Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in Turkeys

Semen of Turkeys between 31 and 52 weeks of age was analyzed to investigate the cause of reduction in Turkey fertility at the end of the reproductive period. Sperm motility and viability, lipid concentration, fatty acid composition and lipid peroxides were evaluated on fresh spermatozoa or spermatozoa stored for 48h at 4 degrees C. Fertility of fresh semen was also evaluated.Fertility obtained with fresh semen decreased at 44-47 weeks of age. Ageing was also accompanied by a decrease in sperm viability (at 47 weeks) and later by a decrease in motility of spermatozoa (at 52 weeks). Polyunsaturated fatty acids (PUFAs) were the first lipids of fresh spermatozoa affected by age, especially n-3 and n-9 PUFAs. Changes in these PUFAs were followed by a 30% increase in lipid peroxidation at 47 and 52 weeks of age and a reduction in phospholipid content at 52 weeks.In vitro storage did not cause lipid peroxidation in sperm obtained during the first half of the reproductive period but malondialdehyde (MDA) levels significantly increased in sperm obtained during the second half of this period. In vitro storage also decreased phospholipid content of spermatozoa from 41 weeks of age, and viability and motility regardless of age.In conclusion, lipid alteration mainly originating from PUFAs peroxidation could partly explain the decrease in semen quality and fertility observed with ageing. In addition, lipid peroxidation was increased during in vitro storage of spermatozoa from older Turkeys.     

Attempts on freezing the Greylag (Anser anser L.) gander semen

Semen of Greylag (Anser anser L.) ganders was frozen according to a method previously elaborated by the authors for freezing the White Koluda gander semen. Semen was collected from five to eight Greylag ganders, twice a week during three succeeding reproductive cycles, by dorso-abdominal massage. Semen samples were diluted in the ratio of 1:1 or 2:1 (two parts semen: one part diluent) with EK diluent, supplemented by 6% DMF, equilibrated and pre-frozen to -140 degrees C at a rate 60 degrees C/min, before being transferred into liquid nitrogen container. Semen samples thawed in a water bath of 60 degrees C were used for twice a week insemination in a volume of 200 microl. Three Greylag and three White Koluda geese were involved in frozen-thawed semen fertilizing ability test. The reproductive cycle of wild geese lasts usually about 6-7 weeks. The ejaculate volume (30-140 microl) and sperm concentration (10x10(6) to 150x10(6) ml(-1)) are much lower than these of domestic ganders, but spermatozoa morphology is similar, particularly while compared to 1-year-old White Koluda ganders semen. There are about 90% of live spermatozoa and about 30% of live morphologically normal cells in Greylag gander fresh semen. The Greylag gander spermatozoa susceptibility to cryopreservation procedure is as high as in domestic ganders. Dilution ratio 2:1 resulted in higher number of live spermatozoa, which withstood cryoinjury stress. In relation to fresh semen about 60% of spermatozoa remained intact (on the basis of light microscope examination) in the frozen-thawed semen. Insemination of frozen-thawed semen resulted in 37.5% of fertile eggs in Greylag and 25.0% in White Koluda geese. Low fertility rate was caused by an insufficient number of live normal spermatozoa used for insemination (about three million in every dose).     

Effects of semen filtration and dilution rate on morphology and fertility of frozen gander spermatozoa.

Feces, urates or dirt originating from feathers often contaminate gander semen during collection, threatening its fertilizing ability. Seminal plasma used as a diluent has a similar effect, particularly on spermatozoa subjected to cryopreservation or short-term storage under refrigeration. The aim of the experiments was to evaluate the effects on spermatozoa motility, morphology and fertilizing ability after minimizing the influence of the contaminants by semen filtration or dilution prior to freezing. Pooled semen, collected twice a week from 9 White Italian ganders by dorso-abdominal massage, was divided into two parts. One sample was filtered and both were diluted in 1:1 or 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with dimethyl-acetamide (DMA) in the final concentration 6% (v/v) and frozen to -140 degrees C in a computerized freezer, at a rate of 60 degrees C/min. In fresh and processed (filtered, freeze-thawed) semen were examined the spermatozoa motility and morphology, and fertilizing ability for freeze-thawed semen, both for unfiltered and filtered. In freeze-thawed semen no tangible differences due to experimental factors were observed in motility and percent of live spermatozoa in total. On average 35 to 42% of the spermatozoa survived the freezing process, but only 10 to 15% were normal, without any damage visible under the light microscope. The fertility of unfiltered freeze-thawed semen inseminated twice a week in a 0.2 mL dose (about 3 to 5 x 10(6) of live normal spermatozoa each) averaged 66.1% and hatchability of the set eggs 57.1 and 86.5% of the fertile eggs. The fertility obtained after the insemination with semen filtered prior to freezing was lower (64.3%), but hatchability was slightly higher (58.6 and 91.1% of set and fertile eggs, respectively). The duration of fertility for filtered semen was longer than that for unfiltered, 10 days after the last insemination the eggs were still fertile. The fertility results of freeze-thawed gander semen were very promising taking into consideration the small amount of inseminated live normal spermatozoa and it is possible to improve this result by increasing the number of spermatozoa in the insemination dose.     

Evaluation of fresh and frozen-thawed semen of individual ganders by assessment of spermatozoa motility and morphology

Individual differences in gander Anser anser L. reaction to semen collection procedure, quality and quantity of fresh semen and its susceptibility to the freezing process are discussed. Semen was collected individually by dorso-abdominal massage, from 1-year old White Koluda ganders (n = 12) every 2-3 days. Ganders' reactions to massage were observed during the entire reproductive cycle (from 11 February to 13 June, from every male 40 semen collections were performed). For individual evaluation and freezing purpose semen was collected 13 times from every male. In the fresh semen, the following parameters were evaluated: ejaculate volume, color, density, blood or fecal contamination, motility, concentration and morphology of spermatozoa. Motility and spermatozoa morphology were evaluated in the frozen-thawed semen. Semen diluted in 2:1 ratio with EK diluent was frozen with 6% of dimethyl-formamide (DMF) to -140 degrees C at a rate 60 degrees C/min. Semen was thawed by placing the straws in a 60 degrees C water-bath for 4-5 s. Ten out of 12 ganders had from 67.5 to 100.0% positive reactions resulting in semen ejaculation. Significant (P < or = 0.01) differences in fresh semen quality of particular ganders were observed for all evaluated traits. In 1-year-old gander semen morphologically intact spermatozoa constitute only 27.8-45.2% of all cells. Therefore, the sperm quality factor (SQF), proposed by the authors, which includes ejaculate volume, sperm concentration and the percentage of live normal spermatozoa, seems to be a good predictor of gander semen fertilizing ability. The SQF of individual ganders varied from 7.7 to 11.5. The percentage of live normal spermatozoa in the frozen-thawed semen depended mainly on fresh semen quality. In relation to the fresh semen average from 57.2 to 63.2% of spermatozoa survived freezing process and from 23.9 to 38.5% remained morphologically intact.     

An effective method for freezing White Italian gander semen

Efficiency of freezing method, worked out for the White Italian gander semen was evaluated by comparing motility, morphology and fertilizing ability of spermatozoa in fresh and frozen-thawed semen. A part of pooled semen, collected from 25 White Italian ganders by dorso-abdominal massage was used immediately for artificial insemination of 10 geese (the control group) with a dose of 80 microl. This insemination was performed six times at weekly intervals. The remainder of the semen was diluted 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with 6% (v/v) of dimethylformamide (DMF) and frozen to -140 degrees C at a rate of 60 degrees C/min. Frozen semen was thawed in a 60 degrees C water-bath and inseminated twice weekly in a dose of 100 microl (10 females of the experimental group, 12 inseminations were made). The freezing process affected spermatozoa motility and morphology, but had no effect on their fertilizing ability. Positive movement was observed in 50-60% of the spermatozoa in fresh semen and about 40% of the frozen-thawed cells. The average percentage of total live and live normal spermatozoa decreased due to freezing from 92.2 to 68.4% and from 34.7 to 14.1%, respectively. After the fresh semen insemination with average 12 million of the live normal spermatozoa per week average fertility was 88.24%; hatchability of set eggs was 80.88% and hatchability of fertile eggs was 91.67%. For frozen-thawed semen inseminated with average 9.5 million of the undamaged spermatozoa per week, the average fertility and hatchability rate was 83.78, 73.87, and 88.17%, respectively. Fecundity rates obtained after insemination with the frozen-thawed gander semen allow for the application of the freezing technique into breeding practice, in place of natural mating or to assist natural mating in periods of lowered fertility level.     

Seasonal timing of sperm production in roe deer: interrelationship among changes in ejaculate parameters,morphology and function of testis and accessory glands

Roe deer are seasonal breeders with a short rutting season from mid-July to mid-August. The seasonality of reproductive activity in males is associated with cyclic changes between growth and involution of both testes and the accessory sex glands. This study characterizes morphological and functional parameters of these organs prior to, during and after breeding season in live adult roe deer bucks. Size and morphology of the reproductive tract was monitored monthly by transcutaneous (testes, epididymis) and transrectal (accessory glands) ultrasonography. Semen was collected by electroejaculation. Concentration, motility and morphological integrity of spermatozoa as well as the content of proteins and testosterone in semen plasma were evaluated. Proportions of haploid, diploid and tetraploid cells were estimated by flow cytometry in testicular tissue biopsies. Serum testosterone was measured by enzyme immunoassay. Most parts of the male reproductive tract showed distinct circannual changes in size and texture. These changes were most pronounced in the testes, seminal vesicles, and prostate. All reproductive organs were highly developed during the rut only. The volume of ejaculates, total sperm number and percentages of motile and intact spermatozoa also showed a maximum during this period and corresponded with high proportions of haploid cells in the testis. The highest percentages of tetraploid cells were found in the prerutting period. The production of motile and intact spermatozoa correlated with both the protein content of semen plasma and the concentration of testosterone in semen plasma and blood serum. These results suggest the importance of combined actions of the testes and accessory sex glands and the crucial role of testosterone in facilitating the optimal timing of intensified semen production to ensure sufficient numbers of normal spermatozoa in seasonal breeders.     

Effect of Dietary Selenium and Vitamin E on Ganders’ Response to Semen Collection and Ejaculate Characteristics

Compared to other domestic bird species, geese exhibit the lowest reproductive efficiency (poor semen quality, low egg production, and poor fertility and hatchability rates). From an economic perspective, it is a necessity of improve these reproductive traits. Studies have demonstrated that the essential trace element—selenium—plays key roles in testicular development and the maintenance of spermatogenesis. The aim of the present study was to determine the effect of feed supplementation with organic selenium and vitamin E on ganders’ response to manual semen collection and semen quality. Sixteen 3-year-old White Koluda ganders were randomly divided into two groups. The control group was provided commercial feed while the experimental group was provided with the same commercial feed supplemented with selenium (0.3 mg/kg) and vitamin E (100 mg/kg). The response of individual ganders from both groups to manual semen collection and the quality of the semen collected were evaluated. The supplements increased (P?≤?0.05) the frequency and decreased the time interval of a complete ejaculatory response of the ganders to manual semen collections (82.7 % supplement vs. 73.5 % control). Males from the supplemented group had significantly higher (P?≤?0.01; P?≤?0.05) ejaculate volumes, sperm concentrations, and percentages of viable sperm and lower percentages of immature sperm (spermatids). Lipids peroxidation, expressed in terms of the malondialdehyde concentration, was lower (P?≤?0.01) in semen of the supplemented group (0.172 nmol/50?×?10⁶) as compared to the controls (0.320 nmol/50?×?10⁶). Moreover, the duration of the reproductive period of the ganders in the experimental group was 1 week longer. The results show that supplemental dietary selenium and vitamin E improved both the ganders’ response to manual semen collection and semen quality. We conclude that such feed supplementation could lead to greater economic benefits through increased reproductive efficiency within the goose production industry.     

Effect of dietary Vitamin E on the cloacal gland, foam and semen characteristics of male Japanese quail

This experiment was to investigate the effects of increasing the level of dietary Vitamin E (Vit. E) on cloacal gland size, foam production and semen characteristics of male Japanese quail (Coturnix coturnix Japonica). One hundred and eighty male Japanese quail chicks (day old) were randomly distributed to three dietary treatments for a period of 25 weeks. Each treatment comprised of three replicates each containing 20 chicks. The basal diet contained 15 IU Vit. E/kg and the two experimental diets were supplemented with 150 and 300 IU Vit. E/kg (diets T2 and T3, respectively). DL alpha-tocopherol acetate was used as the source of Vit. E. All chicks were provided feed and water ad libitum. Foam characteristics, in terms of frequency of foam discharge (24h), cloacal gland index and foam weight were significantly higher (P<0.05) in T2 group. Body weight, testes weight (left and right) and plasma testosterone concentrations did not differ significantly. Semen characteristics (semen volume, sperm motility, % live sperm, % hatchability and sperm concentrations) did not differ significantly (P>0.05). Percentages of abnormal and dead spermatozoa were significantly (P<0.05) lower and fertility was higher (P<0.05) in the T2 group. From this study, it can be concluded that moderate supplementation of dietary Vit. E may be beneficial for foam production, cloacal gland and improve the semen characteristics in male Japanese quail.     

Extending the interval between second vaccination and slaughter: II. Changes in the reproductive capacity of immunocastrated ram lambs

Immunocastration improves the welfare of castrated commercial slaughter lambs; however, the time-point at which this technique influences semen quality and sperm production has not yet been established for various vaccination schedules. Furthermore, the effect of extended intervals between second vaccination and slaughter needs to be investigated regarding continued testosterone suppression in immunocastrated lambs. The effect of extending the interval between second immunocastration vaccination and slaughter from four to six weeks on the reproductive capacity of Dohne Merino lambs was examined. A total of 40 Dohne Merino lambs were stratified according to initial weight (45.4±3.68 kg) and randomly assigned to four treatments that included intact control rams (R), Burdizzo-castrated lambs (B) and lambs immunocastrated with either four (ICS4) or six (ICS6) weeks between second vaccination and slaughter. Blood and semen samples were collected throughout the study period to determine serum testosterone concentrations, evaluate semen quality and assess sperm viability. Semen samples from R showed improvement over the trial. Throughout the collection period, B lambs had low serum testosterone concentrations, poor sperm motility and sperm viability, as expected. However, a slight increase in the percentage of live sperm in semen samples from B lambs towards the end of the collection period indicated poor success rates of the technique in some lambs. Burdizzo-castration also caused testes tissue necrosis and abscessing, indicating physiological stress. Semen appearance scores varied for both immunocastrated treatments, but the mass motility scores decreased over time. The ICS6 lambs showed a consistent and continuous decline in serum testosterone concentrations and sperm viability, with an increased percentage of dead abnormal sperm in the semen samples at the end of the study. The ICS4 treatment was successful in interrupting serum testosterone production and reducing semen quality; however, not as consistently as the ICS6 treatment. Primary immunocastration vaccination influenced serum testosterone concentrations but consistently low levels were only realised for both treatments after secondary vaccination. Although all castration treatments influenced testes size and colour, the six-week vaccination-to-slaughter interval caused a greater decrease in testes cut surface L* (lightness) colour values and in seminiferous tubule circumference. Extending the interval between second immunocastration vaccination and slaughter resulted in a more consistent and reliable influence on reproductive capacity of immunocastrated lambs. Thus, immunocastration is a suitable alternative to Burdizzo-castration regarding the interruption of testosterone production and testis functioning.     

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.     

The possibility of obtaining intergeneric hybrids via White Kołuda (Anser anser L.) goose insemination with fresh and frozen-thawed Canada goose (Branta canadensis L.) gander semen

The objective of the present experiments was to produce the intergeneric hybrids of domesticated and wild goose via artificial insemination with fresh and frozen-thawed semen. The experiments were carried out during two successive goose reproductive seasons, on eight five-year-old Canada Goose (Branta canadensis L.) males used as semen donors and 16 two-year-old White Ko?uda geese designated to fertility tests. Pooled semen was collected twice a week by the dorso-abdominal massage. In freshly collected semen, ejaculate volume, color, consistency, degree of fecal or blood contamination, spermatozoa concentration, motility, and morphology were evaluated. Part of the semen collected in the first year of the experiment (Experiment 1) was used for geese insemination with fresh semen, while the remainder was frozen. In Experiment 2 all samples were subjected exclusively to freezing procedure. Geese were inseminated once a week with fresh semen in a dose of 80 μl or 160 μl, and twice a week with frozen-thawed semen in a dose of 80 μl (160 μl per wk) or 100 μl (200 μl per wk). Eggs were set weekly and incubated up to hatching.The volume of ejaculates varied from 0.100 to 0.470 ml; spermatozoa concentration from 140 to 310 million ml⁻¹; progressive movement was observed in 40 to 60% of spermatozoa; the percentage of total live spermatozoa ranged from 69.3 to 92.0%, the highest percentage (34.0-68.3) was represented by live normal spermatozoa and those with bulb-head (13.3-41.0). Cryopreservation caused a decrease in percentage of motile cells to 30%; total live spermatozoa contribution by 27.2%p, including those live normal by 15.9%p (in relation to the fresh semen), bulb-head spermatozoa by 10.9%p, and increase (by 5.9%p) in number of spermatozoa with other deformations. Goose insemination 1×/week with fresh semen containing about 10.3 million live normal spermatozoa resulted in 66.7% of fertile eggs and with dose higher by 2.8 million spermatozoa (on average) the fertility increased by 20.9%p (up to 87.6% on average). Hatchability from set and fertile eggs was 55.9% and 83.9% vs. 66.3% and 75.6%, respectively. After twice a week insemination with frozen-thawed semen containing about 10.2 million live normal cells 58.2% eggs were fertile; hatchability from set eggs was 42.8% and from fertile eggs 71.7%, while insemination dose increase by 2.7 million spermatozoa per week caused a fertilization increase by 3.8%p (62.0% on average), this increase was not statistically significant, but hatchability from the fertile eggs (95.4%), was significantly (P < 0.05) higher.The use of AI with fresh semen in the creation of intergeneric hybrids of Canada goose males and White Ko?uda females allows a high level of egg fertility to be obtained. Furthermore, one limitation which is the short reproductive season of the Canada goose may be overcome by the use of cryopreserved semen.     

Influence of prostaglandin F2 alpha on sperm production and seminal characteristics of the stallion

Six mature stallions were used to test the effect of prostaglandin F2 alpha (PGF2 alpha ) on sperm production and seminal characteristics. Semen was collected from each stallion twice weekly 1 hr following a 10 mg intramuscular injection of PGF2 alpha or a sham injection. A switchback design was used so that three stallions received PGF2 alpha and three served as controls during the first 9 weeks (period 1). Treatment regimens were reversed during the second 9 weeks (period 2). Treatment of stallions with PGF2 alpha resulted in an increase (P less than .05) in gel free seminal volume and a decrease in sperm cell concentration. Total spermatozoa, sperm cell motility, and percentage of primary and secondary sperm abnormalities of ejaculates were not significantly affected by treatment of stallions with PGF2 alpha before semen collection. All treated stallions exhibited a pronounced sweating response to the drug. During the experiment, two of the six stallions masturbated within 20 to 30 minutes after PGF2 alpha treatment without achieving an erection.     

Autophagy and apoptosis have a role in the survival or death of stallion spermatozoa during conservation in refrigeration

Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described.     

The effect of selection for fertility of frozen-thawed semen on spermatozoa oxygen uptake, motility and concentration, and ejaculate volume in the chicken

The response to 3 generations of selection for duration of fertility of frozen-thawed semen on fertility and hatchability of fresh and frozen-thawed semen, spermatozoa oxygen uptake, motility and concentration, and ejaculate volume was measured in a broiler line of chickens. The selected line, as compared to the control, had significantly higher levels for all fertility traits of frozen-thawed semen but not hatchabilities. For fresh semen, the lines differed only for duration of fertility. The only difference in semen quality traits was in oxygen uptake by spermatozoa of frozen-thawed semen. The differences between lines for the other parameters were not significant but were in a direction that supported the hypothesis that selection had resulted in improved reproductive capacity. All estimates of fertility within an ejaculate were positively correlated (P < 0.01). Correlations between corresponding fertility estimates of fresh and frozen-thawed semen were positive, low and generally non-significant. The proportion of variation as determined by stepwise regression, in fertility estimates accounted for by the measurements of oxygen uptake and motility of fresh and frozen-thawed spermatozoa, ejaculate volume and spermatozoa concentration ranged from 12 to 19 percent.     

Electroejaculation, semen characteristics and serum testosterone concentrations of free-ranging african elephants (Loxodonta africana)

A regimented electroejaculation protocol (120 electrical stimulations; 10-30 V) was used to collect semen and characterize ejaculate quality from 9 adult, free-ranging African elephants under anaesthesia. Eight of the 9 ejaculates contained high concentrations of progressively motile spermatozoa. The overall mean ejaculate volume, sperm concentration/ml ejaculate, sperm motility, sperm status and ejaculate pH were 93.3 ml, 2408.6 X 10(6) spermatozoa/ml, 70%, 3.9 and 7.4, respectively. A high percentage (mean 77.5%) of spermatozoa within each ejaculate was morphologically normal. Of the aberrant spermatozoa, 72% had a cytoplasmic droplet defect. When sperm viability was tested in vitro at 37 degrees C, sperm motility rating declined by at least half of the initial assessment within 3.5 h of semen collection. Generally, spermatozoa maintained motility in vitro for less than 6 h. Serum testosterone ranged from 1.4 to 8.2 ng/ml in 4 males evaluated in the morning (07:30-08:00 h). In 4 of the 5 bulls assessed in the afternoon (15:00-18:00 h), testosterone levels were less than 0.9 ng/ml. The remaining bull, evaluated at 16:00 h, had exceptionally high testosterone concentrations (peak 25.6 ng/ml) and a preputial discharge potentially indicative of 'musth'. The present study demonstrates that high quality semen can be collected consistently from the African elephant and that striking differences exist in serum testosterone amongst free-ranging males which may be due, in part, to a diurnal rhythm.     

In vitro fertility and motility characteristics of frozen-thawed boar epididymal spermatozoa separated by Percoll

Frozen-thawed epididymal spermatozoa from four boars were separated through a Percoll gradient, and motility characteristics and in vitro fertility were assessed. Percoll-separated spermatozoa had a significantly higher percentage of motile and progressively motile spermatozoa than those that were not separated (P < 0.0001). However, there were no clear differences in other motility parameters between Percoll-separated and un-separated spermatozoa. Furthermore, sperm agglutination was decreased by Percoll separation (P < 0.05). The effects of Percoll separation on in vitro fertility of spermatozoa differed among boars. In addition, there were large differences in fertility between sperm samples in vitro. Sperm samples, which indicate highly motile and progressively motile, did not always show high in vitro fertility. Furthermore, there was no distinct pattern between fertility in vitro and motility parameters. There was no difference in fertility in vitro between Percoll-separated and un-separated spermatozoa from two of the four boars. However, in vitro fertility of Percoll-separated spermatozoa was higher than that of un-separated spermatozoa from the other two boars.     

RELATIONSHIPS AMONG MORPHOLOGICAL STATUS, STEROID HORMONES, AND POST-THAWING VIABILITY OF FROZEN SPERMATOZOA OF MALE MINKE WHALES (BALAENOPTERA ACUTOROSTRATA)

Spermatozoa from 21 mature minke whales ( Balaenoptera acutorostrata ) taken in the Antarctic Ocean for Japanese research were recovered from vasa deferentia, diluted 1:9 in a Tris-based diluent, and frozen at - 80°C on board the vessel. After a period ranging from 45 to 125 d, the samples were transferred to liquid nitrogen and transported to the laboratory. After thawing at 37°C the motility (percentage of motile spermatozoa), vitality (proportion of live spermatozoa), and sperm concentration were determined for each sample. These values were tested for correlations with morphological measurements (body size, body weight, testis weight) and serum concentrations of progesterone (Pd), estradiol-17β (E2), and testosterone (T). Ten of 21 samples had motile spermatozoa (2%-40%). Although no motile spermatozoa were observed in 1.1 samples, all sperm samples were examined by eosinnigrosin staining and showed vitality levels of 3%44%. It was found that the motility (Y = 0.54) and vitality (r = 0.53) of the spermatozoa were significantly (P < 0.01) correlated with the E₂ levels (8.50 ± 1.80 pg/ml). Serum T levels (0.07 ± 0.02 ngml) were significantly correlated with the E₂ levels (r = 0.58, P < 0.01>, but sperm concentrations were not correlated with either Ea or T levels. The present study demonstrates that spermatozoa of minke whales can be successfully cryopreserved.     

Comparisons between three different extenders for canine intrauterine insemination with frozen-thawed spermatozoa

Ejaculates from 7 dogs were obtained on the same day and were pooled. This pooled semen was separated into 3 equal fractions and processed simultaneously, the only difference being in the extender used for freezing. The extenders were laiciphos (containing laiciphos, egg yolk, distilled water and glycerol- Group 1); Tes/Tris (containing Tes/Tris, egg yolk, distilled water and glycerol- Group 2); and biociphos (containing biociphos with glycerol in it, egg yolk and distilled water- Group 3). Spermatozoa were conditioned in 0.5ml French straws and presented normal characteristics before freezing and after thawing. The sperm concentration of the pooled was 683 x 10(6) sperm/ml; sperm motility was above 95%, the percentage of live spermatozoa was above 95% and was of good quality and mobility. Characteristics of the spermatozoa after thawing were the same for spermatozoa frozen with laiciphos and Tes/Tris. Mean sperm concentration was 201.5 +/- 4.95 x 10(6) sperm/ml, sperm motility was 65%, the percentage of live spermatozoa was 80% and the quality of motility.was good. Spermtozoa frozen with biociphos had the following post-thaw characteristics: sperm concentration was 201 x 10(6) sperm/ml, sperm motility was 50%, the percentage of live spermatozoa was 78% and the quality of mobility was medium. Abnormalities were less than 15% for all spermatozoa after thawing. Intrauterine artificial inseminations were performed by laparoscopic intrauterine insemination twice at Days 3 and 5 after the estimated LH peak in 15 normally cyclic Beagle bitches (5 per group) presenting normal hormonal profiles. There were no differences between groups. The females were inseminated with 1.0 ml of spermoatozoa (concentration of 200 x 10(6) sperm/ml) diluted with 1.0 ml of extender. A 60% pregnancy rate was obtained in bitches inseminated with frozen-thawed spermatozoa extended with laiciphos or Tes/Tris and 100% in bitches inseminated with spermatozoa extended with biociphos. Females inseminated with laiciphos, Tes/Tris and biociphos had a mean litter size of 5 +/- 2.6, 3 +/- 1 and 3.4 +/- 1.3 pups, respectively. This study demonstrated that post-thaw assessment of sperm characteristics is not the best technique for evaluating sperm fertility after freezing or for assessing different semen extenders.     

Subtle membrane changes in cryopreserved bull semen in relation with sperm viability,chromatin structure,and field fertility

This study investigated the use of annexin-V/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen-thawed semen samples were obtained from 18 Swedish Red and White bulls (one to three semen batches/bull) and fertility data was based on 6900 inseminations. The annexin-V/PI assay revealed that post-thaw semen samples contained on average 41.8+/-7.5% annexin-V-positive cells. Most of the annexin-V-positive cells were dying cells, i.e. also PI-positive. The incidence of annexin-V-positive cells was negatively related (r=-0.59, P<0.01) to the percentage of viable cells, as detected by fluorometry. The incidence of annexin-V-positive spermatozoa significantly correlated to the SCSA variable xalphat (r=0.53, P<0.05). The incidence of annexin-V-negative, dead cells was the only annexin-V/PI assay variable that correlated significantly with fertility both at batch (r=-0.40, P<0.05), and bull (r=-0.56, P<0.05) levels. Among sperm viability variables, subjectively assessed sperm motility (r=0.52-0.59, P<0.01), CASA-assessed sperm motility (r=0.43-0.61, P<0.05), and the incidence of live spermatozoa, expressed as total numbers (r=0.39-0.54, P<0.05), or percentage values (r=0.68-0.68, P<0.01), correlated significantly with field fertility both at batch, and bull levels. Among the SCSA variables, only the COMP alphat correlated significantly (r=0.33-0.51, P<0.05) with fertility results. The results indicate a certain proportion of bull spermatozoa express PS on their surface after thawing, e.g. they have altered membrane function, and that the incidence of such cells is inversely correlated to sperm viability, and positively correlated to abnormal sperm chromatin condensation since they eventually undergo necrosis.     

Improvement of llama (Lama glama) seminal characteristics using collagenase

Llama semen is characterized by great structural viscosity and minimal sperm progressive motility. These characteristics, inherent to South American Camelid ejaculates, have slowed down the development of assisted reproductive techniques in these species. The aim of the present research was to evaluate the effect of different combinations of dilutions and incubation time with H-TALP-BSA medium, with and without adding 0.1% collagenase, on the qualitative and quantitative semen characteristics, for its use in assisted fertility techniques. Ejaculates (n = 8; r = 3) were obtained using electroejaculation. Each ejaculate was evaluated and then split into four aliquots. Two of these were diluted 4:1 and 8:1 in 0.1% collagenase in H-TALP-BSA (treatments 1 and 3) and the other two 4:1 and 8:1 in H-TALP-BSA without collagenase (treatments 2 and 4). Treatments 1 and 2 were incubated 4 min at 37 °C while treatments 3 and 4 were incubated 8 min. All aliquots were centrifuged at 800 × g for 4 min immediately after incubation. Supernatants were pipetted to observe thread formation and pellets were re-diluted in H-TALP-BSA. Supernatants from samples treated with collagenase did not form a thread when pipetted, while the ones from samples that were not treated with the enzyme did. Only semen samples treated with collagenase showed progressive sperm motility, with averages over 40%. There were no significant differences (P > 0.05) for the percentage of live spermatozoa and for the percentage of detached heads between raw and treated semen samples. Percentages of spermatozoa with functional membranes were significantly higher (P ≤ 0.05) in samples treated with collagenase than in raw semen and in samples incubated without collagenase. These results show that treating semen with 0.1% collagenase in H-TALP-BSA improves semen rheological properties while facilitates the separation of spermatozoa from seminal plasma in llama; it also promotes sperm progressive motility, while maintaining sperm membrane functionality and integrity. Consequently, this protocol could be used for in vitro llama embryo production with ejaculated spermatozoa.