Diagnostic Methods for Evaluation of Stallion Subfertility: A Review

Classically, evaluation of the breeding stallion for reduced fertility has relied on physical examination of the reproductive system, as well as evaluation of sperm number, motility, and morphology. Over the past 20 years, a number of other diagnostic methods have become available to facilitate reproductive evaluation of the stallion. Specifically, ultrasound imaging has provided much-improved diagnostic methods for evaluation of the external and internal genitalia of the stallion, and these methods have now become routine in evaluation of the stallion. Biochemical analyses of semen can provide useful information for diagnosis of azoospermia (determination of alkaline phosphatase), detection of urine contamination, or changes in pH. Numerous sperm function assays provide information concerning subcellular compartments of the sperm including the plasma membrane, DNA, acrosome, and mitochondria. Data correlating these functional assays with fertility in the stallion are limited in most cases, with the exception of the sperm chromatin structure assay. Finally, the recent sequencing of the equine genome offers the possibility of both marker-assisted selection for fertility traits and more specific information about genetic mutations that may be associated with differing levels of fertility in the stallion.     

Motility and Fertility Evaluation of Thawed Frozen Stallion Semen After 24 Hours of Cooled Storage

Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were (1) evaluate the longevity of frozen stallion semen once it had been thawed, extended, and maintained at 5°C for 48 hours in a passive cooling container, and (2) determine fertility potential of frozen semen that had been thawed, extended, and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE minus cryoprotectant at a concentration of 50 million total sperm/mL. The remainder of the ejaculate was frozen in CryoMax LE extender at a concentration of 200 million total sperm/mL. Semen was thawed using 1 of 3 thawing protocols, and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE minus cryoprotectant and cooled to 5°C. Sperm motility was evaluated at 24 and 48 hours. Eight mares were inseminated over two estrous cycles using frozen semen that had been thawed, extended in INRA96, and cooled for 24 hours. There was no difference in progressive motility at 24 or 48 hours of cooled-storage post-thaw between the 3 thawing protocols. An overall per cycle pregnancy rate of 56% (9/16 cycles) was achieved using frozen-thawed semen that had been extended and cooled for 24 hours. In summary, frozen stallion sperm was thawed, extended, and cooled to 5°C for 24 hours and still maintained adequate (>30%) sperm motility and fertility.     

Evaluation of stallion semen.

This article outlines a basic method for conducting a stallion semen evaluation. After the removal of the gel fraction of the ejaculate, semen gel-free volume is determined, and any abnormality in appearance is noted. Concentration of sperm cells in semen can be determined with the use of either a hemacytometer or spectrophotometer after appropriate dilution of raw semen. The percentage of progressively motile sperm is evaluated promptly after collection of semen with the use of a phase-contrast microscope. The total numbers of sperm and progressively motile sperm in the ejaculate are calculated. The determination of seminal pH and the classification of sperm morphologic features are additional seminal characteristics evaluated during a semen evaluation. Sperm motion characteristics can be further evaluated with the use of computerized sperm image analysis systems and may add additional information concerning the quality of ejaculated sperm. Unfortunately, no single seminal characteristic has in itself been shown to be highly correlated with fertility, although various seminal characteristics are known to affect fertility. Therefore, to properly interpret the fertility of a semen sample, a complete and thorough semen evaluation must be performed.     

The Effect of Trehalose Supplementation of INRA-82 Extender on Quality and Fertility of Cooled and Frozen-Thawed Stallion Spermatozoa

Stallion semen cryopreservation is often associated with poor post-thaw sperm quality. Sugars act as nonpermeating cryoprotectants. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze–thaw process. Semen samples were collected from six Arabian stallions, divided into five different treatments in a final concentration of 100 × 10⁶ sperm/mL by using INRA-82 extender containing 0, 25, 50, 100, and 200 mM of trehalose then subjected to both cold storage and cryopreservation. Sperm motility, acrosome, plasmatic membrane, and DNA integrity were analyzed, and 57 mares were used to evaluate the field fertility of chilled and frozen-thawed semen. Results showed that the extender containing 100 mM trehalose only increased the functional acrosomal, plasma membrane, and DNA integrities. The inclusion of 50 mM trehalose in semen extender resulted in significantly (P < .05) increased post-thaw total motility compared to the control group, and chilled semen achieved higher pregnancy rates compared to the frozen-thawed one. Pregnancy rate of mares inseminated with frozen-thawed semen (P < .05; 46.15% vs. 36.36%, respectively) was lower than those inseminated with chilled semen (76.47% vs. 68.75%, respectively) but higher than control. In conclusion, addition of 50 mM trehalose yielded the highest quality stallion semen after cooling and post-thawing in terms of motility, integrities of acrosome, membrane, and DNA as well as improved field fertility.     

Pregnancy rates of mares inseminated with semen cooled for 18 hours and then frozen

The ability to ship cooled stallion sperm for subsequent freezing at a facility specializing in cryopreservation would be beneficial to the equine industry. Stallion sperm has been centrifuged, cooled to 5 degrees C for 12 h, and frozen without a detrimental effect on motility in a previous study; however, no fertility data were available. Experiment 1 compared the post-thaw motility of sperm cooled for 18 h at 15 or 5 degrees C at either 400 or 200 x 10(6) sperm/mL and then frozen. Storage temperature, sperm concentration, or the interaction of temperature and concentration had no effect on total (TM) and progressive motility (PM) after cooling. Post-thaw TM and PM were higher for control than (P < 0.05) for treated samples. There was no difference in post-thaw TM and PM due to temperature or concentration. Experiment 2 further evaluated procedures for cooling before freezing. Ejaculates were either cooled to 5 degrees C for 18 h and centrifuged, centrifuged at room temperature and then cooled to 5 degrees C for 18 h before freezing, or centrifuged and frozen immediately (control). There was no difference among treatments on post-thaw TM or PM. In Exp. 3, mares were inseminated with semen that had been extended in skim milk-egg yolk without glycerol, centrifuged, resuspended at 200 x 10(6) sperm/mL, cooled to 5 degrees C for 18 h, and then frozen or not cooled for 18 h before freezing (control). Pregnancy rates did not differ for mares receiving semen cooled and then frozen (21 of 30, 70%) or semen frozen directly without prior cooling (16 of 30, 53%). In summary, a procedure was developed for cooling stallion sperm for 18 h before freezing without a resultant decrease in fertility.     

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.     

In vitro evaluation of frozen-thawed stallion semen: a review

The article reviews methods used for in vitro evaluation of sperm, with particular emphasis on frozen-thawed stallion sperm. The techniques, limitations of the methods and correlations with fertility results are discussed. Very few studies have tried to find correlation between fertility of frozen stallion semen and laboratory tests. It is difficult and expensive to inseminate an adequate number of mares to achieve statistically significant differences. Significant, but low correlations have been demonstrated between the foaling rate and subjective motility of sperm incubated for 2 h and 4 h at 37 degrees C and hypoosmotic swelling test after 0 and 3 h of incubation. Significant correlations have been reported between the pregnancy rate and viability of propidium iodide-stained sperm assessed by flow cytometry as well as for glass wool and Sephadex filtration tests. No correlations have been detected between fertility and motility immediately after thawing. In spite of that, motility estimation by light microscope is the most commonly used method to evaluate frozen-thawed stallion sperm. Computer assisted automatic sperm analyzers have replaced light microscopy in research projects, but so far nobody has been able to demonstrate a correlation between fertility of frozen stallion semen and any of the motility parameters obtained by these instruments.     

The Effects of an Oxygen Scavenger and Coconut Water on Equine Sperm Cryopreservation

Alternative sources of lipoproteins in semen extenders could replace animal by-products. We hypothesized that: (1) post-thaw semen parameters and fertility would not be different in coconut water (CW)–treated samples compared with egg yolk (EY)–treated samples and (2) the use of an oxygen scavenger (Oxyrase) would improve post-thaw sperm motility and membrane integrity and decrease lipid peroxidation. Experiment 1: three ejaculates each from five stallions were split into four treatments: EY, CW, egg yolk with Oxyrase, and coconut water with Oxyrase. Computer-assisted sperm analysis measured progressive and total motility, velocity, and linearity. Membrane integrity, apoptosis, and lipid peroxidation were evaluated using propidium iodide, annexin, and BODIPY fluorescent probes, respectively. Samples were cryopreserved, stored in liquid nitrogen, and then thawed to 37°C and analyzed again. Experiment 2: one ejaculate was divided into two aliquots and cryopreserved using either CW or EY. In a crossover design, 12 mares were bred on two consecutive cycles with either EY or CW. Pregnancy evaluations were at 14-day gestation. No differences were detected in sperm parameters between CW and EY (P > .05). Oxyrase did not improve sperm motility parameters in post-thaw samples, nor did it show protective effects for viability or against membrane damage (P > .05). More mares became pregnant using CW than EY (11/12 vs. 6/12, respectively; P = .013). Use of CW is a viable alternative to animal-based products in the cryopreservation of stallion semen.     

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.     

Artificial insemination and preservation of semen.

Artificial insemination is an effective technique for improving utilization of stallions in breeding programs. When proper semen handling and insemination procedures are used, optimal pregnancy rates are attainable. When AI techniques are employed for mares and stallions with marginal fertility, pregnancy rates may be improved in comparison with natural mating. Preservation of stallion semen in the liquid or frozen state reduces the costs and potential health hazards incurred by transporting mares and provides easier access to genetic material that may otherwise be unavailable. Acceptable pregnancy rates are consistently obtained with cooled semen. Conversely, techniques for cryopreservation of stallion semen will require more refinement before the procedure can be considered commercially viable on a wide scale.     

Upregulation of CRISP-3 and kallikrein in stallion seminal plasma is associated with poor tolerance of cooled storage

For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expression in SP with either positive or negative effects on sperm survival during storage was studied using two-dimensional differential gel electrophoresis and liquid chromatography–mass spectrometry. Lower sperm chromatin integrity was associated with upregulation of the proteins kallikrein, CRISP-3 and HSP-1, while higher chromatin integrity was associated with upregulation of TIMP-2. In the sperm-rich fractions, kallikrein and CRISP-3 differed significantly between SP samples with differing effects on sperm chromatin integrity. In the sperm-poor fractions, TIMP-2 and HSP-1 differed significantly between the two SP groups. Differences in the seminal plasma proteome are associated with sperm longevity during cooled storage.     

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%).     

Relationship of Sperm Quality to Fertility after 4 Days of Cooled Storage of Equine Semen

This study evaluated measures of sperm quality in relation to fertility achieved with fresh semen or semen cooled and stored. Semen from 1 stallion was collected and processed to provide 3 treatments: group 1 received fresh semen; group 2 received cooled semen containing 50% seminal plasma (SP) stored for 4 days; and group 3 received cooled semen containing 50% SP stored for 1 day, then centrifuged and resuspended in fresh extender containing 10% SP on days 1 to 3. Inseminates were evaluated for sperm motion characteristics and the percentage of sperm with intact membranes (SMI). Mares (n = 34) in estrus were treated with an ovulation-inducing drug and inseminated with 100 million membrane-intact sperm on the following day. Pregnancy status was determined via transrectal ultrasonography 2 weeks after ovulation. The mean percentage of SMI was higher in group 1 (81%, initial) than in group 2 (74%, day 4) or group 3 (74%, day 4) (P < .05). The median percentages of total sperm motility differed among the groups (77%, 5%, 59% for groups 1, 2, and 3 respectively; P < .05). Median values for the percentages of progressively motile sperm and curvilinear velocity for group 1 (55%, 216 μm/s) and 3 (37%, 186 μm/s) were higher than for group 2 (1%, 73 μm/s) (P < .05). Pregnancy rates did not differ among groups (5 of 11, 45% in group 1; 5 of 11, 45% in group 2; and 7 of 12, 58%, in group 3; P = .77). These data suggest that, at least for this stallion, sperm membrane integrity may be a more valuable means of assessing potential fertility of cooled-stored semen than sperm motion characteristics.     

The Use of Sex-Sorted Stallion Semen in Embryo Transfer Programs

Sorting stallion semen into two separate populations of enriched X- or Y-bearing sperm can be done successfully. For this, stallion semen can be shipped to a sorting facility, but the mare must be in close to the sorting laboratory. Fertility rates when using 20-40 million sperm are an acceptable 60% per insemination. The procedure can be implemented in embryo transfer programs, with no deleterious effect on the pregnancy rate or embryonic death.     

Fertility of a stallion with low sperm motility and a high incidence of an unusual sperm tail defect

At the beginning of the breeding season an eight-year-old standardbred stallion had semen with virtually zero sperm motility and an approximately 90 per cent incidence of midpiece and tail defects. The motility of the sperm improved to 7 per cent when semen was collected daily but its morphology did not improve. Electron microscopy revealed that the defects consisted mainly of a loss of microtubules in the axoneme and of disorganised midpieces. A pregnancy rate of 24 per cent per cycle and 44 per cent for the season was achieved in 32 mares after the insemination of whole ejaculates collected from the stallion frequently. The fertility was much higher than would have been expected from the characteristics of the semen. It is concluded that this sperm defect, reminiscent of the 'Dag defect' in bulls and the defect in T-locus mice, does not render the animal infertile.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Effect of Seminal Plasma Components on the Quality of Fresh and Cryopreserved Stallion Semen

The importance of seminal plasma (SP) components for stallion semen quality and freezability is little known. This study aimed to evaluate the relationship between SP components and fresh/cryopreserved stallion semen quality. Semen of 30 stallions was collected, and then, SP was recovered and lyophilized. Total protein (TP), vitamin C (CVIT), vitamin E (EVIT), vitamin A (AVIT), iron (Fe), copper (Cu), magnesium, and zinc (Zn) in SP were assessed. Sperm was frozen in an extender supplemented with lyophilized SP. In fresh semen motility, abnormal morphology (AM), sperm vitality (SV), and plasma membrane integrity (PMI) were evaluated. In post-thaw semen, additionally, total motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat cross-frequency (BCF) were assessed. Levels of component of SP were established by a distribution analysis. Generalized linear models were fitted. Comparisons of means were done with Tukey's test. Correlation and regression analyses were performed. Vitamins and ions were found to be related to fresh semen quality. For post-thaw sperm, medium TP showed higher semen quality. Negative regression and correlation coefficients between CVIT and all post-thaw semen parameters were found. Low EVIT yielded the lowest PM, VSL, and VAP values, while a high level of AVIT yielded the best results for sperm quality. A high level of Cu yielded higher results for TM, PM, VCL, and ALH. Moreover, a negative correlation was found between Zn, SV, and PMI. In conclusion, SP composition influences fresh and post-thaw stallion semen quality.     

Current Reproductive Technologies Impacting Equine Embryo Production

Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12–24 hours and transport them to recipient stations. Embryo freezing has become more common using the technique of vitrification of embryos >300 μm or deflating embryos >300 μm before freezing. Oocyte vitrification has resulted in poor pregnancy rates although the technique works well in women. The ability to collect oocytes from mares and fertilize them by sperm injection has revolutionized the veterinarian’s approach to infertility in the mare and/or stallion. A transvaginal approach can be used to collect oocytes from preovulatory follicles and unstimulated follicles 5–25 mm in size. Although traditional in vitro fertilization does not work well in the horse, ICSI can be used to produce blastocysts which, upon nonsurgical transfer into recipients, provide a pregnancy rate similar to fresh embryos collected from donor mares. Sorting sperm by flow cytometry into X- and Y-bearing spermatozoa has been shown to provide about a 50% pregnancy rate with freshly sorted sperm but only 12% with sorted, frozen/thawed stallion sperm. It is likely that more advanced reproductive techniques will be developed in the future. Their acceptance will depend on how well they work, perceived need, cost, and, to some extent, the breed associations.     

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility

This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk–based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one “bad cooler” and one “good cooler” stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.     

Studies on Motility and Fertility of Cooled Stallion Spermatozoa

This study on extended, cooled stallion spermatozoa aimed to compare the ability of three extenders to maintain sperm motility during 24 h of preservation, and to describe pregnancy and foaling rates after artificial insemination (AI) of stallion spermatozoa stored and transported in the extender chosen from the in vitro study. After 6 and 24 h of preservation, motility, both subjective and evaluated by the motility analyzer (total, progressive and rapid), was lower in non-fat, dried skim milk-glucose than in both other extenders: dried skim milk-glucose added to 2% centrifuged egg yolk, and ultra high temperature treated skim milk-sugar-saline solution added to 2% centrifuged egg yolk (INRA82-Y). Rapid spermatozoa and sperm velocity parameters, after 24 h, were significantly higher in INRA82-Y. In the fertility trial, semen collected from three Maremmano stallions, diluted in INRA82-Y, and transported in a refrigerated Styrofoam box, was used to inseminate 56 mares of the same breed. Pregnancy rates after the first cycle and per breeding season were significantly higher for the 31 mares inseminated in three AI centres (54.8 and 80.6%, respectively) than for the 25 mares inseminated at the breeder's facilities (28.0 and 52.0%). Foaling rates were not significantly different between the AI centres mares (54.8%) and the other mares (44.0%). In conclusion, INRA82-Y yielded satisfactory pregnancy and foaling rates, especially when employed in the more controlled situation of an AI centre, and can therefore be included among those available for cooled stallion semen preservation.