Morphometric differences in sperm head dimensions of fertile and subfertile stallions

Gross morphological evaluation of stallion spermatozoa is of clinical value in assessing male fertility in the horse. While of value, methods of subjective sperm classification yield highly variable results. Recent development of computer-assisted sperm morphometry analysis (ASMA) technology has allowed for the objective analysis of sperm head morphometry. In the current study, ASMA was employed to determine morphometric differences in sperm head dimensions between fertile and subfertile stallions. At least 200 spermatozoa from each of 10 fertile and 10 subfertile stallions were analyzed by a commercial ASMA instrument. The mean measurements for length, width, area, perimeter, and width/length for each stallion were recorded and group means compared by a two-sample t-test. The mean measurements for length, area and perimeter were significantly larger in the subfertile than the fertile group (5.77 microm vs 5.33 microm, 12.66 microm vs 11.37 microm and 14.59 microm vs 13.64 microm, respectively). The width of sperm heads from stallions in the subfertile group also tended to be larger than those of fertile stallions. The data suggest that differences in the dimensions of sperm heads may exist between fertile and subfertile stallions.     

Sperm morphology in Estonian and Tori Breed Stallions

The standard procedure for assessing the breeding potential of a stallion includes the parameter total number of spermatozoa classified as morphologically normal. This study investigated sperm morphology of fresh semen in randomly chosen Estonian (E, n = 8) and Tori (T, n = 7) breed stallions with proven fertility. Two ejaculates were examined from each stallion. An aliquot from each ejaculate was fixed in 1 mL formol-saline immediately after collection and examined with phase-contrast microscope at a magnification 1000× for all types of morphological abnormalities. Furthermore smears were prepared and stained according to Williams (carbolfuchsin-eosin) for a more detailed examination of the sperm heads with light microscope at a magnification 1000×. Analysis of variance was applied to the data, and results are presented as LSmeans (± SE). One T stallion that had a disturbance in the spermatogenesis and one 22-year-old E stallion were not included in the analyses. The T stallions had on average 57.5 ± 4.1% and the E-stallions 74.4 ± 3.8% morphologically normal spermatozoa (p = 0.012). In 4 of 7 T stallions and 7 of 8 E stallions both ejaculates had >50% morphologically normal spermatozoa. There was a significant difference between breeds in mean percentage of proximal droplets (17.3 ± 2.7% and 2.9 ± 2.5% for T and E stallions, respectively; p = 0.003).     

Influence of ejaculation frequency of stallions on characteristics of semen and output of spermatozoa.

Approximately 1 week was required to stabilize the extragonadal sperm reserves in stallions ejaculated daily for 10 weeks. The true daily sperm output of a stallion was equal to the mean daily sperm output of seven ejaculates +/- 1-35 X 10(9) spermatozoa. Mean concentrations of spermatozoa/ml and number of spermatozoa/ejaculate were higher (P less than 0-01) for X1 and X3/week ejaculation frequencies than for a X6/week frequency. Sperm output/week was nearly identical for a X6/week frequency. Sperm output/week was nearly identical for the X3 and X6 frequencies and higher (P less than 0-01) than the X1 frequency. Increase of ejaculation frequency from one to two ejaculates/day twice weekly significantly (P less than 0-01) raised the output of spermatozoa/week. Gel-free semen volume, spermatozoa/ml, and number of spermatozoa/ejaculate were higher (P less than 0-01) in the first, than in the second, ejaculate. Collection of semen on alternate days would be a practical ejaculation frequency for inseminating mares. Two ejaculates collected twice a week would be a practical ejaculation frequency for long-term storage of stallion semen.     

Prediction of daily sperm output in stallions

Ten sexually inexperienced Thoroughbred stallions, ranging in age from 4 to 7 yr, were used to determine the relationship between available extra-gonadal reserves (AEGR) and daily sperm output (DSO). Ejaculates were obtained during the months of June through December. Each stallion was ejaculated at 0700, 0800, and 1700 h on Day 1 and 0700, 1200, and 1700 h on Days 2 and 3. A single ejaculate was collected at 0700 h on Days 4 through 7. DSO was calculated by averaging the total spermatozoa obtained on Days 5, 6, and 7. A minimum of 14 d was allotted to each stallion between trials to allow replenishment of AEGR. Weekly trials were classified as 1) primary: ejaculates taken from sexually inexperienced stallions; 2) normal: all weekly ejaculated in which the total spermatozoa in the second ejaculate (0800 h on Day 1) divided by the total spermatozoa in the first ejaculate (0700 h on Day 1) yielded percentages >20 and <70%; and 3) all: all weekly trials. Regression analysis yielded an equation of estimated DSO=0.18 (total spermatozoa in first ejaculate) + 0.93. Measured DSO, mean of total spermatozoa collected on Days 5, 6, and 7, divided by total spermatozoa in the first ejaculate of normal weekly trials averaged 27.5+/-1.9%. When 27.5% was multiplied by the total spermatozoa found in the first ejaculate (0700 h on Day 1) in primary, normal, and all trials, correlation coefficients between measured and estimated DSOs of 0.95, 0.95, and 0.92, respectively, were obtained. In conclusion, there appears to be a relatively stable relationship between AEGR and DSO in Thoroughbred stallions aged 4 to 7 yr. This relationship allowed a famrly accurate (82%) estimation of DSO when the total number of spermatozoa found in the first ejaculate of sexually rested stallions is multiplied by 27.5%.     

Sources of variation in the morphological characteristics of sperm subpopulations assessed objectively by a novel automated sperm morphology analysis system

There is evidence that the mammalian ejaculate contains distinct subpopulations of spermatozoa and that the variability among these subpopulations may have adaptive and functional significance. This study investigated the precision, reproducibility and operating characteristics of a novel automated sperm morphology analysis system, the Hobson Morphology package, establishing protocols to investigate boar sperm characteristics. Five ejaculates were collected from each of three boars from different genetic lines: Landrace-Meishan introgression, Sireline Large White and Damline Large White. Five semen smears per ejaculate were stained with haematoxylin and eosin. Two hundred spermatozoa per slide were analysed. No significant differences among slides within an ejaculate were detected for sperm tail length (P = 0.770), head width (P = 0.736) and head length (P = 0.615), indicating that both staining and morphology analysis were precise and reproducible. Among the boars, variability in tail length was detected (P = 0.001), but head width (P = 0.114) and length (P = 0.069) did not differ significantly. Multivariate pattern analysis (PATN computer package) highlighted three sub-populations of spermatozoa objectively on the basis of tail length (10.0-22.0 microns, 22.1-73.0 microns and 73.1-130.0 microns). The Landrace-Meishan introgression boar possessed more spermatozoa (P < 0.0001) with tails 73.1-130 microns long. Subsequent analysis of morphology parameters in a pure-bred Meishan boar showed similar measurements for tail length (mean +/- SD; 66.36 +/- 24.70 microns) to the Landrace-Meishan introgression boar (mean +/- SD; 67.09 +/- 21.80 microns). Sperm subpopulations originate during spermatogenesis, when heterogeneous genotypic effects determine the structural features of spermatozoa. The findings of this study confirm that tail length differs between boars and that subpopulations of spermatozoa can be detected within a single ejaculate.     

Advanced fertility diagnosis in stallion semen using transmission electron microscopy

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3, infertile stallions (pregnant mares<10%, n=3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% (x ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.     

Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry

Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.     

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa.

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40 degrees C is recommended for stallion semen.     

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40° C is recommended for stallion semen.     

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.     

The effect of age on the morphometric sperm traits of domestic pigs (Sus scrofa domestica)

The experiment was conducted using 20 male domestic pigs, which were in 2 (equal-sized) age groups: under 14 months old and over 18 months old. At least 5 ejaculates from each male were taken, and in each ejaculate, morphometric measurements of 50 spermatozoa were made. The measured parameters were head area, head length and width, and flagellum length. For each ejaculate, the basic physical traits and the frequency of occurrence of developmental anomalies of the spermatozoa were examined. It was found that sperm dimensions tended to increase along with the boars' age. Considerable and statistical differences in head area and flagellum length were proved. Spermatozoa collected from older boars (above 18 months of age) had a head area larger by 0.49 microm(2)(P< or = 0.01) and a flagellum longer by 0.67 microm (P< or =0.01) than spermatozoa collected from younger boars (under 14 months of age). The differences in head length and width between the spermatozoa of the tested boars were considerably smaller and were not statistically provable. Significant correlations between the head area of spermatozoa and the head length (r = 0.56, P< or =0.05) and head width (r = 0.36, P< or =0.05) were found. However, the head length was not significantly directly correlated with its width (r = 0.18, P< or =0.05), and flagellum length was negatively correlated with spermatozoan head width (r = -0.71, P< or =0.05). Slight correlations between the morphometric traits of the sperm and the physical traits of the ejaculates (r = -0.11 to 0.16) were found, although in most cases, the correlations were not statistically provable.     

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2–4 h Giemsa staining at 25–40° C is recommended for stallion semen.     

The effect of vesiculectomy on seminal characteristics in the stallion.

Successful unilateral extirpation of an inflamed seminal vesicle in a stallion led to systematic trials of the influence of a reduction and absence of the secretion of this gland upon semen characteristics. Operations were performed by the method described for the bull. The volume of ejaculates dropped and sperm concentration per ml increased in each of 2 stallions from which the seminal vesicles had been uni- or bi-laterally removed. Total sperm number and motility remained uninfluenced, but the percentage of eosin-stained spermatozoa increased in the unilaterally operated stallion and the percentage of abnormal spermatozoa increased significantly in both. Concentration of citric acid per ml and per ejaculate was significantly reduced after bilateral vesiculectomy. Ergothioneine concentration per ml increased in the unilaterally and in the bilaterally operated stallion.     

Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro

To facilitate the study of interactions between equine spermatozoa and homologous oviduct epithelial cells, we developed an assay to count labelled spermatozoa bound to oviduct epithelial cell (OEC) monolayers and used the assay to compare the binding ability of spermatozoa from different stallions. Washed spermatozoa from three stallions were incubated with the fluorochrome Hoechst 33342 (5 μg/ml) for 1 min. Spermatozoa were then layered over confluent monolayers of oviduct epithelial cells in 2 cm² culture wells. Coculture treatments comprised five concentrations of spermatozoa (10⁵, 5 × 10⁵, 10⁶, 2.5 × 10⁶, and 5 × 10⁶ per well). Cocultures were incubated for 30 min before unattached spermatozoa were aspirated in coculture supernatant. Fluorescent videoimages of attached spermatozoa were digitized, and attached spermatozoa were counted by image processing and analysis. Four wells (replicates) of each concentration were allocated within each ejaculate, and ejaculates were blocked by stallion for ANOVA. The total number of spermatozoa bound was not different between replicate wells (P > 0.1). Stallion, ejaculate, concentration, and all higher level interactions influenced total spermatozoa bound (P < 0.00001). Coefficients of variation between replicates were lowest for inseminate concentrations between 10⁶ and 5 × 10⁶ spermatozoa per well. Within the ejaculate, a log linear relationship exists between the number of bound spermatozoa and a spermatozoal concentration of the inseminate between 5 × 10⁵ and 5 × 10⁶ spermatozoa per well. This assay provides a reliable method of determining numbers of spermatozoa bound to somatic cells in vitro. Furthermore, differences exist in the ability of spermatozoa from different stallions to bind OEC monolayers. © 1996 Wiley-Liss, Inc.     

Reproductive parameters of miniature stallions

Breeding soundness evaluation (BSE) of stallions is a routine component of stud farm practice. Guidelines for assessing satisfactory breeding potential have been developed using data derived from stallions of full-size breeds. In view of the increasing popularity of miniature stallions, knowledge of normal semen parameters of these stallions is important. Therefore, testicular measurements and semen parameters from 216 sexually rested miniature stallions were obtained. Semen was collected twice, 1.5 to 3 h apart, using an artificial vagina. Values were averaged over the 2 collections because of the sexual inexperience of the stallions. The smaller stallions (Group A, 72 to 86 cm; Group B, 87 to 96 cm) had smaller testicles (P<0.05), and Group A stallions had the lowest ejaculate volume (P<0.05) compared with Group C (97 to 104 cm) stallions. Thus, although there was no difference in the concentration of spermatozoa per milliliter between groups of stallions, Group A stallions had fewer total spermatozoa in their ejaculate than Group C stallions (4.31+/-0.47x10(9) vs. 5.41+/-0.30x10(9), P<0.05). Moreover, miniature stallions had smaller testicles and fewer total spermatozoa in their ejaculate than is commonly accepted as normal in full-size stallions. Average total scrotal width of miniature stallions was found to be 7.13, 7.38 and 7.95 cm for Groups A, B and C, respectively. The average total number of spermatozoa in the ejaculates of miniature stallions in this study was 4.94+/-0.22x10(9) cells, with 1.75+/-0.09x10(9) total normal, motile spermatozoa. When only stallions <96.5 cm in height were considered (conforming to requirements of the American Miniature Horse Association Registry), the average total number of spermatozoa in the ejaculates was 4.59+/-0.30x10(9) cells, with 1.70+/-0.11x 10(9) total normal, motile spermatozoa. Based on these findings, different criteria should be used to evaluate the potential breeding soundness of miniature stallions than are commonly applied to full-size stallions.     

Morphometry of normal and teratozoospermic canine sperm heads using an image analyzer: work in progress

Combining the traditional morphologic evaluation of spermatozoa with computer assisted image analysis adds randomness, objectivity, repeatability and accuracy to morphometric measurements. We collected semen from 10 fertile, normospermic dogs aged 1 to 7 yr and from 3 teratozoospermic breed-matched dogs. Sperm head morphology was examined in Giemsa-stained smears by light microscopy, using a computer-assisted image analyzer and by transmission electron microscopy. We found significant variation in sperm head area, length, width and degree of roundness among normospermic individual dogs, indicating that it would be necessary to examine many more dogs before the size and shape of normal dog spermatozoa could be determined. The normospermic dogs were used as controls for the teratozoospermic cases. Case 1: A 2-yr-old subfertile Cavalier King Charles Spaniel had semen with small and narrow-based sperm heads and a proximal cytoplasmic droplet in most of the spermatozoa. With the image analysis system, sperm heads were shown to be smaller and more oval than in normospermic dogs. The variatons in size and shape were similar in magnitude to those of control dogs. An examined infertile half-brother had similar semen quality. Case 2: A 3-yr-old Petit Basset Griffon Vendeen with 2 unsuccesfull matings exhibited spermatozoa with severe abnormalities. Measured by image analyzer, sperm heads were irregular in shape and very small in area. One of the two littermates examined had semen of the same quality as the case dog. Case 3: A 3-yr-old fertile Golden Retriever had semen with giant sperm heads in about 50% of spermatozoa. Image analyzing results revealed 2 populations of different sized sperm heads. Giant heads consisted of 52.2% of all spermatozoa. The results of the study reported here suggest that the image analysis technique may be useful in evaluating structural changes in sperm morphology, supplementing visual assessment that is used in conventional methods.     

Morphometric changes in goat sperm heads induced by cryopreservation

Two experiments were designed to evaluate the effect of cryopreservation on morphometric characteristics of the goat sperm head. To address this question, we evaluated the size of the sperm head in fresh control cells, post-cooling cells after equilibration with the glycerol preservation solution, and post-thawing cells. Assessment was by automated morphometric sperm head analysis (ASMA) using phase-contrast microscopy without staining. In the first experiment, ASMA was performed on heterospermic pooled samples (fresh, post-cooling after equilibration with the glycerol preservation solution and post-thawing): length, width, area and perimeter were measured. In the second experiment, sperm viability was assessed by Hoechst staining and head morphometry was carried out as before, simultaneously during the cryopreservation process, and the head size was identified for both live and dead spermatozoa. The data were analysed by principal component analysis (PCA). The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables (length, width, area and perimeter), that retain as much of the information in the original variables as possible. The main findings that have emerged from this study are that (i) a simple procedure has been developed for measuring spermatozoa heads without staining, which minimises the possibility that sperm head dimensions were influenced by procedural artefacts; (ii) the dimensions of goat sperm heads after cryopreservation in skimmed milk-glucose medium were smaller than in fresh sperm, but this was due to the equilibration phase with the cryoprotectant and not to the cryopreservation process itself; and (iii) dead spermatozoa showed smaller heads than live sperm, consequent upon the loss of membrane function. No differences were observed between post-cooling cells after equilibration with the glycerol preservation solution and post-thawing spermatozoa and only minor osmotic differences between them and fresh sperm were observed.     

In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products.

Formation of a spermatozoa ('sperm') reservoir in the mare is thought to occur through lectin-mediated sperm attachment to the oviductal epithelium. Once attached, prefertilization sperm survival is supported by oviductal factors. Cryopreservation of stallion sperm decreases the number of sperm attaching to oviduct epithelial cells (OEC) and the length of time these sperm survive. Quantification of in vitro interactions between sperm and OEC in a co-culture system may provide an assay for functional integrity of cryopreserved or fresh sperm samples. Additionally, superior additives for in vitro handling of stallion sperm may be isolated from OEC secretory products. Experiment 1 compared first service conception (FSC) rates resulting from the use of cryopreserved sperm of seven stallions, with sperm function in co-culture such as attachment to OEC and subsequent survival time. Stallions were grouped by cumulative FSC rates observed over three seasons as having average (44 +/- 3%) or high (65 +/- 2%) fertility over a total of 217 first services (31 +/- 9 per stallion). Samples from stallions in the high fertility group had more (P = 0.04) sperm attached to OEC and longer subsequent sperm survival in co-culture (P = 0.05) as compared with those from the average fertility group. FSC rates correlated with numbers of sperm attaching to OEC and their survival time in co-culture (r > or = 0.71). In Experiment 2, the function of cryopreserved stallion sperm was evaluated in culture with OEC secretory products from three different sources. After 5 h of culture, sperm incubated with medium conditioned by bovine OEC which had been 'bioactivated' (e.g. previously exposed to sperm in culture) were found to be more (P < or = 0.05) motile and capacitated as compared to sperm in basal TALP medium alone. Sperm in this conditioned medium also survived longer (P = 0.05; 27 +/- 5 h vs. 17 +/- 4 h) than did those in control medium.     

Effect of controlled exercise on semen characteristics in two-year-old stallions

A two-phase study was conducted to evaluate the effect of controlled daily exercise on semen characteristics of 2-yr-old stallions. Eight nonexercised, sexually inexperienced 2-year-old Morgan stallions were randomly assigned to one of two groups. In Phase 1, four stallions were randomly assigned to receive daily controlled exercise for 16 wk. In Phase 2, these stallions were confined to box stalls for 16 wk. The remaining four stallions were confined to box stalls for 16 wk during Phase 1 and received daily controlled exercise for 16 wk during Phase 2. Individual semen collections were obtained from all stallions every 14 days. Daily sperm output (DSO) was determined by daily semen collections for seven consecutive days on Wk 0, 8, 16, 24, and 32. Mean DSO (10(9)) in Phase 1 was 3.52+/-.62 and 2.79+/-.75 for Wk O and 3.12+/-.39 and 2.61+/-.68 for Wk 16 for exercised and nonexercised stallions, respectively. Mean DSO (10(9)) in Phase 2 was 2.61+/-.68 and 3.12+/-.39 for Wk 16 and 2.48+/-.69 and 3.95+/-.51 for Wk 32 for exercised and nonexercised stallions, respectively. No effect (P>.05) of treatment was observed in either phase when parameters of spermatozoa concentration, total spermatozoa per ejaculate, DSO, total volume, gel-free volume, gel volume, percentage motility, percentage live, pH, and percentage normal were measured and analyzed.     

The influence of age and breed on stallion semen

This study reports on the variation in semen quality and in spermatozoal and behavioral characteristics of 168 stallions representing 9 breeds and ranging in age from 2 to 26 yr. Semen samples were collected into an artificial vagina and the number of mounts and urethral pulsations per semen sample were recorded. Semen characteristics were examined for total volume, gel-free volume, gel volume, color score, mass activity, nonmotile spermatozoa, dead spermatozoa, semen density, spermatozoa concentration, total number of spermatozoa and semen pH. Morphological characteristics of the spermatozoa included abnormal heads, abnormal mid-pieces, abaxial mid-pieces, protoplasmic droplets and abnormal tails. Sources of variation were evaluated and the overall means calculated by least-squares analyses of variance for nonorthogonal data. The significance of breed effects and between stallion variability were estimated using mixed-model procedures. All semen characteristics with the exception of color and urethral pulsations had significant variation due to age. Semen quality (gel-free volume, sperm concentration, total sperm numbers and sperm abnormalities) was poorest in stallions under 3 yr of age and over 11 yr. Significant breed variation was apparent in most characteristics except for pH, semen color, abnormal midpieces and urethral pulsations. It is recommended that both the age and breed of stallion be taken into consideration when evaluating stallion semen.