Sperm Production and Sperm Morphology of Swedish Warmblood Stallions

The present study investigated daily sperm output and sperm morphology of fresh semen in eight Swedish Warmblood stallions aged 5–8 years. They were used for artificial insemination, and their fertility during the breeding season of semen collection exceeded 60% per cycle. One ejaculate of semen was collected daily for 10 consecutive days from each stallion. The gel-free volume was measured, and the sperm concentration was assessed with a Bürker chamber. The volume of gel-free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). Sperm morphology was examined in ejaculates collected on days 2, 5 and 10. An aliquot from each ejaculate was fixed in 1 ml formol–saline immediately after collection and examined under a phase-contrast microscope (magnification 1000×) to assess morphological abnormalities. Furthermore smears were prepared and stained according to Williams (carbolfuchsin–eosin) for a more detailed examination of the sperm heads under a light microscope (magnification 1000×). Analysis of variance was applied to data. Total spermatozoa number decreased progressively during the first 8 days of collection, and daily sperm output (DSO) was calculated as mean TSN of collections on days 8–10, being 6.4 × 10⁹ spermatozoa. The overall percentages of morphologically normal spermatozoa in ejaculates collected on days 2, 5 and 10 were above 70%, being significantly lower in ejaculate 2 (68.6%) compared with ejaculates 5 and 10 (72.9% respectively 75.3%).     

Single layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large sample size

Reasons for performing study: An improvement in sperm quality after single layer centrifugation (SLC) has been seen in previous studies using small sample sizes (for example, n = 10 stallions). There is a need to investigate whether this improvement is repeatable over several breeding seasons with a larger number of stallions (n ≥ 30 stallions). Objective: To make a retrospective analysis of the results of SLC performed on more than 250 sperm samples (176 ejaculates) from 31 stallions in 3 consecutive breeding seasons. Methods: Sperm quality (motility, proportion of morphologically normal spermatozoa and the proportion of spermatozoa with undamaged chromatin) was assessed before and after SLC. Results: All parameters of sperm quality examined were significantly better in sperm samples after SLC than in their unselected counterparts (P<0.001 for each parameter). The yield of spermatozoa obtained after SLC was influenced by the type of extender used and also by the concentration of spermatozoa in the original ejaculate, with fewer spermatozoa being recovered when the loading dose contained a high concentration of spermatozoa. The optimal concentration was approximately 100 × 10⁶/ml. Sperm concentration in the samples loaded on to the colloid influenced the sperm yield while the type of semen extender affected sperm quality and survival. Furthermore, the scaled‐up SLC method was found to be suitable for use with a range of ejaculates, with similar sperm kinematics being observed for standard and scaled‐up preparations. Conclusions: SLC consistently improved the quality of stallion sperm samples from a large number of ejaculates. The method could be scaled‐up, allowing larger volumes of ejaculate to be processed easily from a wide range of stallions.     

Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids

The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.     

Clinical observations on changes in concentrations of hormones in plasma of two stallions with thermally-induced testicular degeneration

To investigate effects of thermally-induced testicular degeneration on hormonal and seminal parameters in stallions, the scrotum was insulated for 36 hours in two mature (5-year-old mixed breed and 11-year-old Throughbred) stallions. Semen was collected daily for 10 days (DSO) prior to, and at intervals after, scrotal insulation. When DSO determinations were not being made, semen was collected 3 times weekly. Jugular blood samples were collected at 15-minute intervals for 6 hours from each stallion prior to, and at intervals after, scrotal insulation. A mouse interstitial cell testosterone assay was modified to quantify biologic activity of equine luteinizing hormone (BLH) in plasma samples. Immunoactive luteinizing hormone (ILH) and testosterone (T) concentrations were determined in plasma samples by routine RIA procedures. Percentages of progressively motile and morphologically normal spermatozoa began to decrease by 1 to 2 weeks postinsulation, reached nadir values at 3 to 3-1/2 weeks postinsulation, and returned to preinsulation values by 7 weeks postinsulation. Total number of spermatozoa and total number of progressively motile, morphologically normal spermatozoa in ejaculates at DSO returned to normal by 8 weeks postinsulation in stallion 2 and 12 weeks postinsulation in stallion 1. Concentrations of BLH and ILH increased, and while T concentrations decreased, immediately postinsulation. The increase in ILH concentrations was greater than the increase in BLH concentrations, resulting in a decrease in the BLH:ILH (B:I) ratio. Following the peak in LH secretion immediately postinsulation, LH concentrations gradually decreased while T concentrations increased. The B:I ratio was elevated from 1 to 13 weeks postinsulation compared to immediately postinsulation. In addition to changes in spermatozoal quality in ejaculates, stallion response to scrotal insulation included increased secretion of luteinizing hormone and impaired Leydig cell function (as determined by reduced testosterone concentration in circulating plasma). The proportion of biologically active LH secreted in response to thermal testicular injury increased during the recovery phase.     

Spermatozoa morphology during the breeding season in Thoroughbred stallions in Japan

The morphology of spermatozoa of modern Thoroughbred stallions in Japan was investigated during the breeding season. A total of 299 semen samples were collected from the penises of 16 stallions immediately after service. The rate of abnormalities in sperm heads and tails, spermatozoa with cytoplasmic droplets and slides with medusa cells to total observed slides in each stallion were 3.9 +/- 2.1%, 11.5 +/- 5.9%, 2.4 +/- 2.6% and 20.1%, respectively. The values for the area, length, width and aspect ratio of the stallion sperm head were 12.54 +/- 1.34 microm(2), 5.93 +/- 0.40 microm, 2.69 +/- 0.21 microm and 0.46 +/- 0.05, respectively. With the exception of medusa cells, the features were significantly different among the stallions (P<0.05).     

Prediction of first season stallion fertility of 3-year-old Dutch Warmbloods with prebreeding assessment of percentage of morphologically normal live sperm.

In the selection procedure to acquire a breeding licence, 3-year-old Dutch Warmblood stallions have to undergo a breeding soundness test It is questioned whether this evaluation is predictive of the stallion's fertility results in the first breeding season. Therefore, semen parameters at the beginning of their first breeding season were evaluated and correlated to nonreturn at first cycle and foaling rate of mares bred by stallions (n = 13). The total number of mares inseminated with chilled semen from those stallions was 1055. Semen parameters were recorded on 2 ejaculates, collected 1 h apart. Percentage progressive sperm motility, % morphologically normal from unstained spermatozoa (MNA), % sperm cells with abnormal acrosomes and the total number of spermatozoa were correlated with first cycle nonreturn rate and foaling rate. Mean motility at evaluation was 72 +/- 6%. Mean MNA was 62 +/- 13%. Mean first cycle nonreturn rate and foaling rate were 58 +/- 15% and 69 +/- 12%, respectively. A significantly positive correlation (P<0.05) was found between the MNA and first cycle nonreturn rates. Foaling rates were not significantly correlated with semen characteristics and first cycle nonreturn rates. In conclusion, the breeding soundness test is of predictive value for the breeding results in the breeding season following the test. First cycle nonreturn rates reflect fertilising capacity better than foaling rates.     

A comparison of duck and chicken egg yolk for cryopreservation of stallion sperm

OBJECTIVE: Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. DESIGN: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after thawing. Morphology data were also collected at 0 and 1 hr post thaw. RESULTS: Total and forward progressive motility were higher when the sperm were frozen in the presence of duck rather than chicken egg yolk. Furthermore, the total and forward progressive motility and percentage of morphologically normal sperm were higher when frozen at a concentration of 200 than 20 x 10(6)/mL. CONCLUSION: The results of this study demonstrate that the motility parameters of stallion sperm are improved when the semen is frozen in lactose EDTA extender supplemented with duck egg yolk rather than chicken egg yolk. Moreover, sperm motility and the percentage of morphologically normal sperm were higher after freezing at a concentration of 200 x 10(6)/ml rather than 20 x 10(6)/ml.     

Characterisation of movement pattern and velocities of stallion spermatozoa depending on donor, season and cryopreservation

The aim of the study was to compare different types of movement pattern and velocities of stallion spermatozoa depending on cryopreservation during breeding and non-breeding season. Ejaculates were collected from four stallions during May (n = 24) and December (n = 24). Parameters of sperm movement were evaluated by computer-aided sperm analysis (CASA) system, and included percentages of motile spermatozoa, different patterns of motility, the velocity, linearity (LIN), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF). In winter the average percentages of motility were slightly higher compared to the breeding season in May (70.8 +/- 12.7% vs. 66.8 +/- 12.2%, respectively). Cryopreservation and thawing led to a significant decrease in the number of motile sperm to 11.3 +/- 5.8% in May and 15.6 +/- 7.0% in December. The pattern of motility was also changed. Detailed analysis by CASA demonstrated that cryopreservation resulted in a shift from the proportions of linear to more non-linear motile spermatozoa and to a significant increase of local motile and hyperactivated spermatozoa. Mean velocity of fresh motile spermatozoa differed between May and December (119.1 +/- 43.9 vs. 164.4 +/- 66.4 microm/sec, respectively; P < 0.05). Cryopreservation and thawing led to a slight increase of curvilinear velocity (VCL) and straight line velocity (VSL). The motility analysis has shown that the parameters BCF and ALH were highly correlated in stallion spermatozoa (r = -0.67; P < 0.001). The BCF of stallion spermatozoa was slightly reduced in the non-breeding season. Altogether, the influence of factors on the motility of stallion spermatozoa has the following rank order: cryopreservation (P < 0.0001) > stallion (P < 0.001) > season (P < 0.05).     

Semen collection in rhinoceroses (Rhinoceros unicornis, Diceros bicornis, Ceratotherium simum) by electroejaculation with a uniquely designed probe.

Electroejaculation in rhinoceroses has historically yielded inconsistent results, with the collection of high-quality, sperm-rich samples rare. The goal of this study was to develop a reliable method of electroejaculation in the rhinoceros by designing a rectal probe that appropriately fits the anatomy of this taxon and refining the procedure. A curved probe handle ending in an oblate, ellipsoid head was built using readily available supplies. A combination of rectal massage, penile massage, and electrical stimulation with a specially designed probe was employed in attempts to collect semen on 14 occasions from greater one-horned rhinoceroses (Rhinoceros unicornis; n = 4), black rhinoceroses (Diceros bicornis; n = 2) and a southern white rhinoceros (Ceratotherium simum; n = 1). During 13 of the 14 attempts, ejaculates were collected in multiple fractions. All but one of the ejaculates contained spermatozoa, and seven ejaculates contained good-quality fractions of semen (-60% sperm motility; > or =20 x 106 spermatozoa/ml) suitable for sperm banking and assisted reproduction procedures. Mean (+/-SEM) values for volume, pH, osmolality, and total sperm number for ejaculates containing good-quality fractions (98.2 +/-21.8 ml, 8.5+/-0.1, 290.4+/-6.7 mOsm, and 37.1+/-12.0 x 10(9), respectively) did not differ (P > 0.05) from those containing only poor-quality samples. Urine and/or erythrocyte contamination was not uncommon in fractions of both ejaculate types. Males producing good-quality samples ranged in age from 7 to 34 yr. None of the samples contained > or =75% morphologically normal spermatozoa. Electroejaculation with a uniquely designed probe consistently produced ejaculates in the rhinoceros. However, the production of high-quality samples continued to be challenging, occurring in only 50% of collection attempts. Regardless, the technology has progressed to a stage at which good-quality semen samples can be produced for sperm banking and assisted reproduction, and thereby can be integrated into intensive rhinoceros management strategies for the ultimate survival of this taxon.     

Determination of the relationship between sperm morphologic classifications and fertility in stallions: 66 cases (1987-1988)

The analysis of breeding records and sperm morphologic classifications from ejaculated semen during 99 stallion seasons, over a 2-year period, revealed a significant correlation (r = 0.34, P less than 0.01) between the percentage of morphologically normal sperm in ejaculates and the per cycle fertility estimate of the stallions studied. In addition, the percentage of sperm classified as having major defects (abnormal heads, proximal droplets, and abnormal midpieces) was significantly inversely correlated (r = -0.36, P less than 0.01) with the same fertility estimates. Multiple variable regression demonstrated that the variation in 2 morphologic features classified as major defects, abnormal heads, and proximal droplets, accounted for the largest amount of variation in fertility. It appears that in stallions, a large percentage of ejaculated sperm with major defects or other defects in combination with major defects is associated with a larger reduction in fertility than is associated with other defects.     

Does the Microbial Flora in the Ejaculate Affect the Freezeability of Stallion Sperm?

In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze, three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were: Staphylococcus spp. and Micrococcus spp. (in all the stallions), β-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3–5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in stallion number 1) and Klebsiella spp. (in stallions 1, 3 and 5). The presence and richness of Klebsiella and β-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw, namely the proportion of dead spermatozoa (ethidium+ cells; r = 0.55, p < 0.05) and the amplitude of lateral displacement of the sperm head (ALH, μm; r = −0.56, p < 0.05), respectively. The degree of growth of Corynebacterium spp. in the ejaculate was positively correlated with the percentage of spermatozoa showing high caspase activity post-thaw (r = 0.62, p < 0.05). The presence and number of colonies of β-haemolytic Streptococcus were negatively correlated (r = −0.55, p < 0.05) with low sperm caspase activity. It is concluded that the microbial flora of the equine ejaculate may be responsible for some of the sublethal damage experimented by the spermatozoa during cryopreservation.     

Single Layer Centrifugation of Stallion Spermatozoa through Androcoll™‐E does not Adversely Affect their Capacitation‐Like Status,as Measured by CTC Staining

This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation‐like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6°C. Sperm capacitation‐like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation‐like status between colloid‐selected and non‐selected spermatozoa. Sperm motility decreased significantly during cold storage, whereas the proportion of apparently capacitated spermatozoa increased. There was no change in the proportion of acrosome‐reacted spermatozoa. In conclusion, SLC through Androcoll?‐E does not adversely affect the capacitation‐like status of stallion spermatozoa, although it did increase with time during cold storage.     

Effect of Removing Seminal Plasma Using a Sperm Filter on the Viability of Refrigerated Stallion Semen

Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed “bad coolers” were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration.     

Characterization and cooled storage of semen from corn snakes (Elaphe guttata).

The phylogenetic order Squamata has many representatives that could benefit from the use of semen preservation as a tool for assisting conservation. To date, few studies have been made evaluating the potential for collecting and preserving semen from snakes. The objectives of this study were to characterize semen parameters of the corn snake (Elaphe guttata), including appearance, volume, concentration, sperm motility, and sperm morphology, and to determine the longevity of corn snake sperm motility stored at 4 degrees C. Single semen samples were collected from 22 adult corn snakes. The appearance of the corn snake semen was generally cloudy, and the color was white to tan. Corn snake spermatozoa initially exhibited a median motility of 92.5%. Corn snakes were found to produce small-volume ejaculates (median 0.01 ml). However, the overall concentration of the snake ejaculate was high (chi = 852 x 10(6) +/- 585 x 10(6) spermatozoa/ml). Morphologically, a mean of 75.7 +/- 9.3% of the sperm cells in an ejaculate were normal. Snake ejaculate with a white appearance had significantly higher sperm concentrations (chi = 1,859 x 10(6) +/- 1,008 x 106 sperm cells/ml; F = 15.74, P = 0.001) than tan ejaculates (chi = 601 x 10(6) +/- 439 x 106 sperm cells/ml). Sperm motility decreased significantly in samples that were stored at 4 degrees C for greater than 48 hr in a refrigerator or Equitainer I. This is the first study to characterize semen volume, appearance, and concentration; sperm motility; and sperm morphology in captive corn snakes. The information derived from this study can be used to develop a model for a collection, cooled storage, and shipping program for semen from endangered or threatened captive and wild snakes.     

Seminal characteristics of two- and three-year-old quarter horse stallions

Seminal and testicular characteristics were compared in 19 three-year-old and 23 two-year-old Quarter Horse stallions. Semen was collected, and testicular evaluations made on all stallions once in April or May and again 60 days later. Semen was collected from stallions twice, one hour apart, at each evaluation. Average testicular tone and scrotal width were greater in three-year-olds than in two-year-olds. Ejaculates of three-year-olds had higher motility scores (66% vs 47%) sperm movement (3.2 vs 2.4), normality (77 vs 71), and total number of normal, motile spermatozoa per ejaculate (4.3 × 10⁹ vs 2.2 × 10⁹). Two-year-olds had twice as many cytoplasmic droplets (11% vs 5%) as three-year-olds. Average sperm concentrations per ml of gel-free semen and gel-free volume of ejaculates were not different. Volume of ejaculate and motility of spermatozoa were positively correlated with pregnancy rate, whereas percent cytoplasmic droplets was negatively correlated with pregnancy rate in both groups of stallions. Pre- and post-ejaculatory urethral, seminal, and preputial cultures were examined for pathologic organisms on all stallions. Eighteen of 42 had positive cultures for Klebsiella spp., Pseudomonas spp., β-Strep spp., or E. coli. Nine two-year-olds and 3 three-year-olds had positive cultures for Klebsiella spp. All but one of these stallions were housed on wood shavings and allowed limited exercise. Three-year-old stallions had superior seminal quality as compared with two-year-olds.     

Comparison of spermatozoal movement and semen characteristics with fertility in stallions: 64 cases (1987-1988).

Information pertaining to evaluation of single ejaculates of semen and records for 2 consecutive breeding seasons were obtained. In all, data for 99 individual breeding seasons (n = 43 Standardbreds and 56 Thoroughbreds) were evaluated. Included in each semen evaluation was examination of semen characteristics and computer-aided analysis of spermatozoal movement characteristics. On the basis of the analysis of breeding records for 4,175 mares (7,017 estrous cycles), a per-estrous cycle fertility rate was calculated from data for 96 of the breeding seasons. Stallions with lower fertility than the mean overall season fertility had significantly (P less than 0.01) lower mean values for subjective appraisal of the percentage of motile and progressively motile spermatozoa and for percentage of morphologically normal spermatozoa. Lower mean values were obtained for computer-aided movement analysis of the percentage of motile and progressively motile spermatozoa, and for mean velocity of motile spermatozoa. Semen characteristics, including spermatozoal movement characteristics, and fertility were significantly (P less than 0.05) correlated for Thoroughbred and Standardbred stallions when analyzed individually and when data for both breeds were combined. Characteristics most highly correlated (P less than 0.01) with fertility data for both breeds combined were: subjective appraisal of the percentage of motile (r = 0.40) and progressively motile (r = 0.46) spermatozoa; percentage of morphologically normal spermatozoa (r = 0.36); and computer-aided analysis of percentage of motile spermatozoa (r = 0.34). However, on the basis of evaluation of a single ejaculate for each stallion, the variation in these characteristics only accounted for approximately 20% of the observed variation in fertility rate.     

Directional Freezing of Equine Semen in Large Volumes

Despite its potential impact on the horse industry, sperm cryopreservation is not an established technology throughout the industry, for a number of reasons that include a reduction in pregnancy rate and increased cost per pregnancy. We have evaluated a novel directional freezing technique, based on a multi-thermal gradient (MTG), by comparing it with the conventional, controlled-rate cryopreservation method (CRCM). Ninety-seven ejaculates with > or =50% motility, collected from 31 stallions were each divided into two parts and subsequently frozen by either MTG or CRCM. Frozen samples were then stored in liquid nitrogen until thawing. The two treatments were evaluated by three methods: progressive linear motility (PLM), viability stain and hypoosmotic swelling (HOS) test. High correlation was found between the three evaluation methods for all post-thaw samples. Eighty-eight per cent of the ejaculates frozen by MTG had post-thaw PLM > or =35%, whereas only 59% of the ejaculates frozen by CRCM had such motility. Post-thaw evaluations of samples frozen by MTG and CRCM were: PLM - 50.2 +/- 1.5% and 37.4 +/- 1.5%, respectively; viability - 53.6 +/- 1.5% and 39.5 +/- 1.4%, respectively; membrane integrity, as evaluated by HOS - 36.2 +/- 1.3% and 26.5 +/- 1.1%, respectively. The differences according to all the evaluation methods were highly significant (p < 0.001), and the results indicate that freezing stallion semen by MTG is superior to CRCM.     

New Methods for Selecting Stallion Spermatozoa for Assisted Reproduction

Improved sperm selection techniques are needed to increase the efficiency of equine-assisted reproduction. Single layer centrifugation (SLC) of spermatozoa has been shown to improve the quality of stallion sperm samples. In this study, the functionality of selected stallion spermatozoa was tested by intracytoplasmic sperm injection of equine oocytes after selection by SLC through Androcoll-E or by discontinuous density gradient centrifugation (DGC) through Redigrad and Tyrode's medium with added albumin, lactate, and pyruvate. The mean cleavage rates of the injected oocytes from SLC- and DGC-selected spermatozoa were 67% and 66%, respectively, whereas the proportion of blastocysts developing from cleaved oocytes was 28% and 22%, respectively (P > .05, not significant). An incidental finding was that there was a tendency for SLC-selected spermatozoa to have a higher percentage of spermatozoa with normal morphology than DGC (70% ± 22% vs. 58% ± 38%) and for more blastocysts to be obtained from subfertile ejaculates (21 [19.6%] vs. 15 [14.4%], respectively). In further experiments, stallion spermatozoa bound to hyaluronan, although binding may depend on the semen extender and sperm treatment as well as incubation time. In conclusion, SLC-selected stallion spermatozoa function normally when injected into oocytes. SLC may potentially be better than DGC at selecting spermatozoa from subfertile ejaculates, but this effect needs rigorous investigation with a much larger sample size. Use of the hyaluronan-binding assay for assessing the potential fertility of stallion spermatozoa may be useful but requires further evaluation.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

Pregnancies following artificial insemination with spermatozoa from problem stallion ejaculates processed by single layer centrifugation with Androcoll-E

Some stallions produce ejaculates of low quality and/or low fertility when used for artificial insemination (AI). The purpose of these five case studies was to use Single Layer Centrifugation (SLC) to select the best spermatozoa from 'problem' ejaculates for subsequent use in AI. Sperm quality, in terms of motility, morphology and chromatin integrity, was improved in the SLC-selected samples compared to the corresponding uncentrifuged samples, with the exception of one stallion thought to have ampullary stasis. In this stallion, neither the incidence of spermatozoa with detached heads nor the proportion of damaged chromatin was decreased by SLC, in contrast to previous results. Pregnancies were obtained after using SLC-selected spermatozoa from the five stallions for AI, indicating that the spermatozoa were functional after SLC. Overall, the results suggest that SLC may be useful when preparing AI doses from some 'problem' ejaculates.