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.     

Rectally Guided or Hysteroscopic Insemination: Is there a difference?

The increasing use of frozen semen, the high cost of a dose of semen from some stallions, and the inability of some stallions to cope with their demand has triggered an incentive to reduce the number of spermatozoa from the traditional 500 PMS to significantly lower numbers per insemination. Two techniques have been described to deposit low sperm numbers in the uterus. Although experimental results have been reported for the rectally guided deep horn insemination (RGDHI) and endoscopic deep horn insemination (EDHI) techniques, little information is available in clinical settings with respect to the results of either method. This article reports on a comparison of the rectally guided insemination and hysteroscopic techniques in a clinical setting over three breeding seasons. Semen frozen in 0.5-mL straws and concentrations of 40 to 200 million per straw was available from 88 stallions whose fertility with frozen semen ranged between 0% and 100%. Of the 2544 inseminations performed 1279 were done by RGDHI, which resulted in a 43% seasonal pregnancy rate compared with 45% for the 1265 inseminations using the EDHI technique. There was an overall, but nonsignificant, advantage for the EDHI technique over the RGDHI and semen usage was slightly lower when using the EDHI procedure. There was no difference in fertility among breeding seasons for either technique. However, fertility of some stallions was increased by increasing the sperm numbers in the inseminates. From these data and results from others, it is evident that lower sperm numbers deposited by rectally guiding a flexible pipette to the tip of the horn or by using an endoscope to deliver the semen will result in acceptable pregnancy rates with frozen semen in commercial settings.     

Epigallocatechin‐3‐gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24–72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin‐3‐gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.     

Removal of cystic calculi using a transinguinal laparoscopic‐assisted technique in two stallions

A 12‐year‐old donkey and a 6‐year‐old Connemara pony, both sexually intact males, were admitted for surgical removal of urinary calculi. Both underwent a laparoscopic‐assisted cystotomy technique to exteriorise the bladder and remove uroliths via the inguinal canal after hemi‐castration. Both surgeries were successful and no complications occurred. This modified technique, which is minimally invasive, may be a safe and efficient way to remove bladder stones from adult stallions.     

Sperm quality during extra‐gonadal sperm reserve depletion in stallions

To estimate when, during stallions’ extra‐gonadal reserves (EGR) depletion period, sperm quality would reach its highest quality, six light breed sexually rested stallions were collected daily for 7 days to deplete EGR. On collection days 1, 3, 5, and 7, sperm output, total (TM) and progressive (PM) motility, morphology, and plasma membrane (PLM) integrity were evaluated. Sperm output decreased as EGR depletion advanced, stabilizing on days 5–7. Sperm motility (TM and PM) and morphology were not different during EGR depletion. Plasma membrane integrity improved from day 1 to 3; however, no further improvement observed on days 5 and 7. Sperm of sexually rested stallions reach the highest quality on day 3 of the EGR depletion period.     

Single‐Layer Centrifugation Reduces Equine Arteritis Virus Titre in the Semen of Shedding Stallions

Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single‐layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll‐E, a species‐specific colloid were evaluated for their ability to separate spermatozoa from virus in ejaculates from carrier stallions. The three types of SLC significantly reduced the virus titre in fresh semen at 0 h and in stored semen at 24 h (p < 0.001) but did not completely eliminate the virus. Sperm motility parameters such as total motility and progressive motility were significantly increased after colloid centrifugation, whereas curvilinear velocity and amplitude of lateral head deviation were decreased, and the remainder (straight line velocity, average path velocity, straightness, linearity, wobble and beat cross‐frequency) were not significantly affected by the processing. Although virus titres were reduced in the SLC samples, significant levels of infectivity still remained, especially in stallions shedding large amounts of virus. It remains to be determined whether SLC‐processed sperm samples from stallions shedding low virus titres retain sufficient equine arteritis virus to cause infection in mares through artificial insemination.     

Semen parameters and hormone concentrations in stallions subjected to long-term estrogen administration

Eight pony stallions were paired by estimated daily sperm output (DSO) and randomly assigned to one of two treatments in a randomized block experiment. Stallions received 44 μg/kg BW estradiol cypionate (ECP) or an equivalent volume of physiological saline solution on alternate days during the breeding season. Blood samples collected immediately preceding each injection were assayed for luteinizing hormone (LH), estradiol-17β (E₂) and testosterone (T). Semen was collected twice weekly, 3.5 days apart, to evaluate sperm motility and total number of sperm per ejaculate. Prior to and after 4, 8 and 12 weeks on treatment, semen was collected once daily for 7 days to determine DSO. Data were separated into 9 periods (10 days each) for statistical analysis and subjected to analysis of variance for a randomized block design to determine treatment effects.There were no differences (p>.05) between groups for DSO or LH prior to initiation of treatment. Testosterone was higher (p<.05) in ECP stallions compared with C stallions prior to treatment and at all time points measured. As expected, E₂ was higher (p<.05) in the ECP stallions compared to C stallions after 20 days (period 2) of treatment and for the remainder of the experiment. However, E₂ was higher (p<.05) in the C group prior to treatment, but there was no difference between the groups after 10 d of treatment (period 1). ECP stallions had higher (p<.05) DSO than C stallions after 30 d on treatment. After 40 and 50 d (periods 4 and 5), ECP stallions demonstrated higher (p<.05) total sperm per period than C stallions. This was preceded by higher (p<.05) LH values for ECP stallions than for C stallions after 10 and 20 d (periods 1 and 2). No differences were found between the ECP and C groups for LH between 30 and 60 d. Although numerically higher, no significant differences (p>.05) were seen after 60 days for DSO or after 60, 70 or 80 days for total sperm per period. ECP stallions had higher (p<.05) DSO and total sperm per period after 90 d than C stallions. Additionally, LH remained significantly higher (p<.05) in the ECP group after 60 days (periods 7, 8 and 9). Elevated LH concentrations in ECP stallions demonstrated that estrogen treatment did not inhibit LH secretion in this study.     

Alkaline and Acid Phosphatase, β‐Glucuronidase and Electrolyte Levels in Fractionated Stallion Ejaculates

Seminal plasma (SP) is a mixture of contents from the testes, epididymides and accessory sex glands. The sperm concentration is highest in the first few jets, or fractions, of the ejaculate, and the composition of SP varies between these fractions because accessory gland secretions are released in a specific order. The aim of this study was to compare the levels of Na, Cl, K, Mg, Ca, inorganic phosphate (Pi) and the enzymes alkaline phosphatase (AP), acid phosphatase (ACP) and β‐glucuronidase (BG) in the different fractions of the ejaculate and in different stallions. All semen collections were done using a computer‐controlled phantom that collects the ejaculatory jets separately in five cups. The cups with the highest (HIGH) and the lowest (LOW) sperm concentration were analysed. In Trial I, semen was collected from three reproductively normal stallions. In Trial II, ejaculates of two reproductively normal stallions were compared to those of two subfertile stallions. In Trial III, semen was collected from seven stallions with varying reproductive history. The sperm‐rich fractions contained the highest levels of AP, ACP, BG and inorganic phosphate, and the values were positively correlated to the sperm concentration. Significant differences between the subfertile and the fertile stallions pairs in HIGH : LOW ratios were found in Pi and Cl concentrations. The highest concentrations of Ca and Mg were found in the last fractions with low sperm concentrations, with no significant differences between the fertile and the subfertile stallion pairs. The concentrations of K, Na and Cl were similar in HIGH and LOW fractions and in whole ejaculate samples. Pre‐sperm fluid contained the highest concentrations of Na and Cl. Some of the possible variation in storage tolerance between ejaculates and ejaculatory fractions could perhaps be explained by differences in the composition of SP.     

Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

Genetic Parameters and Breeding Values for Semen Characteristics in Hanoverian Stallions

The objectives of this study were to show whether semen traits of 30 Hanoverian stallions regularly used in AI may be useful for breeding purposes. Semen characteristics were studied using 15 149 ejaculates from 30 Hanoverian stallions of the State Stud Celle of Lower Saxony. Semen samples were collected between 2005 and 2009. Traits analysed were gel‐free volume, sperm concentration, total and motile sperm number and progressive motility. A linear multivariate animal model was employed to estimate heritabilities and permanent environmental variances for stallions. The same model was used to predict breeding values for all traits simultaneously. Heritabilities were high for gel‐free volume (h² = 0.43) and moderate for total number of sperm (h² = 0.29) and progressive motility (h² = 0.20). Gel‐free volume, sperm concentration and total number of sperm were genetically negatively correlated with progressive motility. The effect of the permanent environment for stallions accounted for 9–55% of the trait variance. The total variance among stallions explained 37–69% of the trait variance. The average reliabilities of the breeding values were 0.43–0.76 for the 30 Hanoverian stallions. In conclusion, the study could demonstrate large effects of stallions, routinely employed in a breeding programme, on semen characteristics analysed here. We could demonstrate that estimated breeding values (EBV) with sufficient high reliabilities can be predicted using data from these stallions and these EBV are useful in horse breeding programmes to achieve genetic improvement in semen quality.     

Fertility Assessment in Sorraia Stallions by Sperm‐Fish and Fkbp6 Genotyping

The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm‐FISH and molecular analysis of FKBP6 – a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A‐A/A) for IAR. Instead, all animals had a G/G‐A/A genotype – a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm.     

Testosterone serum profile,semen characteristics and testicular biometry of Mangalarga Marchador stallions in a tropical environment

This study was conducted to characterize the daily profile of testosterone secretion and its mean concentrations in the four seasons as well as to evaluate the semen characteristics and testicular biometry of Mangalarga Marchador stallions throughout the year in a tropical region. Three stallions were submitted to semen collections and evaluation of testicular biometry every 14 days along a year. Blood samples were collected once at the middle of each season, in a 20‐min interval during 24 hr in order to evaluate the testosterone secretion profiles among seasons. Testosterone concentrations along the day were higher at the beginning of the afternoon (from 12:00 to 15:00 hr), but a circadian secretion was not clearly observed. Mean testosterone concentrations did not differ among seasons (p > .05), but a pattern of secretion along the day showed variations with higher concentrations in the afternoon during the winter. Ejaculate volume was higher during summer; however, sperm motility decreased in summer and spring. Total sperm in ejaculate, sperm morphology and testicular biometry kept constant along the year showing no differences among the seasons. The results demonstrated that in a tropical region, reproductive aspects of stallions did not show a clearly defined seasonal variation, and months of autumn and winter were not unsuitable for reproduction of the males.     

Testicular Measurements and Daily Sperm Output of Tori and Estonian Breed Stallions

Evaluation of testicular measurements and daily sperm output (DSO) yields valuable information for predicting the reproductive capacity of stallions. The present study evaluated testicular measurements (height, length, width and circumference) and DSO of eight Tori and eight Estonian breed stallions. One ejaculate of semen was collected daily for 10 subsequent days from each stallion. The gel‐free volume of semen was measured with a graduated glass cylinder and the sperm concentration was assessed with a Chorjajev chamber. The volume of gel‐free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). The DSO was calculated as mean TSN of collection on days 8–10 in Tori breed stallions and on days 4–10 in Estonian breed stallions. The DSO of Tori breed stallions was 12.9 × 10⁹ spermatozoa and of Estonian breed stallions 4.5 × 10⁹ spermatozoa (p < 0.001). Testicular measurements (in cm) 1 day after the last semen collection were as follows: left testis– height 7.3, length 10.4 and width 7.3 in Tori breed stallions, and 5.9, 8.1 and 5.9, respectively, in Estonian breed stallions; right testis– height 7.4, length 10.6 and width 7.4 in Tori breed stallions, and 5.5, 7.4 and 5.3, respectively, in Estonian breed stallions. All these testicular measurements were significantly smaller in Estonian than in Tori breed stallions (p < 0.001). Testicular circumference was 45.4 and 35.4 cm in Tori and Estonian breed stallions, respectively (p < 0.001). The testicular circumference was correlated with DSO in both Estonian (p < 0.05) and Tori breed stallions (p = 0.071). The results give us valuable information on the reproductive capacity of Tori and Estonian breed stallions.     

Morphology and head morphometric characters of sperm in Thai native crossbred stallions

Background

One of the semen quality parameters use to determine fertility is the percentage of sperm that express normal morphology. Sperm head morphometry is also correlated with fertility. The objectives of this study were 1) to investigate the sperm morphology and normal sperm head morphometry of Thai native crossbred stallions, and 2) to compare our results with the characteristics of proven fertile sperm from purebred stallions.

Methods

Semen samples were collected monthly from nine stallions, of which five were Thai native crossbred (T) and four were purebred of proven fertility (F: F1 was a Standard-bred; F2 was a Warm-blood; F3 and F4 were Thoroughbreds). All the animals were aged between 5 and 12 years. Sperm morphological examination was performed using formaldehyde-fixed samples under phase-contrast microscopy (1000×). Normal sperm head morphometry characteristics were measured by Computer-Assisted Semen Analysis (Hamilton Thorne, USA.) after applying the Harris'' haematoxylin staining technique.

Results

The percentages of morphologically normal and abnormal sperm varied among individual stallions in both the T and F groups. The mean percentage of morphologically normal sperm was not significantly different (P > 0.05) between T and F stallions (mean ± SE, 49.7 ± 1.3 and 48.1 ± 2.8, respectively). A comparison between the T and F sperm heads revealed that all the dimensional parameters were significantly different (P < 0.05). The coefficients of within-animal variation (CVs) ranged from 2.6 (shape factor 1) to 7.5 (elongation) and 2.9 (shape factor 1) to 8.1 (elongation) in T and F, respectively. In the case of the T group, those sperm head parameters that featured a low within-animal CV and a high between-animal CV were perimeter (2.9, 19.1), shape factor 1 (2.6, 25.8) and shape factor 3 (3.8, 32.0). In the case of the F group, only shape factor 1 (2.9, 26.1) featured such characteristics.

Conclusion

We found variability in the percentage of morphologically normal and abnormal sperm, as well as in sperm head dimensions among Thai native crossbred stallions, and these results were similar to those of purebred stallions. Our findings demonstrate that the heads of the T sperm specimens were larger and rounder than that of the F sperm. Perimeter, shape factor 1 and shape factor 3 could be used as parameters for the identification of individual T stallions based on a sperm sample.     

The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility

Pregnancy rates in managed horse populations depend on the innate fertility of the mares and stallions involved and on the quality of breeding management. Of course, because a single stallion usually mates many mares, stallion fertility is a critical factor in the overall success of a breeding program. Unfortunately, accurate evaluation of stallion fertility per se requires a large number of normal mares to be mated and is necessarily retrospective. Rather, the ideal is to predict fertility in advance of the stallion's breeding career, and this is currently attempted by way of a thorough physical examination and a routine analysis of semen quality. However, while such a ‘breeding soundness examination’ identifies stallions that clearly lack the capacity for adequate fertility, it is of limited use for predicting the level of fertility and fails to identify some seriously sub‐fertile animals. Similarly, while various sperm function tests (e.g., sperm head morphometry, the hypoosmotic swelling test, glass wool‐sephadex filtration, progesterone receptor exposure) have been shown to correlate fairly well with fertility in the field, most examine only a single or a narrow range of the attributes that a sperm must possess if it is to fertilize an oocyte in vivo, and are thus more useful for identifying specific causes of sub‐fertility than for predicting the level of fertility. On the other hand, combining the results of the various sperm function tests does improve the reliability of fertility estimation and current research is therefore concentrated on identifying a range of tests that covers as many important sperm attributes as possible but that can be performed rapidly and cheaply. In this respect, flow‐cytometry has proven to be an ideal tool because it allows the objective, rapid and simultaneous analysis of a number of properties in a large number of sperm. Moreover, stains are available for an increasing range of sperm characteristics including viability, capacitation and acrosome status, mitochondrial activity and chromatin integrity. Flow‐cytometric analysis of sperm with appropriate probes thus offers considerable promise for the prediction of stallion fertility.     

Sperm mitochondrial regulation in motility and fertility in horses

The biological nature of age‐related declines in fertility in males of any species, including stallions, has been elusive. In horses, the economic costs to the breeding industry are frequently extensive. Mitochondrial function in ejaculated sperm, which is essential for sperm motility, is reflected by adenosine triphosphate production, mitochondrial oxidative efficiency and production of reactive oxygen species, and that this balance may become compromised in ageing stallions and during the process of cryopreservation. This presentation will focus on mitochondrial integrity and function as an avenue for understanding the pathophysiology of sperm when undergoing cryopreservation and male ageing. We discuss the importance of understanding the differences and similarities of sperm mitochondria to that of somatic cells regarding structure and mitochondrial biochemistry relating to sperm function. The roles of oxidative phosphorylation and glycolysis in sperm mitochondria are outlined as is the method of determining oxygen consumption and calcium homoeostasis in sperm mitochondria. Further, we outline the role of oxidative stress and reactive oxygen species.     

Relationship of cysteine‐rich secretory protein‐3 gene and protein with semen quality in stallions

Cysteine‐rich secretory protein‐3 (CRISP‐3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP‐3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP‐3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP‐3 was identified and haplotypes were derived. Semen quality was assessed through the total motility (MOT), sperm vitality (SV), normal morphology (NM), functional integrity of membrane (MI) and a seminal quality index (SQi). CRISP‐3 protein content of seminal plasma (SP) was determined by ELISA. The effect of the genotype, the haplotype and the concentration of the CRISP‐3 protein on the seminal quality were evaluated through generalized linear models and linear regression analyses. Homozygous genotypes for SNP1, SNP2 and SNP3 and the heterozygous genotype for SNP4 showed a positive effect on seminal quality. Different haplotypes with positive effect on MOT, SV, NM, MI and SQi were identified. The allelic substitution analysis resulted in positive regression coefficients for MOT (SNP2) and MI (SNP2 and SNP3). A high level of CRISP‐3 resulted in a higher MOT and SQi. It is concluded that the quality of stallion semen is influenced by the genotype of CRISP‐3 and the concentration of CRISP‐3 protein in SP.     

Effect of GnRH immunisation on hormonal levels, sexual behaviour, semen quality and testicular morphology in mature stallions

The aim of this study was to investigate the effect of gonadotrophin-releasing hormone (GnRH) immunisation on mature stallions that had been used for breeding. Four Standardbred stallions were used in the study: 3 experimental animals and 1 control animal. Semen was collected regularly, i.e. twice/week, during the 4 months prior to the experimental period. The stallions were immunised against GnRH with a GnRH-BSA conjugate. Equimune was used as the adjuvant. The stallions were immunised on 5 occasions, 4 at 2 week intervals, and the fifth 4 weeks after the fourth. Blood samples were taken once a week for analysis of GnRH antibody titre and every third week for testosterone and oestrone sulphate analyses. Semen was collected once a week, and libido and sexual behaviour were observed. Ejaculate volume, sperm concentration, total number of sperm in the ejaculate, sperm motility and sperm morphology were evaluated. Testicular size was measured once a week. At the end of the study, the stallions were castrated, and a histological examination of the testes performed. All immunised stallions produced antibodies against GnRH, and plasma testosterone concentration decreased. However, the effect of immunisation varied between stallions. In 2 of the stallions, high levels of antibodies were found, while in the third, the level was moderate. Four weeks after the first immunisation, a decrease in libido was observed. Two months after the first immunisation, marked changes in semen quality were observed in the 2 stallions with high antibody titres. Fourteen weeks after the first immunisation, the total number of sperm/ejaculate had decreased from >8.6 x 10(9) to <2.7 x 10(9), sperm motility from >59 to <10% and the frequency of morphological normal spermatozoa had decreased from >60 to <14%. The dominating abnormalities were abnormal head shapes, proximal cytoplasmic droplets and detached heads. In the third stallion, only slight changes in semen quality were found. No changes were observed in the control stallion. Decreases in testicular size were noted in all of the experimental stallions. Pronounced histological alterations in the testes were observed in 2 of the stallions. It is concluded that the vaccine was effective in stimulating production of GnRH antibodies and in suppressing testicular function and androgen secretion. However, there was an individual variation in the responses among the stallions and, further, libido was not totally suppressed.     

Trypan Blue/Giemsa Staining to Assess Sperm Membrane Integrity in Salernitano Stallions and its Relationship to Pregnancy Rates

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty‐one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty‐five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June–July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June–July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut‐off value of 48% LSIA. Trypan blue‐Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor‐quality ejaculates.