Freezability of spermatozoa from Finn and Dorset rams in multiple semen extenders

Ten semen extenders were tested in two experiments for cryopreservation of semen collected from four Finn and four Dorset rams. Two ejaculates of semen were combined from each ram for testing each extender treatment. The extenders consisted of a series of commonly used egg yolk-TRIS media with and without sodium and triethanolamine lauryl sulfate (STLS), a similar extender with 3-N-morpholino propane sulfonic acid (MOPS), and milk and whey extenders. In Experiment 1, extender treatments were replicated with three sets of collections from the eight rams, and in Experiment 2 with two sets. The egg yolk-TRIS-glycerol-STLS (EY(1)TSTLS) extender was significantly superior to other extenders except whole milk in protecting the sperm during freezing and thawing. In Experiment 1, a 20% egg yolk-TRIS-glycerol-STLS extender preserved 71% of the progressively motile Finn sperm (post-thaw divided by pre-freeze percentage of motile sperm), and 76% of the Dorset sperm. In Experiment 2, the corresponding values for the same EY(1)TSTLS extender used with Finn and Dorset sperm were 86 and 64%, respectively. Without STLS the egg yolk extenders were significantly less effective in protecting cryopreserved ram sperm. This egg yolk-TRIS extender, containing STLS and glycerol, may hold promise for freezing ram sperm that could be used successfully for intracervical insemination.     

The influence of breed,season and photoperiod on semen characteristics,testicular size,libido and plasma hormone concentrations in rams

Twenty-seven adult rams (9 Suffolk, 9 Texel and 9 Dorset Horn) were raised under natural photoperiod and were trained to serve into an artificial vagina. On 1 April they were abruptly exposed to 3 different photoperiods as follows: (i) 8 hours light and 16 hours darkness (8L : 16D); (ii) 16 hours light and 8 hours darkness (16L : 8D); (iii) natural photoperiod. All rams were kept at pasture daily between 09.30 h and 16.00 h except when required indoors for experimental work. Rams on artificial photoperiod had appropriate supplemental lighting in an environmental chamber. Semen collection was attempted from each ram on alternate weeks during the experiment which lasted for 6 months. Semen was evaluated for volume, density, motility and abnormalities. Testicular length and circumference were recorded at 2-week intervals and libido was recorded at 4-week intervals. Three blood samples were collected from each ram at 30-min intervals on a weekly basis and the plasma was stored at ?20°C until assayed for testosterone and prolactin.Photoperiod had no significant effect on semen volume, motility and percentage dead or abnormal cells. Breed of ram had a significant effect on semen motility (P < 0.05) with Dorset Horn rams producing semen with the highest motility. Volume and motility scores both increased as the breeding season approached (P < 0.05), while the percentage of abnormal cells decreased (P < 0.01). Breed or photoperiod did not significantly affect scrotal measurements although animals exposed to 8L : 16D had the highest measurements. Month affected testicular measurements which generally increased from April to September. Suffolk rams had higher testosterone concentrations, and this breed also completed the highest number of mounts within an allocated test time (P < 0.05). Dorset Horn rams reached a peak in testosterone concentrations in June/ July whereas Suffolks and Texels reached a similar peak in August. Prolactin concentrations decreased from a maximum at the start and rams on natural photoperiod tended to have highest levels. These results show that month can have a bigger influence on semen characteristics than imposed artificial photoperiods in rams which have been exposed to increasing natural daylength for some months.     

Seasonality of semen production and plasma luteinizing hormone,testosterone and prolactin levels in Romney,Merino and polled dorset rams

An attempt was made to define the seasonality of reproduction in rams in the southern hemisphere by repeated measurement of semen characteristics and of plasma luteinizing hormone (LH), testosterone and prolactin concentrations. These parameters were studied for 16 months in Romney rams on pasture, with Merino and Polled Dorset rams included for comparison.Semen from all three breeds showed regular seasonal changes in ejaculate volumes, with peak values being recorded during March. A similar autumnal peak of seminal fructose levels was noted for ejaculates from Romney and Merino rams, but not for those from Polled Dorsets. Most of the other semen parameters measured showed little tendency for seasonal variations. However, a change in semen collection technique, from predominantly artificial vagina to entirely electroejaculation, may have masked some seasonal changes.Plasma hormone levels also varied in a regular manner, with peak levels occurring in summer and autumn: highest levels for prolactin were recorded in November–March, for LH in December–February and for testosterone in January–March. An exception to this pattern was recorded from the Merino rams, for which there was no definite peak of LH secretion.It is suggested that these seasonal changes resulted primarily from changes in daily photoperiod.     

Effects of exposing boars to different artificial light regimens on semen plasma markers and "in vivo" fertilizing capacity

This study was designed to assess the effects of exposing boars to an artificial photoperiod on semen quality in terms of sperm concentration, sperm vitality, sperm motility and acrosome integrity. We also determined biochemical semen plasma variables, such as total protein concentration, phosphorylated tyrosine residues and fructose, glucose and sorbitol contents, along with their effects on the fertility, prolificacy and libido of the boars. Three groups of 10 males were kept for 3 months under experimental conditions of 24, 12 and 0 h of artificial light, and a constant temperature of 21 +/- 1 degrees C and 60-75% humidity. The animals were fed a nutritious diet and subjected to semen collection twice per week. Semen samples were analyzed throughout the entire experimental period. Our results indicate that, while the extreme photoperiods (0 and 24 h of light) affected semen quality in terms of sperm concentration, acrosome integrity and semen volume, its fertilizing capacity was only significantly reduced under conditions of absolute darkness. Sperm motility was found to be a poor indicator of fertilizing ability, while other sperm factors, such as acrosome integrity or other functional variables seemed to behave better. The photoperiod was found to affect the production of accessory sex gland secretions more than their composition. In addition, light effects on fertility, prolificacy and libido seemed to be achieved through independent mechanisms.     

Comparison of ram sperm interaction with bovine cervical mucus

Ram sperm penetration in estrous bovine cervical mucus was evaluated from ejaculates collected during long (16L:8D) and short (8L:16D) photoperiods with varying ambient temperatures. The distance traveled by vanguard sperm was affected by an interaction of the photoperiod and temperature (P<0.001). Sperm migration distance in a capillary tube filled with mucus (22.5 to 23.2 mm) was greater when sperm were collected from rams on short days and when the ambient temperature was between 10 and 31 degrees C than when sperm were collected under either long or short days (15.5 to 17.8 mm), when ambient temperatures were between 1 to 9 degrees C. Incidence of head-to-head agglutination of sperm differed by temperature (P<0.05) and photoperiodic (P<0.09) conditions. The percentage of ejaculates with evidence of sperm agglutination in the mucus was higher in long (62.5%) vs short (45.2) days, and it was greater in sperm collected in warm (61%) vs cold (44%) days. Physical interaction of cervical mucus with spermatozoa was examined. The binding of an iodinated protein from lyophilized mucus to a detergent soluble extract of washed or unwashed sperm was observed. These data show that both photoperiod and temperature affect the interaction of ram sperm with bovine cervical mucus.     

Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions

This study analyzed effects of different methods and intervals of semen collection on the quantity and quality of fresh, cool-stored, and frozen-thawed sperm and fertility of AI stallions. In Experiment 1, ejaculates were obtained from six stallions (72 ejaculates per stallion) using fractionated versus non-fractionated semen collection techniques. Initial sperm quality of the first three jets of the ejaculate was not different from that of total ejaculates. Centrifugation of sperm-rich fractions before freezing improved post-thaw motility and sperm membrane integrity when compared to non-centrifuged sperm-rich fractions or non-fractionated centrifuged ejaculates (P<0.05). In Experiment 2, semen from four stallions (60-70 ejaculates per stallion) was collected either once daily or two times 1h apart every 48 h. The first ejaculates of double collections had significantly higher sperm concentrations, percentages of progressively motile sperm (PMS) after storage for 24h at 5 degrees C and lower percentages of midpiece alterations than single daily ejaculates. Semen collected once daily showed significantly lower values of live sperm after freezing and thawing than the first ejaculate of two ejaculates collected 1h apart every 48 h. In Experiment 3, semen was collected from 36 stallions (> or =12 ejaculates per stallion) during the non-breeding season and the time to ejaculation and the number of mounts was recorded. When time to ejaculation and the number of mounts increased, volume and total sperm count (TSC) also increased (P<0.05), whereas a decrease was observed in sperm concentration, percentage of PMS after storage for 24 h at 5 degrees C, percentage of membrane-intact sperm in fresh semen (P<0.05) as well as motility and percentage of membrane-intact sperm of frozen-thawed sperm (P<0.05). In Experiment 4, AI data of 71 stallions were retrospectively analyzed for the effect of number of mounts per ejaculation and frequency, time interval of semen collections on pregnancy, and foaling rates (FRs) of mares. Semen volume increased, but sperm concentration and percentage of PMS after 24-h cool-storage decreased with increasing number of mounts on the phantom (P<0.05). A statistically significant inter-relationship was demonstrated between frequency and interval of semen collection and FR. Mares inseminated with stallions from which semen was collected frequently (> or =1 on an average per day) showed significantly higher FRs than mares inseminated with semen from stallions with a daily collection frequency of 0.5-1 or <0.5. FR of mares inseminated with stallions having 0.5-1 days between semen collections was significantly better than FR of mares that were inseminated with stallions having semen collection intervals of 1-1.5 days or >2.5 days.     

Semen quality of postpubertal boars during increasing and decreasing natural photoperiods

The present study analyses the effects of increasing and decreasing photoperiods on the semen quality of 20 selected postpubertal Landrace boars. The boars were exposed, throughout 75 days, to increasing and decreasing photoperiods of natural light, a constant temperature of 21 +/- 1 degrees C and 60-70% of humidity, fed with a nutritious diet and, submitted to a rhythm of semen collection of twice a week. During the last 2 weeks of each treatment, semen samples were analysed and the parameters measured were: ejaculate volume and pH, sperm concentration, sperm production and the number of semen doses per ejaculate, sperm vitality, sperm motility, osmotic resistance of spermatozoa and sperm morphology. The comparative analysis between increasing and decreasing photoperiods indicated that the semen quality of boars exposed to a decreasing photoperiod was reduced as a consequence of decreases in sperm concentration, sperm production and the number of semen doses. There was no difference between increasing and decreasing photoperiods in terms of sperm vitality and sperm motility, nor in the osmotic resistance of spermatozoa to isotonic and hypotonic media. The analysis of sperm morphology showed significantly lower frequencies of mature and immature spermatozoa with a distal cytoplasmic droplet, and significantly higher frequencies of immature spermatozoa with a proximal droplet in boars exposed to the decreasing photoperiod. These results indicate that the sperm quality of the selected boars decreased during decreasing photoperiods, in comparison with increasing photoperiods, mainly due to impaired testicular function.     

Sperm production, testicular size, serum gonadotropins and testosterone levels in Merino and Corriedale breeds

The relationships between testis size, hormone secretion and sperm production were studied during the spring (December) and autumn (May) in rams of two breeds with different breeding seasons and body weights (Corriedale and Australian Merino) maintained on native pastures and under natural photoperiods in Uruguay. Blood samples were collected at 20-min intervals during a 260-360-min period in 13 rams (four Corriedale, nine Australian Merino) during the late spring and autumn. Rams were weighed and testis size was estimated by orchimetry at each time period. Sperm production was estimated during a 2-week period, 2 months before blood collection and during each week following every blood collection. There was no relationship between testicular size and sperm production measured at the same time, nor between live weight and sperm production. In contrast, testicular volume during the late spring was correlated with sperm production in the autumn (r = 0.65; P = 0.02). The autumn serum LH was higher in Corriedale than in Merino rams. LH pulsatility was unaffected by season, but LH pulse frequency tended to be higher in Corriedale than in Merino rams, particularly in the late spring (2.37 versus 1.56 pulses/6 h; P = 0.08). Serum testosterone concentration was similar in both breeds and seasons. FSH levels were higher in the late spring than in the autumn in both breeds (Corriedale: 2.83 +/- 0.48 versus 2.17 +/- 0.24 ng x mL(-1); Merino: 2.23 +/- 0.24 versus 1.88 +/- 0.17 ng x mL(-1)). FSH and testosterone concentrations during the late spring were positively correlated with autumn sperm production (P = 0.07 and P = 0.03, respectively). In conclusion, the present experiment suggests that LH secretion is not a good parameter for the prediction of sperm production. In contrast, in our conditions (breeds and native pastures) testicular size and testosterone or FSH concentrations from the late spring may be used to predict sperm production in the autumn.     

Testosterone secretion and semen plasma enzyme activity in rams with genital pathology stimulated with GnRH

Testosterone secretion in response to GnRH stimulation and enzymatic activity of semen plasma was evaluated comparatively in rams with or without genital abnormality. Scrota, testes and epididymides of 128 rams between 1.5 and 6 years old from various breeds were examined clinically and ultrasonographically. Bilaterally cryptorchid rams (n = 2), and rams with focal testicular degeneration (n = 3) or unilateral sperm granuloma localized in the caput (n = 3) epididymis or the cauda epididymis (n = 3), diagnosed by either clinical or ultrasonographic examination, were selected for the further investigation of spermatologic parameters, testosterone secretion in response to GnRH stimulation, and enzymatic activity of semen plasma before histopathologic confirmation of lesions. Except for the cryptorchid rams, sperm parameters determined in ejaculates were similar to intact controls (n = 3). GnRH administration increased plasma testosterone levels significantly irrespective of the type of genital pathology (P < 0.01). The testosterone response calculated based on area under the curve following GnRH administration in rams having genital abnormality was not significantly different from the controls. Aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activity in the semen plasma varied between rams, with the lowest mean values in the bilaterally cryptorchid group (P < 0.05). Spermatic granuloma localized either in the caput or cauda of the epididymis was associated with a significant reduction in the semen plasma AST activity compared to controls (P < 0.01). In conclusion, our results indicated that the ability of testicular tissue to secrete testosterone in response to GnRH stimulation in rams with bilateral cryptorchidism, focal testicular degeneration and unilateral sperm granuloma was similar to that of intact controls, and that reduced semen plasma AST activity may have a diagnostic value in the diagnosis of the epididymal obstruction in rams. Focal testicular degeneration did not influence AST, alkaline phosphatase (ALP) and LDH activity in semen plasma.     

Increasing storage time of extended boar semen reduces sperm DNA integrity

There is an extensive use of artificial insemination (AI) in the pig industry. Extended liquid boar semen may be used for insemination for up to 5 days after collection. The objective of this study was to determine the changes in sperm quality, when boar semen was extended and stored at 18 degrees C for up to 72 h post-collection. The study included three ejaculates from five boars, for each of the four breeds: Duroc, Hampshire, Landrace and Danish Large White (n=60 ejaculates). The sperm chromatin structure assay (SCSA) showed an increase in DNA fragmentation index (DFI) after 72 h of incubation (P<0.001), with no differences between breeds (P=0.07). For two Hampshire boars, all ejaculates had a large increase in DFI after 24 h of incubation. The standard deviation of DFI (SD-DFI) differed between breeds, with the SD-DFI for Hampshire being significantly greater than for the other breeds. The SD-DFI did not change during the 72 h of storage. Sperm viability was determined using SYBR-14 and propidium iodide in combination with flow cytometry. The sperm viability did not differ between breeds (P=0.21), but a difference in viability during storage (P<0.001) was detected. In conclusion, the SCSA cytogram patterns were consistent for different ejaculates within boars and storage of extended boar semen at 18 degrees C for 72 h significantly decreased the integrity of sperm DNA.     

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.     

Seasonal thyroidal activity and reproductive characteristics of Iranian fat-tailed rams

An experiment was conducted to study seasonal variations of thyroidal activity, serum testosterone concentration and seminal characteristics of two breeds of Iranian fat-tailed sheep. Eight 3 to 4-year-old rams of Ghezel and Mehraban breeds (4 rams/breed) were randomly selected from a flock of fertile rams. Semen was collected by using an artificial vagina, and blood samples were obtained via jugular vein. Seminal characteristics, scrotal length, width, and circumference, and blood parameters were measured at 3-week intervals. Time of the year significantly (P < 0.05) affected the volume of semen, sperm concentration, percent live sperm, percent normal sperm, total sperm number, seminal pH, seminal lactate dehydrogenase (LDH) concentration, scrotal circumference, scrotal width, and serum levels of testosterone, cholesterol, triiodothyronine (T(3)), tetraiodothyronine (T(4)), free T(4) index (FT(4)I), and TSH. Scrotal length was not affected by the time of sampling (P > 0.05). The effect of breed on the serum concentrations of hormones and cholesterol was not significant. Volume of semen, percent normal sperm, and total number of normal sperm were significantly (P < 0.05) higher in Mehraban but scrotal circumference, scrotal width and scrotal length were greater in Ghezel rams. Breedxtime interaction effect was significant for T(4), FT(4)I, sperm concentration, and total sperm number. The largest values for TSH, T(4), FT(4)I, testosterone, total sperm number, percent normal sperm, percent live sperm, sperm concentration, volume of semen and scrotal circumference were found from early summer to winter and the lowest values were recorded at the end of spring and in early summer. In both breeds, high and negative correlations (r = -0.7 to -0.8) were found between LDH levels in semen and percent live sperm, percent normal sperm and total sperm number (P < 0.01). It is unlikely that such variations in seminal quality would affect the fertility under natural mating.     

Opposite variations of two epididymal components and blood plasma testosterone in two breeds of rams.

1. Testicular volume (T Vol), blood plasma testosterone (T) concentration, seminal plasma alpha-glucosidase (alpha-G) specific activity, L-carnitine (L-C) concentration as well as semen characteristics were compared in eight Finnish Landrace (F) and eight Suffolk (S) rams throughout 21 months. 2. Only T Vol and T exhibited a typical seasonal variation in both breeds, whereas L-C, alpha-G and live sperm output presented a seasonal profile only in S rams. 3. L-C and alpha-G variations were opposite to those of T in S rams, while they fluctuated in F rams throughout the entire experiment, as did live sperm output. 4. Only the number of ejaculates and T were significantly higher in F rams (3.50 +/- 0.08 in 5 min and 7.62 +/- 0.40 ng/ml) than in S rams (2.30 +/- 0.05 in 5 min and 5.5 +/- 0.30 ng/ml); these two characteristics might therefore be considered as two indexes of sexual activity in rams. 5. By contrast, among all characteristics measured, only alpha-G was significantly higher in S rams than in F rams (1.33 +/- 0.04 vs 0.77 +/- 0.03 mU/mg proteins); this result, as well as seasonal alpha-G profile present in only S rams, allowed us to conclude that alpha-G might be considered as an additional index of seasonal reproduction in rams.     

Seminal traits, suitability for semen preservation and fertility in the native Portuguese horse breeds Puro Sangue Lusitano and Sorraia: Implications for stallion classification and assisted reproduction

The Puro Sangue Lusitano (PSL) is the major national breed of horse in Portugal, but no studies exist on its seminal characteristics, or on the possibility of conserving semen for future use. The aim of this study was to evaluate semen parameters, fertility and the aptness to semen preservation in Lusitano Stallions. In order to compare characteristics defined by a single or by multiple semen collections per stallion 152 ejaculates obtained from 152 Lusitano stallions presented at an annual breeding soundness examination as well as data related to 371 ejaculates obtained from 9 PSL were analyzed. These latter samples were also evaluated in terms of their possible use in assisted reproduction and were compared with 113 ejaculates obtained from 4 Sorraia horses, a rare and endangered Portuguese breed. The percentage of motile spermatozoa (PMS) was assessed after collection (AC), after semen dilution (AD) and at 24h of cool-storage. Mean values obtained for sperm motility and morphology and semen pH observed after semen collection differ significantly (P<0.05) between single collection/multiple stallions and multiple collections/limited stallions, and no age related effects were detected. Overall, Lusitano semen quality was comparable to that of related breeds, while Sorraia stallions had very poor semen quality. The response to cool-storage of diluted semen samples differed among stallions and breeds, and the best results for progressive motile sperm cells at 24h were in a range of 35-53% for PSL stallions and were lower for Sorraia stallions. Fertility rates obtained with artificial insemination (AI) averaged at 85% for PSL. With the exception of PMS AC, sperm vitality and semen pH no other seminal trait seemed to influence fertility rates in the Lusitano breed.     

Semen production and fertility issues related to differences in genetic lines of boars

Boars have been reported to differ in various characteristics important to productivity of AI units. These aspects include sensitivity to seasonal infertility, ejaculate volume x age interactions, optimum collection frequency, discard (trash) rate of ejaculates, age of puberty, libido and trainability, and semen shelf life. In addition to differences among individual boars, there were also identifiable differences among breeds, between purebreds and crossbreds, and between lines. The percentage of ejaculates discarded varied among seasons and among breeds within season, with maternal purebred lines being the most sensitive to warmer temperatures. These same lines were also slower to mature and produced fewer sperm per ejaculate. Breeds differed in the sustainability of motility in stored extended semen, and in farrowing rates. Boar centers need to factor breed differences into their decision-making processes to ensure adequate boar power and customer satisfaction.     

Use of the point-score system for breeding soundness examination in yearling Dorset, Hampshire and Suffolk rams

Performance tests were conducted on 583 purebred Dorset, Hampshire and Suffolk yearling rams at the Virginia Ram Test Station from 1986 to 1989. Birth dates at entry and weights (lbs) at entry and end-of-test were recorded for each ram. Entry and exit scrotal circumference (SC; cm) data were recorded for each year of the study. Breeding soundness examination (BSE) data at entry were obtained for only the last two years (1988-1989). The BSE followed the basic format recommended by the Society for Theriogenology. The number of seminal white blood cells per (100x) microscope field (WBC/LPF) were also recorded for each ram's ejaculate. Classification of rams into breeding groups (satisfactory, questionable and unsatisfactory) were made using a point-scale system based upon values obtained from SC, sperm motility and morphology assessments. Between-breed differences were noted for age at entry to the test station, weight per day of age, final weight at the end of the test period and average daily gain. Suffolk rams were younger in age (P0.05). Overall the percentage of rams classified as unsatisfactory, questionable and satisfactory was 11.8, 16.5 and 71.7, respectively. Rams with more than 10 WBC/LPF had significantly smaller SC at entry (P<0.01) than rams with less than 10 WBC/LPF. Most of the differences (75%) in BSE scores in this study were contributed by differences in semen quality (spermatozoal motility and morphology) not by differences in SC.     

Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing

The aim of this experimental study was to evaluate the effectiveness of sperm selection using single-layer centrifugation (SLC) prior to freezing on the sperm cryosurvival of boar ejaculates. Twenty-four sperm rich ejaculate fractions (SREF), collected from 24 boars (one per boar), were divided into two groups according to their initial semen traits: standard (n = 15) and substandard (n = 9). Semen samples from each SREF were split in two aliquots, one remained untreated (control samples) and the other was single-layer centrifuged (500g for 20 min) using 15 mL of Androcoll-P Large (SLC samples). The yield of total, motile (assessed by CASA) and viable (cytometrically evaluated after staining with H-42, propidium iodide (PI) and FITC-PNA) sperm after SLC was higher (P < 0.05) in standard than substandard semen samples. The semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw sperm motility and viability (assessed at 30 and 150 min post-thawing) were higher (P < 0.05) in SLC than in control samples, regardless of the initial semen traits of the ejaculates. Additionally, thawed spermatozoa from SLC samples were more resistant (P < 0.05) to lipid peroxidation (BIOXYTECH MDA-586 Assay Kit) than those from control samples, regardless of the initial semen traits of the ejaculates. The SLC-treatment also influenced the functionality of thawed spermatozoa undergoing an in vitro capacitation process. The percentage of viable sperm showing high membrane fluidity (assessed with merocyanine 540) was lower (P < 0.05) in the SLC than in the control samples, regardless of the initial semen traits of the ejaculates. Thawed viable spermatozoa of SLC samples generated less (P < 0.05) reactive oxygen species (assessed with CM-H₂DCFDA) than those of control samples in the substandard ejaculates. These findings indicate that the sperm selection before freezing using SLC improves the freezability of boar sperm.     

Effects of acetylsalicylic acid and metamizol on hyaluronidase activity and sperm characteristics in rams

The effects of acetylsalicylic acid and metamizol on hyaluronidase activity of semen and sperm characteristics in rams were investigated. Acetylsalicylic acid and metamizol at the doses of 75 and 50 mg/kg were administered to the rams, respectively and then semen samples were taken at 1, 2, 4, 24, 48, 96, 120 and 144 h. The hyaluronidase activities of semen in rams treated with acetylsalicylic acid and metamizol were determined to increase significantly (P<0.001) when compared with control groups at all times. Additionally, the spermatozoa motilities in both groups were measured to increase significantly (P<0.05) when compared with control group. Furthermore, there were significant (P<0.01, <0.05) decreases in the sperm concentrations and semen volumes of rams treated with acetylsalicylic acid and metamizol at all times, respectively. In conclusion, although the use of acetylsalicylic acid and metamizol cause an increase in the hyaluronidase activities and spermatozoa motilities, these drugs decrease the sperm concentrations and semen volumes along 6 days. For these reason, the use of these drugs in breeding rams during ramming season is not suitable.     

Mating behavior, serum testosterone and semen characteristics in vasectomized and short scrotum rams

Ten similar rams were either vasectomized (VAS) or altered by securing the testes near the abdomen using elastrator bands (short scrotum, SS). Semen characteristics (electroejaculation) were similar (P>0.10) between groups before treatment and ejaculate volumes remained relatively constant (P>0.10) throughout the trial. Remaining sperm cells were nonmotile and 95% abnormal by 6 days after vasectomy. By 12 days after surgery, ejaculates contained no cells. Two SS rams had detectable sperm numbers at 12 days after treatment, but 95% were abnormal and all but 1% were nonmotile. Ejaculates collected 3 months after surgery were similar between treatments, in that all existing cells were nonmotile and abnormal. Partial spermatogenic function was regained in SS rams 15 months after treatment as indicated by the presence of low concentrations of motile sperm. Three and 15 months after treatment, sexual activity was estimated by exposing rams to a group of four estrous ewes. During the 15-minute exposure, VAS males exhibited more (P<0.10) nonmating sexual approaches than did SS animals during the first year. However, nonmating approaches and incomplete and completed matings were similar (P>0.10) between VAS and SS rams during the second year. No treatment differences in serum testosterone were observed before, during or after either libido trial. Shortening the scrotum of 6-month-old ram lambs has little effect on libido or testosterone when compared with VAS rams. However, the SS method of alteration does not result in complete sterility as indicated by the presence of motile spermatozoa in ejaculates 15 months after SS treatment.     

Acrosome reaction and concentration of prostaglandin E2 in semen of rams treated with flunixin meglumine (Banamine)

The objectives of this study were to determine 1) the effect of Banamine on the seminal concentration of PGE2 and 2) the ability of sperm cells from treated rams to undergo acrosome reaction in vitro as an indirect measure of their fertilizing capacity. Seven rams, approximately 55 kg bodyweight and 2 to 5 yr of age, were divided into two groups: Group 1, treated (n = 4) and Group 2, controls (n = 3). Treatment consisted of administration of 75 mg i.m. Banamine twice daily, 6 to 8 h apart, for 45 d. On Day 0 (first day of treatment) and on Days 2, 9, 11, 16, 23, 25, 28, 30, 36, 39, 43 and 46 semen samples were collected from both groups using an electroejaculator. Blood samples were obtained for determination of serum levels of Banamine using high-performance liquid chromatography. Semen samples were examined for motility and morphology. Highly motile (>/=85%), normal-appearing semen samples were pooled on each day of collection and 25 ul of the pooled sample (1x10(6)/ml) of each group were induced to undergo acrosome reaction in vitro using ionophore A23187. Acrosome reaction was demonstrated using a staining technique designed for demonstrating the process in bull sperm cells. The percentage of acrosome-reacted and non-acrosome-reacted sperm was determined by random microscopic examination of 100 sperm cells using a double-blind approach. The supernatants of the remainder of the semen samples were assayed for levels of PGE2 using RIA. Values for acrosome-reacted sperm cells and PGE2 levels on the first day of treatment from both groups were compared with corresponding values from each day of sampling using Wilcoxon Rank Sums test (P<0.05). In Group 1, the mean serum level of Banamine was 3.02+/-0.58ug/ml. There was a significant increase in the ability of sperm cells from rams in Group 1 to undergo acrosome reaction as treatment progressed compared with the sperm cells from rams in Group 2. However, there was a significant decrease in concentration of PGE2 in semen from rams in Group 1 compared with those from Group 2. The results of this study suggest an inverse relationship between the capacity of sperm cells to undergo acrosome reaction and concentration of PGE2 in semen of rams treated with a nonsteroidal anti-inflammatory drug.