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.     

Cryopreservation of Stallion Spermatozoa with INRA96 and Glycerol

The aim of the present study was to improve success of cryopreservation of stallion spermatozoa. Semen from eleven stallions was collected and frozen in INRA 96 with two different concentrations of glycerol (3.5% and 6.0%) and compared with a control freezing process. The mean post-thaw motility for the eleven stallions of 57.93% (3.5% glycerol) and 66.50% (6.0% glycerol), which was statistically higher (P < 0.05) when compared with the mean post-thaw motility (39.7%) for semen in a control egg-yolk extender (Equipro^® CryoGuard™ Complete, Minitube). The Equipro^® CryoGuard™ Complete is a commercial semen freezing protocol that has been one of the standard processes used in our laboratory for freezing equine spermatozoa. INRA 96 with 6% added glycerol was used in the fertility trial as it provided the highest spermatozoa survival. To evaluate fertility of the frozen semen, eight mares were bred over two cycles with both fresh and frozen semen. The pregnancy rate of mares bred with frozen semen (55.6%) was not statistically different (P > 0.05) from the pregnancy rate of mares bred with fresh semen (55.6%). INRA 96 with 6.0% glycerol improved the survivability of stallion spermatozoa through the cryopreservation process, and subsequent fertility was not different (P > 0.05) from fresh, extended semen.     

Applicability of a New Cell Culture Device for Cooled‐Storage of Stallion Semen

A new device for storage and shipping of cell cultures – the Petaka G3 cell management device – was tested for its applicability for cooled‐storage of equine semen. Semen from three stallions was processed with EquiPro extender either without antibiotics (three ejaculates per stallion) or with gentamicin (250 mg / l; three ejaculates per stallion). Semen was either stored at five (anaerobic conditions) or 15°C (aerobic conditions) in syringes or cell culture devices. Total and progressive motility, as well as membrane integrity of spermatozoa, were evaluated from days 1 to 7 after collection with computer‐assisted semen analysis. In experiment 1 (extender without antibiotics), total motility, progressive motility and viability of spermatozoa significantly decreased over time (p < 0.05). The decrease was significantly faster at 15°C than at 5°C (p < 0.05). In the presence of gentamicin (experiment 2), this difference was no longer present. It can be concluded that cooled‐storage of equine semen in sophisticated devices for cell culture is not advantageous to syringes for successful maintenance of semen longevity.     

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.     

Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa

Two experiments were conducted to test whether stallionand/or semen processing techniques influenced spermatozoal motility and acrosomal status following cold storage. Ejaculates from each of 18 stallions (N=54) were collected and split. In Experiment I, a skim milk-glucose extender (SKMG) was added to the semen following a 5, 15 or 30 minute delay post-collection. Following each delay, sperm were packaged at a final concentration of 25 million progressively motile sperm per ml (PMS/ml) in a commercially available skim milk-glucose extender (SKMG). In Experiment II, sperm were packaged at concentrations of 25, 50, and 75 million PMS/ml both in the presence and absence of seminal plasma (SP) utilizing SKMG and SKMG plus PBS, respectively. In both experiments, aliquots were cooled, stored, and the percentage of progressively motile and acrosome intact spermatozoa were determined at 24 and 48 hours post-collection. In Experiment 1, delayed dilution resulted in a lower recovery of PMS. In Experiment II, removal of SP resulted in higher percentages of PMS following cold storage. Increasing the concentration of spermatozoa during packaging decreased the percentage of PMS; however, removal of SP reduced the harmful effects on spermatozoa motility. These data suggest that reducing the time that spermatozoa remain in an undiluted state and removal of SP maximize recovery of progressively motile, acrosome-intact spermatozoa. In addition, individualizing the processing techniques for each stallion may enhance spermatozoal survival following cold storage.     

Fertility of stallion spermatozoa isolated on albumin gradients

Fertility of stallion semen extended with bovine serum albumin (BSA) sucrose extender or cream-gelatin extender was compared. Pregnancy rates were 95% of 47 mares and 86% of 46 mares inseminated with BSA-sucrose and creamgelatin extended semen, respectively. Foaling rates and cycles per conception were not significantly different between treatment groups.Semen from 5 mature stallions was used in an attempt to isolate a population of highly motile spermatozoa. Immediately following collection, samples were evaluated for motility and forward movement. Seven to 10 ml of semen, extended 1:1 with BSA-sucrose extender, were pipetted onto BSA medium separation columns. After 1 hour incubation at 37°C, the top, middle and bottom layers were separately withdrawn from each separation column and pooled, respectively. A marked decrease (P<.001) was noted in the mean motility of spermatozoa in the top layer (35%) as compared to the mean pre-incubation motility (59%). Spermatozoa from middle and bottom layers were significantly (P<.001) more motile (70.6 and 87%) than those from the top layer and pre-incubation samples. Rate of forward movement (RFM) of spermatozoa in lower fractions was higher (P<.001) than RFM of spermatozoa in the top layer. Concentration of spermatozoa decreased (P<.001) as the concentration of BSA in the medium increased.     

Insemination Results with Slow-Cooled Stallion Semen Stored for approximately 40 Hours

Heiskanen, M.-L., M. Huhtinen, A. Pirhonen and P. H. Mäenpää: Insemination results with slow-cooled stallion semen stored for approximately 40 hours. Acta vet. scand. 1994,35,257-262.– Semen from 3 stallions was extended using 2 methods (Kenney extender and a modified Kenney extender), slowly cooled, and stored for 41 ± 6 (s.d.) h before insemination. An insemination dose (40 ml) contained 1.5-2 billion spermatozoa. In the experiment, 26 mares were inseminated in 30 cycles. The pregnancy rate per cycle obtained with sperm stored in the Kenney extender was 87% (n=15). When the semen was extended with the modified extender, centrifuged and stored, the pregnancy rate was 60% (n=15). Inseminations were done every other day until ovulation was detected. If a mare ovulated more than 24 h after the last insemination, she was inseminated also after ovulation. The single-cycle pregnancy rate was 58% when the mares were inseminated only before ovulation (n=19) but the rate was 100% when the inseminations were done both before and after ovulation (n=9) or only after ovulation (n=2). The difference in pregnancy rates was significant (p<0.05), indicating that postovula-tory inseminations probably serve to ensure the pregnancies. The extending and handling methods used in this study resulted in a combined pregnancy rate of 73%, and appear thus to be useful for storing stallion semen for approximately 2 days.     

Freezability and Fertility Results with Uncentrifuged Stallion Semen

The first (1 to 3) sperm-rich fractions of the ejaculate were collected from 4 stallions using an open-ended vagina. The volume of the collected fractions was 12 ± 8 ml with a density of 475 ± 200 million spermatozoa/ml. Before freezing, the semen was diluted with a skim-milk based extender 1:1 to 1: 8 (volume of semen: volume of extender), depending on the initial sperm concentration to achieve a final concentration of 100 million/ml. The total number of spermatozoa in an insemination dose ranged from 0.7 to 1 billion spermatozoa. Within 12 h after ovulation, 48 mares were inseminated in 70 cycles. The total single-cycle pregnancy rate at day 21 was 24%, but varied from 10% to 33% per cycle among the stallions.     

Influence of Semen Storage and Cryoprotectant on Post-thaw Viability and Fertility of Stallion Spermatozoa

The aim of the current study was to verify that stallion spermatozoa could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryopreservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio^® extender containing one of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5°C for 20 minutes, then frozen in nitrogen vapor. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty-three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine semen for 24 hours before freezing, while maintaining sperm viability and fertility, is possible.     

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.     

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.     

The Use of Cefquinome in Equine Semen Extender

Antibiotics are commonly used in equine semen extender for conservation, if semen has to be stored cooled for a maximum of 48 hours or frozen, to eliminate pathogenic or potentially pathogenic bacteria from semen and reduce the risk of postmating endometritis. Little is known about the effect of antibiotics on spermatozoa when semen is stored over a longer period. Cefquinome, a broad spectrum antibiotic and fourth-generation cephalosporin, has been proven to be a powerful drug for the treatment of endometritis and mastitis in different species. Recently in equine studies, it was found to localize in high concentrations in the endometrium. Therefore, cefquinome was used as the antibiotic in semen extender and compared with a commercial semen extender containing gentamicin for effects on motility and membrane integrity of spermatozoa. During the breeding season, ejaculates from nine light horse stallions were collected and half of each ejaculate was stored for 48 hours in modified Kenney type semen extender containing either cefquinome or gentamicin. At 0, 24, and 48 hours, aliquots (20 μL) of the stored semen were evaluated for (progressive) motility and membrane integrity, as well as for various motility parameters by computer assisted sperm analysis. No differences (P > .05) were found in total motility or progressive motility between extenders at any time point. However, there were differences (P < .05) in velocity parameters, although the effect of velocity parameters on fertility is not clear. In general, semen parameters after storage in non-fat dried skim milk semen extender containing cefquinome are comparable with those after storage in semen extender containing gentamicin. The wider spectrum of bactericidal activity possessed by cefquinome may prove to be beneficial in some cases.     

Stallion Semen Cooling Using Native Phosphocaseinate-based Extender and Sodium Caseinate Cholesterol-loaded Cyclodextrin-based Extender

The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspended at 100 million sperm/mL with their respective extender. Semen was then cooled and stored as in experiment 1. In both experiments, sperm motility parameters, plasma membrane integrity, and high mitochondrial membrane potential were assessed at 0, 24, and 48 hours post cooling. For experiment 3, 12 mares (n = 24 cycles) were bred with 48 hour–cooled semen from one stallion. Semen was processed as described in experiment 1. Mares had embryo flushing performed by 8-day post-ovulation. In experiment 1, BotuSemen Gold displayed superior total and progressive motility relative to INRA 96 (P < .05). There were no significant differences between the types of containers in any experiment. In experiment 2, INRA 96 and BotuSemen Gold extenders had similar total and progressive motility, but BotuSemen Gold had superior sperm velocity parameters at all timepoints. Embryo recovery was identical for both extenders (50%). Finally, the results obtained herein suggest that BotuSemen Gold is a suitable alternative to be included in semen cooling tests against INRA 96 in clinical practice.     

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.     

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

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

Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction

REASONS FOR PERFORMING STUDY: To compensate for the wide variation in the freezability of stallion spermatozoa, it has become common veterinary practice to carry out repeated ultrasonography of the ovaries of oestrous mares in order to be able to inseminate them within 6-12 h of ovulation with a minimum of 300-500 x 10(6) frozen-thawed spermatozoa. Furthermore, in order to achieve satisfactory fertility, this requirement for relatively high numbers of spermatozoa currently limits our ability to exploit recently available artificial breeding technologies, such as sex-sorted semen, for which only 5-20 x 10(6) spermatozoa are available for insemination. OBJECTIVES: This study was designed to evaluate and compare the efficacy of hysteroscopic vs. conventional insemination when low numbers of spermatozoa are used at a single fixed time after administration of an ovulation-inducing agent. METHODS: In the present study, pregnancy rates were compared in 86 mares inseminated once only with low numbers of frozen-thawed spermatozoa (3-14 x 10(6)) at 32 h after treatment with human chorionic gonadotrophin (hCG), either conventionally into the body of the uterus or hysteroscopically by depositing a small volume of the inseminate directly onto the uterotubal papilla ipsilateral to the ovary containing the pre-ovulatory follicle. RESULTS: Pregnancy rates were similarly high in mares inseminated conventionally or hysteroscopically with 14 x 10(6) motile frozen-thawed spermatozoa (67% vs. 64%). However, when the insemination dose was reduced to 3 x 10(6) spermatozoa, the pregnancy rate was significantly higher in the mares inseminated hysteroscopically onto the uterotubal junction compared to those inseminated into the uterine body (47 vs. 15%, P < 0.05). CONCLUSIONS: When inseminating mares with <10 x 10(6) frozen-thawed stallion spermatozoa, hysteroscopic uterotubal junction deposition of the inseminate is the preferred method. POTENTIAL CLINICAL RELEVANCE: Satisfactory pregnancy rates are achievable after insemination of mares with frozen-thawed semen from fertile stallions 32 h after administration of human chorionic gonadotrophin (Chorulon). Furthermore, these results were obtained when mares were inseminated with 14 x 10(6) progressively motile frozen-thawed spermatozoa from 2 stallions of proven fertility.     

Comparison of Different Extenders with Defined Protein Composition for Storage of Stallion Spermatozoa at 5°C

To maintain the fertility of stallion spermatozoa during cooled storage, extender media are added to semen. In this study, three semen extenders were compared: EquiPro which contains defined caseinates and whey proteins instead of dried skim milk. The extender is provided in dry form and dissolved in distilled water prior to use. EquiPro TM has the same composition as EquiPro but is provided in a sterilized ready-to-use liquid form. AndroMed-E contains soybean lecithin as protein source. Semen was collected from seven stallions. Ejaculates were divided into three aliquots, diluted with the different extenders and stored at 5 degrees C for 4 days. Total motility, membrane integrity, average path velocity (VAP), curvilinear-velocity (VCL), straight-line velocity (VSL), distance average path (DAP), distance curved line (DCL) and distance straight line (DSL) were determined by computer-assisted analysis. Total motility decreased in all extenders during storage. The parameters VAP, VCL, VSL, DAP, DCL and DSL in semen diluted in EquiPro TM at most times and in semen diluted in AndroMed-E at some times were lower than in semen diluted in EquiPro (p < 0.05). Viability on days 0 and 4 was lowest in semen diluted in AndroMed-E (p < 0.05). Velocity decreased faster when semen had been diluted in the sterilized liquid extender EquiPro TM or in AndroMed-E compared with the dry formula of EquiPro. Therefore the liquid sterilized EquiPro despite no difference in its chemical composition differs from the dry, non-sterilized EquiPro extender. Heat sterilization apparently changes effects of the extender on spermatozoa.     

Use of Cholesterol‐Loaded Cyclodextrin in Donkey Semen Cryopreservation Improves Sperm Viability but Results in Low Fertility in Mares

The use of cholesterol‐loaded cyclodextrin (CLC) on semen cryopreservation has been related with better sperm viability in several species; however, the effect on fertility is not known in donkey semen. Ejaculates (n = 25) from five donkeys were diluted in S‐MEDIUM with 0, 1, 2 or 3 mg of CLC/120 × 10⁶ spermatozoa. Semen was frozen, and thawed samples were evaluated by computer‐assisted sperm analyser system (CASA), supravital test, hyposmotic swelling test and fluorescent dyes to assess the integrity of sperm membranes. Mares (n = 60) were inseminated with frozen‐thawed semen treated with the doses of 0 or 1 mg CLC. Percentages of sperm with progressive motility and with functional plasma membrane were greater (p < 0.05) in the CLC‐treated groups than in the control. Percentages of intact plasma membrane and intact plasma membrane and acrosome detected by fluorescent dyes were also greater (p < 0.05) in CLC‐treated groups. Although no difference (p > 0.05) in conception rates was detected between groups (control, 3/30, 10%; CLC‐treated, 1/30, 3.3%), fertility was low for artificial insemination programs in mares. Therefore, we firstly demonstrated that frozen semen treated with CLC in S‐MEDIA extender before freezing improves the in vitro sperm viability, but semen treated or not with CLC in S‐MEDIUM extender results in a very low conception rate in mares inseminated with thawed donkey semen.     

Male, female and management risk factors for non-return to service in Dutch mares

The "effect" of stallion, mare and management-related factors on the odds of pregnancy per cycle in the horse were identified and quantified from the breeding records of Dutch Warmblood (n=4491), Friesian (n=1467) and Shetland-pony mares (n=3267) mated either naturally or by artificial insemination to one of the 88 stallions between 1992 and 1996. A mare was considered to be pregnant when she did not return to oestrous within 28 days of the last insemination. For Dutch Warmblood horses, the percentage of mares that did not return for service within 28 days (NR28) varied between studfarms and ranged from 61 to 82%. The NR28 for mares inseminated with fresh semen ranged from 67 to 74% and for mares inseminated with frozen/thawed semen this percentage was 59. Mares served at a second cycle had lower odds not to return than mares served at the third or subsequent cycle (OR=0.84). For Friesian horses, the NR28 for young mares was higher than that for older mares. Mares served before 1 May in any year had lower odds of non-return than mares served after 1 July (OR=0.69). The NR28 of mares inseminated once per cycle was 6% lower than that of mares inseminated three times or more per cycle. For Shetland ponies, the NR28 also varied between studfarms and ranged from 62 to 78%. Stallions < or =3 years old had lower odds of non-return compared to older stallion (> or =11) (OR=0.57). Mares served before 1 July had lower odds of non-return. Other significant factors for this breed were age of the mare, cycle number and insemination frequency. Stallion factors accounted for 5.9, 2.0 and 14.7% of the variation in the NR28 for Dutch Warmblood, Friesian horses and the Shetland ponies, respectively.     

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

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