Sexual behavior following introduction of a stallion into a group of mares

A rested stallion was introduced daily for 30 days into each of three herds of 20 mares. Observations of sexual and mating behavior were made for one hour. The stallion remained with a herd until another stallion was introduced the following day. Other mares were isolated from stallions and were bred by artificial insemination. The diameter or growth rate of the preovulatory follicle for the six days preceding ovulation and the length of the interovulatory interval for mares which did not become pregnant were not affected significantly by the presence of a stallion. The number of breedings per hour of observation (2.4 ±0.2) and the length of the interval from introduction of a stallion into the herd of mares to first breeding (12 ±1 min) were significantly different among stallions, but the length of the interval between breedings (17 ±2 min) was not. The mean number of breedings per stallion per hour was not affected significantly by the number of posturing (estrous) mares. The number of times that the stallion rebred the same mare when more than one mare postured during the observation hour (49%) was greater (P<0.01) than what would be expected to occur by chance (30%). The hypothesis that breeding occurs preferentially in those estrous mares that are closest to ovulation was not supported, except for significantly lower breeding activity in posturing mares on days 8 and 7 before ovulation and on days 0 (day of ovulation) and ?1 (26% bred) than on days 6 to 1 (52%).     

Morphometry of stallion spermatozoa by computer-assisted image analysis

Metric measurements of stallion spermatozoal heads were determined for live, unfixed spermatozoa and for Feulgen-stained spermatozoa by videomicroscopy and computerized image analysis. Two ejaculates were collected from each of five stallions of normal fertility. Air-dried semen smears were Feulgen-stained, and live, unfixed spermatozoa were examined as wet-mount preparations. For Feulgen-stained spermatozoa, videoimages (x3850) were captured, and sperm heads were detected via image segmentation and particle analysis. For live, unfixed spermatozoa, phase contrast videoimages (x3850) were measured to determine width and length of the sperm head. For Feulgen-stained spermatozoa, there were significant effects (P < 0.001) of stallion and ejaculate on measured parameters of area, circumference, and the length and width of the sperm head. For live, unfixed spermatozoa, there were significant effects of stallion on length and width and of ejaculate on length of the sperm heads. There was a very poor correlation between length and width of sperm heads between Feulgen-stained and live, unfixed spermatozoa. Two indices of sperm shape (oval factor and aspect ratio) were also determined. Both aspect ratio and oval factor were significantly affected by stallion (P < 0.001); however, oval factor was not affected by ejaculate and therefore may represent a less variable determination of sperm head shape across stallions. Overall, length and width of stallion sperm heads were larger (P < 0.01) for live, unfixed spermatozoa than for Feulgen-stained spermatozoa (length: 6.3 +/- 0.4 vs 5.08 +/- 0.44; width: 3.08 +/- 0.34 vs 2.71 +/- 0.28 mum, respectively). Computerized image analysis may be useful as a means to objectively measure sperm head dimensions in the stallion and could be useful in future studies to determine associations with stallion fertility.     

Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187

Experiments evaluated the ability of follicular fluid (FF), dilauroylphosphatidylcholine (PC12) and the calcium ionophore A23187 (A23187) to induce capacitation in stallion and bull spermatozoa, determined by the ability of the spermatozoa to penetrate zona-free hamster, bovine and equine oocytes. Spermatozoa suspensions were incubated at 37 degrees C in one of the following treatments: 1) a modified Tyrode's medium (BGM3) alone; 2) BGM3 + FF; 3) BGM3 + PC12; 4) BGM3 + FF + PC12; 5) BGM3 + A23187; and 6) BGM3 + FF + A23187. Treated spermatozoa were incubated with zona-free hamster, bovine and equine oocytes for 3 h, after which oocytes were stained to assess spermatozoa penetration. The number of hamster oocytes penetrated by spermatozoa incubated in BGM3 alone (1/30) or in presence of FF (2/31) was significantly lower (P < 0.05) than by spermatozoa treated with PC12 or A23187 (16/30 and 17/30, respectively). Processing stallion spermatozoa either by a swim-up procedure or by centrifugation through a Percoll gradient increased the percentages of motile spermatozoa in the final sample, and spermatozoa collected by both processes penetrated similar numbers of zona-free hamster oocytes (P > 0.05). Although treating spermatozoa with PC12 or A23187 enabled both stallion and bull spermatozoa to penetrate oocytes, higher numbers of bovine oocytes were penetrated by bull spermatozoa (25/30) than by stallion spermatozoa (4/30) regardless of spermatozoal treatment. However, the number of zona-free hamster and equine oocytes penetrated by bull spermatozoa (25/30 and 12/18 respectively) and stallion spermatozoa (17/30 and 15/21 respectively) were similar (P > 0.05). We conclude that both PC12 and A23187 capacitate stallion and bull spermatozoa sufficiently to permit the acrosome reaction to occur, enabling spermatozoa to penetrate homologous and heterologous zona-free oocytes.     

Pregnancy rates and sexual behavior under pasture breeding conditions in mares

Pony mares (n=480) and 16 stallions were assigned to four herds of 60 mares and one stallion (large herds) and to 12 herds of 20 mares and one stallion (small herds). The stallions remained with the herds continuously for all of the large herds and seven of the small herds. In the five remaining small herds the stallion was put into a herd for three hours every two days for 12 observation periods. Pregnancy rates and day of ovulation were estimated by size of embryonal enlargements. Mean pregnancy rates of 51% and 54% were obtained in the small herds and 42% in the large herds during a 48-day period (equivalent to two estrous cycles). Pregnancy rates for herds with the stallion present continuously were higher (P<0.01) for the small herds than for the large herds for days 1-24 (42% versus 19%). There was no effect of herd size on number of mares becoming pregnant per herd on days 1-24, but more mares (P<0.01) became pregnant during days 25-48 in the large herds (13.2 mares per herd versus 1.8). In the herds in which the stallion was present intermittently, the number of times that the stallion rebred the same mare when more than one mare was in estrus was greater (P<0.01) than what would be expected to occur by chance (observed, 21%; expected, 11%). Repeated breeding of the same mare seemed related to the availability or activity of the mare, since such mares more frequently followed and positioned themselves in the vicinity of the stallion. Most of the interferences by a mare which involved keeping the stallion and another mare apart were directed at the mare, whereas most of the interferences during mounting were directed at the stallion (P<0.01). Mares were more likely (P<0.01) to interfere when in estrus than when in nonestrus. When interfering mares were in nonestrus, their hostility was usually directed at the stallion (92%), whereas when in estrus their interference was more frequently directed at a mare (73%, P<0.01).     

Herding and snaking by the harem stallion in domestic herds

Four herds of pony mares, each consisting of a stallion and six mares, were used to characterize the nature of herding by the stallion and the factors that induced the herding behavior. Herding behaviors were compared among four successive treatments (six mares alone, stallion added, two new mares added, and entire herd moved to a new pasture). A new treatment was initiated every 7 days and behavior was studied for 5 consecutive days (Days 1-5) for each treatment. Observations were made every 10 min during a 2-h period for each day. The extent of herding was quantitated by the mean distances between mares. The extent of snaking (herding with the head and neck extended and ears held back) was scored 0, 1, 2, or 3 (nil, minimal, intermediate, and maximal, respectively). The mean distance among the original mares on Day 1 when the mares were alone was 5.0 mare lengths and was reduced (P < 0.05) to 1.9 mare lengths when the stallion was added. The mean distance among the original mares of an established stallion/mare herd (3.8 mare lengths) was reduced (P < 0.05) on the day the herd was moved to a new pasture (1.9 mare lengths), similar to the effect of the introduction of the stallion. Scores for the extent of snaking, as well as the extent of herding, were highest (P < 0.05) on Day 1 when the stallion was added or the stallion/mare herd was moved to a new pasture. The extent of herding and snaking decreased (P < 0.05) by Day 2 and was seen only occasionally on Days 3-5. The addition of new mares to the herd did not induce herding of the original mares. However, the new mares maintained mean distances of 8-12 mare lengths from the original mares, resulting primarily from chasing by the stallion. By Day 4, the distances between the new and original mares were not different (P > 0.05) from the distances among the original mares.     

Penile paralysis and paraphimosis associated with reserpine administration in a stallion

Reserpine was administered to an 8-yr-old Thoroughbred stallion at a dosage of 5 mg subcutaneously (s.c.) every 2 wk for a 2-mo period to control unmanageable behavior. Reserpine produced a satisfactory calming effect that lasted for about 2 wk. After the last injection, the stallion developed penile paralysis and was unable to retract his penis, resulting in paraphimosis and attendant penile edema. The prolapsed penis was reduced and kept within the prepuce by placing a purse string suture in the preputial orifice. Phenylbutazone was given orally (1 gm) and the stallion was exercised for 20 to 30 min twice daily. This treatment was continued for 20 d with little improvement. The purse string retention suture cut through the skin 5 d after the stallion was discharged from the clinic. The penis was then supported against the abdominal wall and the stallion was exercised by hand for 30 min each day. The stallion was not used for breeding within 34 mo after the last injection of reserpine. A breeding soundness examination was performed approximately 3 yr after the initial injury. At this time the stallion's penis was noted to extend 5 to 8 cm from the prepuce when in a detumescent state. Although the stallion protruded his penis when exposed to a mare in estrus, a full rigid erection was never attained. Examination of the penis revealed partial engorgement of the corpus cavernosum penis and a 2.5-cm-wide dorsal semi-circumferential depression of the penile shaft approximately 10 to 12 cm proximal to the glans penis. The penile shaft and glans penis distal to this depression were cooler than the proximal portion of the penis. Semen collection was attempted, aided by manual insertion of the penis into the artificial vagina. When serving the artificial vagina, no "belling" of the glans penis was observed, although ejaculation occurred. Semen evaluation indicated normal spermatozoal motility and morphology parameters. The stallion was able to breed several mares with manual assistance to guide the penis into the vagina and one mare was diagnosed pregnant.     

Sociosexual behavior and the ovulatory cycle of ponies (Equus caballus) observed in harem groups

Observations of sociosexual behavior of adult ponies, made on two harem groups (each comprised of one vasectomized male and three females), were correlated with follicular development and ovulation for a total of 15 cycles (minimum of 2 cycles per female). Mean cycle length (interovulatory interval) was found to be 19.7 days, with behavioral estrus lasting 7–8 days (5.5 days preovulatory; 2.3 days postovulatory). Estrous females typically showed increased frequencies of approaching and following the stallion, urinating, presenting, clitoral winking, and tail raising. Approaching and following the stallion appeared earlier and persisted longer than other estrous responses. Deviations from the modal estrous pattern included cycles with subestrus, continual estrus, behavioral estrus in the absence of ovulation, and displays of female mounting. Dominance tests revealed that a mare's status was unaffected by the phases of the estrous cycle. The presence of more than one estrous female affected the copulatory performance of both stallions, most notably in reduced latencies to first mount, intromission, and ejaculation, in spite of differences between the stallions in sexual vigor. Each stallion usually selected the dominant mare for copulation when there were multiple estrous females present, but mounts were not displayed exclusively to one female per test. The social testing situation made apparent the importance of use of space in sociosexual communication in this species, particularly in avoidance of the stallion by diestrous mares and standing alone or in proximity to him by estrous mares.     

Novel purification method for mammalian seminal plasma phospholipid-binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid

A family of bull seminal plasma (BSP) phospholipid-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid-binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low-density lipoprotein fraction (LDF). In order to demonstrate the feasibility of the method, we incubated LDF with alcohol precipitates of bull, boar, and stallion seminal plasma. LDF were re-isolated by ultracentrifugation along with bound proteins. LDF with associated proteins were dialyzed, lyophilized, and delipidated. BSP homologous proteins were finally purified by p-aminophenyl phosphorylcholine (PPC)-agarose and/or gelatin-agarose chromatographies, and analyzed by SDS-PAGE. With this new protocol, phospholipid-binding proteins of bull, boar, and stallion seminal plasma were recovered almost 100%. A new 12 kDa stallion seminal plasma protein of the same family was also isolated and partially sequenced. The radio-immunoassay (RIA) data showed that 10 mg of LDF can bind all BSP proteins present in 120 mg of alcohol precipitated BSP proteins. These results confirm the efficiency of the method and that the LDF step could be used for the isolation of all BSP proteins homologs from different mammalian species.     

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5° and 22 °C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 °C than 22 °C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC’s to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.     

Penetration of zona-free hamster eggs by liposome-treated sperm from the bull, ram, stallion, and boar

Spermatozoa from each of four rams, four stallions, and three boars (six semen samples) were treated with dilauroylphosphatidylcholine (PC12) liposomes and compared with control bull sperm to induce the acrosome reaction (AR) and study possible penetration of the sperm into zona-free hamster eggs. Diluted sperm were incubated with several concentrations of PC12 for 7 min at 39 degrees C prior to insemination of the hamster eggs in vitro. The sperm from the bull were diluted to 10(6) cells/ml, as previously studied. Sperm from the ram, stallion, and boar were diluted to 6 X 10(6) and 20 X 10(6) cells/ml. After addition to the eggs, the sperm concentration was reduced by 75 percent. Inseminated eggs were incubated with sperm for 3 h at 39 degrees C prior to being fixed, stained, and observed for sperm penetration. At an initial concentration of 6 X 10(6) cells/ml, bull sperm treated with 36.7 microM PC12 achieved an egg penetration rate of 92%, whereas under nearly identical conditions stallion spermatozoa achieved only 54% egg penetration. Under similar conditions, ram spermatozoa failed to penetrate eggs, but when the initial sperm concentration was increased to 20 X 10(6) cells/ml, sperm incubated with 51.1 microM PC12 achieved 52% egg penetration. Boar spermatozoa treated with PC12 at either sperm concentration failed to exhibit an AR or penetrate hamster eggs. In general, as PC12 concentration increased the percentage of sperm with an AR increased and sperm motility decreased. It is concluded that 1) PC12 liposomes are effective in inducing the AR in sperm from the bull, ram, and stallion, but under conditions tested are ineffective with boar sperm;(ABSTRACT TRUNCATED AT 250 WORDS)     

Exposure to stallion accelerates the onset of mares' cyclicity

Horses (Equus caballus) belong to the group of seasonally polyestrous mammals. Estrous cycles typically start with increasing daylight length after winter, but mares can differ greatly in the timing of onset of regular estrus cycles. Here, we test whether spatial proximity to a stallion also plays a role. Twenty-two anestrous mares were either exposed to one of two stallions (without direct physical contact) or not exposed (controls) under experimental conditions during two consecutive springs (February to April). Ovarian activity was monitored via transrectal ultrasound and stallion's direct contact time with each mare was determined three times per week for one hour each. We found that mares exposed to a stallion ovulated earlier and more often during the observational period than mares that were not exposed to stallions. Neither stallion identity nor direct contact time, mare age, body condition, size of her largest follicle at the onset of the experiment, or parasite burden significantly affected the onset of cyclicity. In conclusion, the timing of estrous cycles and cycle frequency, i.e., crucial aspects of female reproductive strategy, strongly depend on how the mares perceive their social environment. Exposing mares to the proximity of a stallion can therefore be an alternative to, for example, light programs or elaborated hormonal therapies to start the breeding season earlier and increase the number of estrous cycles in horses.     

Insemination results with slow-cooled stallion semen stored for 70 or 80 hours

An insemination trial was conducted to evaluate the fertility of extended slow-cooled stallion spermatozoa stored for 70 h or 80 h at 5 to 7 degrees C before insemination. Then, 1 or 2 of the first sperm-rich fractions were collected with an open-ended vagina from 4 stallions. Semen from each stallion was diluted within 2 to 3 min after collection with a modified Kenney skim milk extender (6). The proportion of raw semen in the insemination doses was 24+/-6%. One insemination dose (25 to 50 ml) consisted of approximately 2 billion total spermatozoa. In the trial, palpation per rectum and ultrasonography of 34 mares (40 cycles) were performed every 12 h. The pregnancy rate per cycle (30-d) with semen stored for 70 h before insemination was 77% (17 cycles) and, with semen stored for 80 h, 57% (23 cycles). The difference was not statistically significant. The combined pregnancy rate per cycle was 65%. These results indicate that stallion semen can retain its fertilizing capacity for up to 80 h when collected and diluted using this procedure and when the inseminations are done less than 12 h after ovulation.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum

An enzyme-linked immunosorbent assay (ELISA) was developed and evaluated to detect equine antisperm antibodies (ASA) in horse serum. Six maiden mares between 12 and 18 mo of age were immunized with stallion sperm cells (SC group, N=2), seminal plasma (SP group, N=2), or phosphate-buffered saline (PBS) as a control (C group, N=2). Horses received a second injection of the same antigen 2 wk after the first. Blood was collected weekly for 10 wk after initial immunization and again at Week 15. Serum ASA levels (IgG and IgA) were measured by ELISA using two assay systems, one containing stallion SC as the plate antigen and another containing SP.

In horses immunized with SC, peak IgG levels were detected by ELISA during Wk 2 and 3 after first injection using either plate antigen. The antibody levels persisted through Week 5 and then slowly declined until Week 15. Horses immunized with SP had IgG levels that did not differ from control horses using either ELISA plate antigen. The only significant elevation in serum IgA ASA occured during Week 5 after initial immunization and only in mares immunized with SC as detected by ELISA using SC as the plate antigen. Attachment of ASA to stallion spermatozoa was confirmed by an indirect immunofluorescence assay.     

Fertility control using intrauterine devices: an alternative for population control in wild horses

The purpose of this study was to develop a contraceptive method for feral horses. The feral horse population has increased significantly in recent years despite attempts to control numbers. As in most wild animal population control programs, contraceptive methods must be easy to apply, cause minimal disruption to the social structure and be fully reversible. In the present study, we tested the effectiveness of an intrauterine device (IUD) for fertility control in mares. Six mares were fitted with a silastic O-ring-shaped IUD on July 1 of Year 1. The IUD-treated mares were turned out with 12 nontreated mares and a fertile stallion in a large pasture until October 20 (112 d). None of the IUD-treated mares and all the nontreated mares became pregnant. The IUD-treated mares were maintained separately from the stallion during the winter. Following removal of the IUD on April 27 of Year 2, the mares were again introduced to the pasture with the stallion together with 6 nontreated mares. For the 6 mares previously treated with an IUD, the mean interval from introduction to the stallion to conception was 17.5 +/- 5 d or 1.3 cycles per pregnancy, and all mares produced a normal foal at term. Subsequently, 19 recorded mare breeding seasons resulted in 18 foals. Uterine cytology and histopathology indicate that the IUD causes mild chronic endometritis without permanent changes in the endometrium. We conclude that based on our observations, the O-ring-shaped IUD is an effective, safe and practical contraceptive method for mares.     

Studies on the frequency and associations of equine leucocyte antigens in sarcoid and summer dermatitis

The equine leucocyte antigen (ELA) types and the clinical diagnosis for equine sarcoid and summer dermatitis were evaluated in 2026 horses representing five breeds. Data were analysed in unrelated animals and in family material. In the case of equine sarcoid, a strong association was observed between the ELA class II DW13 antigen and its effect on Swiss (cP < 0·001), French (cP < 0·0001) and Irish (cP < 0·01) Warmblood horses. The class I antigen A3 occurred more frequently in sarcoid-affected French horses (cP < 0·001). These results confirm our earlier findings (Gerber et al. 1988). Among Freiberger horses, which lack the ELA DW13 and A3 specificities, a breed-specific class I antigen, ABe108, displayed an increased frequency (cP < 0·05) in the affected group. Among Arabian horses, a tendency for increased frequency of the A1 antigen was observed in the affected animals, but the number of affected horses is too small for statistical significance. The Mendelian segregation in diseased half-siblings by ELA DW13 heterozygous stallions showed a strong association (P < 0·0001) between the inherited DW13 antigen and susceptibility to the sarcoid effect. In the case of summer dermatitis, previously published data (Marti et al. 1992) have been extended. The ELA types in four multiple-case families, founded by the same stallion, were analysed for an association with the effect of sarcoid. Eight out of nine ELA-typed affected offspring inherited the paternal haplotype A15, DW23 in contrast to nonaffected offspring where three out of 12 displayed these antigens (P < 0·005). Moreover, the ELA haplotypes of 11 out of 12 informative affected half-siblings sired by another stallion inherited the paternal haplotype A3, W12, DW23 (P < 0·05). Our findings demonstrate statistically significant associations between certain ELA antigens and two equine diseases. It is still unknown if the major histocompatibility complex (MHC) molecules themselves or another linked gene(s) play a role in the pathogenesis of these conditions.     

Comparison between glycerol and ethylene glycol for the cryopreservation of equine spermatozoa: semen quality assessment with standard analyses and with the hypoosmotic swelling test

The aims of this study were to compare glycerol (G) at customary concentrations and ethylene glycol (EG) as cryoprotectants for stallion semen in a skimmed milk (SM) extender, to test different EG concentrations and to compare the results of manual and computerized analysis with the hypoosmotic swelling (HOS) test. Ejaculates from two stallions were collected over 3 weeks (6 ejaculates per stallion), diluted in a SM based extender, divided into 4 fractions, centrifuged and diluted again to a concentration of 100 x 10(6) mL(-1) progressive motile spermatozoa (PMS) in addition with the cryoprotectant (3% G, 3% EG, 6% EG, 9% EG). Sperm motility was assessed both by microscopy (in raw and frozen-thawed semen immediately after thawing) and with an HTM-IVOS analyzer (Hamilton-Thorne Research, MA, USA), at 0, 1, 4, 6, and 12 h after thawing and storage at 21 degrees C. Raw and frozen-thawed (0 h) semen samples for G and EG at 3% were also submitted to the HOS test with a 100 mOsm sucrose solution and were evaluated to detect the presence of swollen tails. The higher EG concentrations (i.e. 6% EG and 9% EG) significantly reduced the percentage of motile and PMS, immediately after thawing. At the same concentration, i.e. 3%, G resulted in a higher percentage of PMS than EG (36.2 vs. 30%, P < 0.05), but at 12 h after thawing and storage at 21 degrees C, no significant differences were detected between G and EG at 3%. The correlations between progressive motility (assessed by direct microscope observation or measured through the HTM analyzer) and the HOS test results for 3%G and EG were r = 0.61 and r = 0.35, respectively. The HOS test confirmed its suitability as a complementary method of analysis for stallion semen. We conclude that with the SM extender used, EG could substitute G as the cryoprotectant for stallion semen if used at the same or lower concentration.     

Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential

Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sperm parameters evaluated included cell volume, membrane integrity, lipid peroxidation, caspase 3, 7, and 8 activation, mitochondrial membrane potential, and integrity of the cytoskeleton. Glycerol exerted toxicity at concentrations ≥ 3.5% and the maximal toxicity was observed at 5%. The actin cytoskeleton was especially sensitive to glycerol presence, inducing rapid F actin depolymerization at concentrations over 1.5%. The sperm membrane and the mitochondria were other structures affected. The toxicity of glycerol is apparently related to osmotic and nonosmotic effects. In view of our results the concentration of glycerol in the freezing media for stallion spermatozoa should not surpass 2.5%.     

Effects of egg yolk and glycerol levels in lactose-EDTA-egg yolk extender and of freezing rate on the motility of frozen-thawed stallion spermatozoa

Two experiments were designed to evaluate the effects of egg yolk and glycerol concentrations, freezing rate, and clarification of a lactose-EDTA-egg yolk extender on the post-thaw motility of stallion spermatozoa. In both experiments there was no influence of freezing rate (vapor vs controlled) on the percentage of progressively motile spermatozoa after thawing. Furthermore, no significant interaction among treatments was detected. In Experiment 1, clarified (centrifuged at 34,400 × g for 30 min) lactose-EDTA-egg yolk extenders containing 16 or 20% egg yolk and 3 or 4% glycerol were superior to those containing 12% egg yolk or 2% glycerol, based on the percentage of progressively motile stallion spermatozoa at 0, 30, 60, and 90 min after thawing. However, in Experiment 2, clarification of the lactose-EDTA-egg yolk extender was detrimental to the ability of the stallion spermatozoa to survive after thawing; 4% glycerol was superior to 2% glycerol. The best extender based on the percentage of progressively motile spermatozoa after thawing was nonclarified lactose-EDTA-egg yolk extender containing 20% egg yolk and 4% glycerol.     

Factors affecting the success of surgical and nonsurgical equine embryo transfer

Nonsurgical embryo recovery was attempted from light-horse and draft mares. Embryo recovery rates were not affected (P>.05) by technician or stallion but were lower (P<.05) from draft mares (44%) than light-horse mares (67%). Sham transfer of embryos on day 8 post-ovulation did not (P>.05) increase the number of mares returning to estrus by 22 days post-ovulation. Method of embryo transfer greatly affected pregnancy rates. Embryos transferred surgically during March–June resulted in 0 of 12 pregnancies versus 13 of 25 pregnancies obtained during July–September, This strongly suggests a seasonal influence on pregnancy rates. Technician influenced (P<.05) the success of nonsurgical transfer (46.2% vs. 7.7%). In addition, protection of the insemination rod with a sheath (guarded method) appeared to provide some advantage over an unguarded method of nonsurgical transfer (54% vs. 23%). Lastly, a preliminary experiment was conducted to evaluate transfer of embryos via flank incision. Four of 5 embryos transferred by this method resulted in a pregnancy at 50 days post estrus.     

Use of an androgenized mare as an aid in detection of estrus in mares

A study was conducted over a 2-mo period to compare estrus detection results obtained using an androgenized teaser mare with those obtained with a stallion, using the same group of 10 normally cyclic mares. The teaser mare was androgenized by administration of boldenone undecylenate (500 mg i.m. every 1 to 2 wk), and allowed to run loose with the mare group. Estrus was determined by observation of the group for a 30-min period daily. In the second month of the experiment, a marking harness was used on the androgenized mare to help detect mares mounted when in estrus. Estrous periods detected by each teasing method were 1) first month: stallion, 18; androgenized mare, 5; 2) second month: stallion, 16; androgenized mare, 9. There were no estrous periods detected by the androgenized mare that were not also detected by the stallion. Under these conditions, the androgenized mare was not an adequate estrus detection aid. Also discussed are the successful results of an independent trial on a breeding farm using an androgenized mare as an estrus detection aid.