabbit semen appears to be inherited.

Reactive oxygen species, as hydrogen peroxide, can be damaging to sperm. Most species have little protective catalase in their semen, but rabbit semen contains substantial amounts of catalase. The objective of the present study was to characterize a substantial number of Dutch rabbits for seminal content of catalase and determine whether differences were inherited. Usually, 4 or more semen samples were analyzed per male. Catalase was measured by a gasometric procedure. In Experiment 1, the correlation between duplicate determinations was r = .99, and between 2 sets of semen samples from 55 males it was r = .95. There was a significant difference (P < .05) among pairs of males from 6 litters. In Experiment 2, semen from each of 11 males collected at an interval of 1 year contained an average of 13 and 12 units of catalase per mL of semen in consecutive years. The correlation between pairs of samples was r = .85. This indicated that the condition was permanent, and possibly genetically controlled. Experiment 3 analyzed the catalase content of semen from 7 sires and 32 sons. The heritability of seminal catalase concentration was 0.48. These studies indicate that rabbit seminal plasma is high in catalase, and that a substantial portion of the differences among males are under genetic control.

Vaginal delivery of new formulations of nonoxynol-9 coprecipitated with polyvinylpyrrolidone in rabbits: Comparisons between two formulation delivery systems

The objective of this study was to assess the efficacy of two formulation delivery systems (FDS) in preventing the onset of pregnancy in rabbits. Nonoxynol-9 (N-9) was coprecipitated with polyvinylpyrrolidone (PVP), which yields PVP N-9 , and prepared as capsules or tablets. Semen specimens were collected (from eight male rabbits), pooled, and used for in vitro spermicidal assessment or artificial insemination (AI). In vitro spermicidal assessment was performed by introducing and mixing the FDS containing PVP or PVP N-9 with 1.0 mL of semen, followed by incubation at 37°C for 6 h. Semen samples were taken at various time intervals to determine killing of spermatozoa and dissolution of the FDS. The efficacy of the FDS in preventing the onset of pregnancy was assessed by inserting the FDS vaginally. The does were artificially inseminated at 0, 0.5, and 6 h after insertion of the FDS vaginally. The number of pregnant does and newborn rabbits was recorded. In the in vitro spermicidal trial, semen specimens exposed to both FDS containing PVP N-9 were killed within 10 to 15 min of incubation. Tablets containing PVP only or PVP N-9 dissolved completely after 3 h of incubation. However, capsules did not dissolve completely by 6 h of incubation. The results obtained in the in vivo trial showed that both FDS exhibited some variations in preventing the onset of pregnancy over the various time intervals following the insertion of tablets or capsules and AI. The tablet seemed to be a more efficient delivery system than the capsule, yielding significantly lower pregnancy rates at all three time intervals assessed. The tablet FDS, as applied in this study, was found to be the most efficient mode of delivery of the tested spermicidal formulations.

Enumeration of semen leucocytes by fluorescence in situ hybridisation technique

Aim-To determine whether the fluorescent in situ hybridisation technique (FISH) using a total human DNA genomic probe can be used to enumerate semen leucocytes.Methods-Semen samples from five donors were subjected to a mild KC1 solution. These samples were then biotin labelled under FISH conditions using a total human DNA genomic probe and the leucocyte counts were determined. To check the accuracy of the technique a monoclonal antibody against the common leucocyte antigen CD45 [KC56(T-200)] served as a control. An isotypic control for [KC56(T-200)], the immunoglobulin [MsIgG1], served as a secondary control.Results-Semen leucocytes stained by the FISH technique were easily detected because of their distinct bright yellow colour, while the sperm cells were red. The leucocyte count ranged from 0.5 to 4.9 x 10(6) per ml of semen. KC56(T-200) and its isotypic control MsIgG1, which served as control for the FISH technique, accurately identified 94% and 97% of the semen leucocytes of a control donor, respectively.Conclusions-The FISH technique using a total human DNA probe can accurately and effectively enumerate the overall leucocyte population in semen.

Biology of sperm and artificial reproduction in carp

Although the common carp, Cyprinus carpio, has been cultivated for several thousand years and is produced in large quantities, research on reproduction has been very limited. Traditionally, spawning occurred naturally in situ in rearing ponds. In slightly improved methods large breeding ponds stocked with brood fish were devoted to reproduction with fry collection in autumn, or in small spawning ponds with collection of larvae a few days after hatching. Controlled reproduction in hatcheries started only in the 1950s. This paper reviews some of the basic work on carp sperm and describes the technologies of artificial reproduction. The sperm is of a primitive type with uncondensed chromatin and a small midpiece. As in most teleost fish, the sperm is immotile in the male genital tract and in the semen and is activated after release into fresh water. The initiation of motility is due to the decrease in osmotic pressure. The structure of the flagellum is rapidly disorganized in fresh water and sperm stop moving after 30 s. When dilution occurs in a 50 mM NaCl solution, the osmotic change is sufficient to initiate motility, but the flagellum is not disorganized and swimming lasts a few minutes. Motility depends mainly on endogenous ATP stores, about 12 nmol 10 8 spermatozoa, and stops when 50–80% of the ATP is exhausted by hydrolysis. The procedure of artificial insemination includes collection of gametes (from “hypophysised” males and females), mixing in an extender (45 mM NaCl, 5 mM KCl, Tris 2.5 mM, glycine 19 mM, pH 8) in a ratio 1 litre of eggs to 1 litre of diluent and 1 ml of semen. Sperm motility can be triggered during collection by contamination of the semen with urine; this could interfere with fertilization and, if present, urine should be discarded by pouring out the top part of the tube. The sticky layer of the egg is removed by adding a “dissolving solution” (20 g urea/1 + 4g NaCl/1 of water) or milk (diluted 1 5 in water) to the fertilized egg and stirring. One hour later the swollen eggs are transferred to incubators (usually Zug bottles, sometimes 200 litre circular jars).

The effect of transparent fluid on fertility, hatchability and embryonic mortality following insemination of a normal number of stored fowl spermatozoa

Pure semen and semen containing 30% transparent fluid (TF) were diluted twofold in Van Wambeke's diluent and stored for 24 h at 2–5 °C. Caged dwarf breeders were inseminated weekly for 5 consecutive weeks with: (a) 0.08 mL of pure, stored semen per hen (310 × 106 sp.), (b) 0.08 mL of semen containing TF (217 × 106 spermatozoa (sp.)), (c) 0.092 mL of semen containing TF (250 × 106 sp.), or (d) 0.025 mL of pure, fresh semen (194 × 106 sp.). There were no significant differences (P &gt; 0.05) in fertility, hatchability and embryonic mortality between the fresh control and the pure, stored semen nor with the supplementation of semen with TF. Key words: Fowl semen, transparent fluid, storage, fertility, hatchability

Chick production by in vitro fertilization of the fowl ovum.

The aim of this study was to produce viable chicks by in vitro fertilization and transfer of fertilized ova to the oviduct of recipient hens. Out of a total of 76 transferred ova, 53 were laid with fully calcified shells, 31 of which were fertile (58%). Despite the high rate of embryonic loss, six live chicks were hatched from 12 fertile ova exposed to 0.05 ml of semen (1:200 dilution). Nine healthy chicks were hatched from ten control ova which were recovered from the oviduct following artificial insemination and subsequent transfer to recipient hens. This experimental approach provides a useful model for production of transgenic chicks.

Decrease and Recovery of Ejaculated Sperm Concentration after Acute Testicular Gamma-Ray Irradiation in Cynomolgus Monkeys (Macaca fascicularis).

After acute testicular irradiation in adult male cynomolgus monkey semen was periodically collected, and sperm concentration was examined as an index of radiation damage to the seminiferous epithelium. Sperm concentrations decreased after irradiation with 1, 2, or 3 Gy. Concentrations fell below the minimum normal level for unirradiated monkeys, (107 sperm /ml of semen [13]) at 16.0, 8.5, and 8.0 weeks after irradiation, respectively. The lowest sperm concentra-tion was observed 17.0, 12.5, and 14.0 weeks after irradiation, and the percentages of the lowest sperm concentration relative to the pre-irradiation concentration were 1.20, 1.37, and 0.20%, respectively. The sperm concentration recovered to pre-irradiation levels after 35.5, 36.5 and 41.0 weeks at doses of 1, 2 and 3 Gy, respectively. These results indicate that spermatogenesis decreases markedly after irradiation with 1-3 Gy but resumes after a definite period that depends on the radiation dose. Sperm concentration can be utilized as an effective index of radiation damage to the seminiferous epithelium and as an index of recovery.

Detection of bovine herpesvirus-1 in bovine semen by a nested PCR assay

A nested PCR assay targeting a portion of the glycoprotein IV gene has been developed for the detection of Bovine Herpesvirus-1 (BHV-1). Rapid and sensitive detection of the PCR products was achieved using a nonisotopic reverse dot-blot format with a visible color readout. Cross-reactivity of this PCR assay was not observed with the closely related BHV-3. The sensitivity of this assay when tested on a supernatant from a BHV-1 cell culture was approximately 4.5 TCID 50 (50% tissue culture infectious dose). A procedure using the chelating resin Chelex 100 was used to prepare viral DNA from artificially inoculated samples of extended and raw semen for use in the PCR assay. In combination with nested PCR and reverse dot blot, this method allowed the detection of 5 × 10 3 TCID per 0.5 ml of semen, which is comparable to the detection in the Cornell Semen Test. The whole procedure can be completed in approximately 8 h. This assay has therefore the potential of replacing the currently available yet time consuming and costly detection methods for BHV-1 in bovine semen.

Conception rates following intrauterine insemination of European (Dama dama dama) fallow deer does with fresh or frozen‐thawed Mesopotamian (Dama dama mesopotamica) fallow deer spermatoza

A total of 121 European fallow deer does, being either parous (n= 15) or nulliparous (n= 106), were treated with intravaginal progesterone impregnated controlled internal drug release (CIDR) devices for 14 days. The does were divided into three treatment groups and inseminated in utero by laparoscopy, at approximately 65 hours after CIDR device removal, with 25 × 106 fresh Mesopotamian (n= 40), 25–35 × 106 frozen‐thawed Mesopotamian (n= 41) or 30–32.5 × 106 frozen‐thawed European (n= 40) fallow deer spermatozoa. The semen used had been collected, from two Mesopotamian and two European fallow deer bucks, by electroejaculation under general anaesthesia. Pregnancy was diagnosed by rectal ultrasonogrdphy on Day 50 after insemination.There were no apparent differences in the quality of ejaculates between the two subspecies of fallow deer. The volume of semen and the total number of spermatozoa ranged between 0.6–1.2 ml and 2.11–4.95 × 109 per ml of semen, respectively. Evaluation of frozen‐thawed spermatozoa revealed post‐thaw motility rates between 50–70%. The overall conception rate was 65.3%. A higher conception rate was observed following insemination with European than Mesopotamian frozen‐thawed spermatozoa (75% vs. 53.7%, respectively, P &lt; 0.05). Insemination with fresh Mesopotamian spermatozoa increased the conception rate to a level not significantly different from that observed following insemination with European frozen‐thawed spermatozoa (67.5% vs. 75%, for fresh Mesopotamian and frozen‐thawed European semen, respectively).

Seasonal variation in fructose and citric acid in seminal plasma of Murciano-Granadina goats

Ejaculates were collected by artificial vagina from nine, 13-month-old Murciano-Granadina bucks twice a week. Semen collection continued for 12 months. Mean (±SE) fructose and citric acid concentration was 792.56 (± 13.14) and 295.73 (± 5.04) mg/ 100 ml of semen, respectively. High concentrations ( P < 0.05) of fructose and citric acid were found during the summer (867.57 and 311.98 mg/ 100 ml of semen, respectively) and autumn (931.42 and 327.66 mg/ 100 ml of semen, respectively) but low values during the spring (567.95 and 249.11 mg/ 100 ml of semen, respectively). This seasonality may be attributed to circannual changes in day length. However, we consider that these seasonal differences do not affect semen quality, because these seminal components did not show any significant correlation with the sperm quality parameters studied (motility, sperm morphology and acrosome deterioration).

In vivo Fertilizing Capacity of Deep Frozen Boar Semen Packaged in Plastic Bags and Maxi‐Straws

Pooled ejaculates from six fertile boars were frozen under controlled conditions in Teflon FEP-film plastic bags (5 ml) and maxi-straws (2.5 ml) using 3% glycerol as cryoprotectant. The percentages of both post-thaw motility and normal apical ridges were significantly higher (P less than 0.001) for the bags (54.5 and 75%) than for the maxi-straws (40.1 and 59.4%) respectively. For evaluation of the in vivo fertilizing capacity of the frozen-thawed spermatozoa, 26 gilts were inseminated once 24 h after the first observation of standing reflex in their second oestrus, with 5 ml of semen (containing 5 billion spermatozoa) reconstituted in 80 ml of BTS from either bags or maxi-straws. Ova were recovered from the oviducts/uteri 2-4 days following insemination and examined for cleavage and sperm binding to the zona pellucida (ZP). Significantly higher rates (P less than 0.02) of fertilized ova were found in the bag-inseminated (75%) than in maxi-straw inseminated gilts (63%); and similarly their ova had significantly more spermatozoa in the ZP, irrespective of whether they were fertilized or nonfertilized. This study confirmed that the plastic bags are suitable and may be used for packaging single insemination doses of deep frozen boar semen for routine A.I. work.

Identification of male-factor semen samples prior to insemination and in vitro fertilization

Semen analyses carried out as part of the clinical in vitro fertilization or intrauterine insemination protocols provide important information that determine the type of clinical treatment of the male partner and the sperm processing method. It is postulated that the sperm of male-factor patients cannot survive hypoosmotic stress conditions because of defective sperm membrane function. To test this, 0.1 ml of semen from each of 102 patients was placed in 1.0 ml of 150 mosmol/liter eosin citrate fructose solution and incubated for 30 min at 37 degrees C. The percentage viability of the sperm cells was then determined. The results indicated that patients with two or more abnormal semen parameters had a significantly lower percentage viability while in the hypoosmotic solution (40.6 +/- 4.7%), in contrast to non-male-factor patients (69.0 +/- 1.6%). Donor sperm (N = 32) serving as controls (73.3 +/- 2.1%) had a viability in hypoosmotic solution similar to that of non-male-factor patients. The data suggest that sperm of male-factor patients are less able to survive the hypoosmotic stress conditions as shown by the percentage viability in hypoosmotic solution and emphasize the importance of using less stressful sperm processing methods for in vitro fertilization or insemination in these patients.

Radioimmunoassay detection limits for 19-OH [formula omitted] prostaglandin in normal, infertile and vasectomized semen stains. Analysis of saliva, sweat and urine for possible non-specific or matrix effects

The sensitivity of a simple radioimmunoassay (RIA) for the detection of 19-OH prostaglandin F 1α F 2α (PG F) has been evaluated on a number of semen samples from vasectomized, infertile and fertile donors. The specificity of the test has been examined by testing saliva, sweat and urine from a number of male and female donors. The assay technique could readily detect the PG in semen stains prepared from 0.2 μl of semen from normal, infertile and vasectomized donors. The detection limit of the assay system, based on the observed displacement, was calculated to be approximately 0.05 μl semen. The assay could be conducted over a pH range of 7.5–10.5 even after the PG has been heated to 100°C. The 19-OH series of PG were absent from sweat, saliva and female urine using the normal assay protocol; volumes in excess of 100 μl of some urines particularly from women in labour and those with acute urinary tract infection showed some displacement. Low levels of PG were detected in 50% of the male urine analysed. However, urine samples from men who had engaged in recent sexual activity contained relatively high concentrations of PG which could be readily detected in 10 μl of urine. These results emphasise the potential of these compounds as specific and sensitive markers for the presence of human semen.

Fertility of stallion spermatozoa isolated on albumin gradients

Fertility of stallion semen extended with bovine serum albumin (BSA) sucrose extender or cream-gelatin extender was compared. Pregnancy rates were 95% of 47 mares and 86% of 46 mares inseminated with BSA-sucrose and creamgelatin extended semen, respectively. Foaling rates and cycles per conception were not significantly different between treatment groups. Semen from 5 mature stallions was used in an attempt to isolate a population of highly motile spermatozoa. Immediately following collection, samples were evaluated for motility and forward movement. Seven to 10 ml of semen, extended 1:1 with BSA-sucrose extender, were pipetted onto BSA medium separation columns. After 1 hour incubation at 37°C, the top, middle and bottom layers were separately withdrawn from each separation column and pooled, respectively. A marked decrease (P<.001) was noted in the mean motility of spermatozoa in the top layer (35%) as compared to the mean pre-incubation motility (59%). Spermatozoa from middle and bottom layers were significantly (P<.001) more motile (70.6 and 87%) than those from the top layer and pre-incubation samples. Rate of forward movement (RFM) of spermatozoa in lower fractions was higher (P<.001) than RFM of spermatozoa in the top layer. Concentration of spermatozoa decreased (P<.001) as the concentration of BSA in the medium increased.

Factors Affecting Broiler Breeder Performance.: 7. Effect of Varying Levels of Dietary Protein on the Development and Reproductive Performance of the Dwarf Broiler Breeder

Vedette dwarf broiler breeder female chicks were raised in floor pens and fed a standard starter ration. At 3 wk of age, 1,032 pullets were divided into four dietary treatment groups (258 bird/treatment). Birds were transferred into individual cages at 18 wk of age and at 21 wk of age assigned to one of four laying rations. Dietary treatments consisted of a 4 × 4 factorial design with four developer diets (12, 14, 16, and 18% protein) and four layer diets (12, 14, 16, and 18% protein).Body weight was determined at 6, 12, and 17 wk of age and thereafter at 4 wk intervals. Hen-day egg production was determined weekly from 21 to 58 wk of age. Specific gravity and egg weights were measured at 4-wk intervals on eggs collected over 4-day periods. Fertility and hatchability were measured at 32, 34, and 44 wk of age on the one-half of the birds in each treatment that had been artificially inseminated weekly with .05 mL of semen from Ross males. Embryonic mortality and chick weights of offspring were recorded when the breeder flock was 32 and 44 wk of age.Dietary treatment had a significant effect on body weight (P>.05) at Week 12, (12% and 14% vs. 16% and 18%) but by Week 17 differences were no longer significant. Significant differences in body and egg weights during the laying cycle were detected (P<.05), with hens receiving the higher protein (16% and 18%) laying diets exhibiting heavier body and egg weights. Significant interactions between dietary treatments were seen for age at sexual maturity, hatchability, chick weights, and progeny performance (P<.05). Fertility and hen-day egg production were not significantly different among dietary treatments (P>.05).

Composition et emploi d'un dilueur d'insémination chez la carpe, Cyprinus carpio

A sperm diluent with the composition: NaCl 45 m M, KCl 5 m M, Tris 20 m M pH 8.0, was developed for the artificial fertilization of carp. Osmotic pressure was around 120 mOsm. In this medium, spermatozoan motility was markedly longer (1.5 min vs. 30 s in fresh water) and ova could be fertilized for 4–5 min vs. 1–2 min in fresh water. The dilution rate was 10 −3, corresponding to an extender volume of 0.5 l/l of ova with 0.5 ml of semen. Semen diluted with this extender did not exhibit any morphological change detectable with a microscope, whereas in fresh water a coiling of the flagellum was observed 5 s after dilution. Before transfer into an incubator, fertilized eggs should not remain in the presence of the extender for longer than 15–17 min. The extender used in this experiment did not prevent eggs from agglutinating after transfer to fresh water. A specific medium including urea may be used, but the concentration of urea should not exceed 400 m M.

Individual Variation for In Vitro Fertilization Success in Dairy Bulls

Individual semen samples from 29 bulls in routine artificial breeding service were tested for their capability to achieve in vitro fertilization. Ejaculated semen was diluted, .1ml of semen in 2ml of a modified Tyrode's medium with an osmolality of 340 mOsmol/kg, washed thrice, incubated 3h at 37°C before being used for in vitro fertilization, or incubated 4h and 8h before assessment of motility, capacitation, and acrosome integrity. The degree of variability in percentage of oocytes fertilized was assessed along with several factors that might contribute to this variation. Variation among bulls was not significantly different. Variation from one replicate to another was high. Variation was found in motility, capacitation, and frequency of acrosome reaction, but these variables were not significantly correlated to fertilization rate in vitro.