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In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.

The objective of this study was to determine if prolonged pulsatile administration of homologous gonadotropin-releasing hormone (GnRH) at therapeutic or 5x therapeutic doses would cause down-regulation of the stallion's hypothalamic-pituitary-testicular axis. Fifteen stallions were randomly assigned to three treatment groups (n=5/group) and received a 0.5 ml subcutaneous dose of saline (group 1), 50 microg GnRH (group 2), or 250 microg GnRH (group 3) every 2 hours for 75 days. Weekly evaluations of follicle stimulating hormone, luteinizing hormone, and testosterone and monthly evaluations of daily sperm output and spermatozoal motility failed to demonstrate any decreased pituitary or gonadal responsiveness within or among treatment groups (P > 0.1) as a result of treatment with GnRH. Results of this study demonstrate that the hypothalamic-pituitary-testicularaxis of the stallion, unlike that of other domestic species, is remarkably refractory to GnRH-induced down-regulation.

Five experiments were conducted to evaluate damage incurred in each processing step for cryopreservation of stallion spermatozoa. In Experiment 1, semen was centrifuged for 9 centrifugation times and the percentage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed. Recovery of spermatozoa was > or = 80% when spermatozoa were centrifuged for > or = 10 min. Experiment 2 evaluated spermatozoa cryopreserved at 5 different concentrations in each of 2 extenders (skim milk-egg yolk-glycerol, SM-EYG; and lactose-EDTA, LAC). In SM-EYG, TMOT and PMOT were higher at spermatozoal concentrations of 20, 200 and 400 x 10(6)/ml (51%/41%, 52%/44%, 50%/43%, respectively) than for samples frozen at > or = 800 x 10(6) spermatozoa/ml (41%/35%, 32%/27%; P < 0.05). Spermatozoa frozen in LAC at a concentration of 20 x 10(6)/ml resulted in the highest TMOT and PMOT (43% and 30%, respectively, P < 0.05). The effect of freezing rate on motion characteristics of spermatozoa was evaluated in Experiment 3. The VCL of spermatozoa frozen in SM-EYG was the only parameter affected by freezing rate (P < 0.05). Experiment 4 evaluated motion characteristics after cryopreservation of spermatozoa in different sized straws (0.5 or 2.5 ml) in each of 2 extenders (SM-EYG and LAC). In SM-EYG, PMOT (38%) and VCL (109 microns/s) were highest when spermatozoa were frozen in 0.5 ml straws (P < 0.05). In Experiment 5, spermatozoa thawed immediately after cryopreservation or thawed after storage in liquid nitrogen for 24-48 h were evaluated. There was no effect of length of storage in liquid nitrogen on spermatozoal motion characteristics (P < 0.05). Experiment 6 evaluated the effects of cooling time to 5 degrees C (0, 2.5 and 5 h) on motion characteristics of spermatozoa cryopreserved in 2 extenders (SM-EYG and LAC). TMOT and PMOT were effected by cooling time, and there was a cooling-time-by-extender interaction (P < 0.05). In SM-EYG, TMOT and PMOT were higher if spermatozoa were cooled to 5 degrees C prior to initiation of freezing than if freezing was initiated at 20 degrees C (P < 0.05). A suggested protocol for cryopreservation of stallion spermatozoa would include: 1) centrifugation at 400 g for 14 to 16 min; 2) extension at 23 degrees C with SM-EYG to 400 x 10(6) spermatozoa/ml; 3) cool to 5 degrees C for 2.5 h; 4) package in 0.5 ml straws at 5 degrees C; 5) freeze in liquid nitrogen vapour at -160 degrees C; and 6) thaw for 30 s in 37 degrees C water.

Three experiments were conducted to evaluate the effects of egg yolk and(or) glycerol added to a nonfat dried skim milk-glucose (NDSMG) extender on motion characteristics and fertility of stallion spermatozoa. In Experiment 1, ejaculates from each of 8 stallions were exposed to each of 4 extender treatments: 1) NDSMG, 2) NDSMG + 4% egg yolk (EY), 3) NDSMG + 4% glycerol (GL), and 4) NDSMG + 4% egg yolk + 4% glycerol (EY + GL). Samples were cooled at -0.7 degrees C/min from 37 to 20 degrees C; subsamples were then cooled at -0.05 or -0.5 degrees C/min from 20 to 5 degrees C. Percentages of motile spermatozoa (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, 24 and 48 h after initiation of cooling. There was no overall effect (P > 0.05) of cooling rate. PMOT was highest (P < 0.05) for spermatozoa extended in NDSMG + GL at 48 h. At 24 and 48 h, MOT and PMOT were lowest (P < 0.05) for spermatozoa extended in NDSMG + EY. In Experiment 2, ejaculates from 8 stallions were exposed to each of 4 treatments: 1) NDSMG, 2) NDSMG + EY, 3) semen centrifuged in NDSMG and resuspended in NDSMG, and 4) semen centrifuged in NDSMG and resuspended in NDSMG + EY. Samples were cooled from 20 to 5 degrees C at each of 2 rates (-0.05, -0.5 degrees C/min). A detrimental interaction between seminal plasma and egg yolk was noted for PMOT at 6 h and for both MOT and PMOT at > or = 24 h postcooling. Experiment 3 determined if egg yolk or glycerol affected fertility. The seminal treatments were 1) NDSMG, 2) NDSMG + EY with previous removal of seminal plasma, and 3) NDSMG + GL. All samples were cooled to 5 degrees C and stored 24 h before insemination. Embryo recovery rates 7 d after ovulation were lower for mares inseminated with spermatozoa cooled in NDSMG + EY (17%, 4/24) or NDSMG + GL (13%, 3/24) extenders, than semen cooled in NDSMG (50%, 12/24). We concluded that egg yolk (with seminal plasma removal) or glycerol added to NDSMG extender did not depress MOT or PMOT of cooled stallion spermatozoa but adversely affected fertility.

Motion characteristics of cooled stallion spermatozoa in 2 freezing extenders were studied. Ejaculates from 8 stallions were split into treatments and cooled in thermoelectric cooling units at each of 2 rates. Cooling started at 37 degrees C for Experiments 1 and 3 and at 23 degrees C for Experiments 2 and 4, at a rate of -0.7 degrees C/min to 20 degrees C and from 20 to 5 degrees C, at either -0.05 degrees C/min (Rate I) or -0.5 degrees C/min (Rate II). Percentages of motile (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, 24 and 48 h. Treatments in Experiment 1 were modified skim milk extender (SM); SM + 4% egg yolk (EY); SM + 4% glycerol (GL); and SM + 4% egg yolk + 4% glycerol (EY + GL). At 24 and 48 h, MOT and PMOT were lowest (P < 0.05) for spermatozoa extended in SM + EY; spermatozoa in SM + GL had the highest MOT and PMOT. Thus, glycerol partially protected spermatozoa against the effects of cooling after long-term storage. Treatments in Experiment 2 were SM, semen centrifuged and pellet resuspended in SM (SMc), SM + EY, and semen centrifuged and pellet resuspended in SM + EY (EYc). Spermatozoa in SM + EYc had the highest (P < 0.05) PMOT at 24 h and MOT and PMOT at 48 hours. Spermatozoa in SM + EY (not centrifuged) had the lowest MOT and PMOT at 24 and 48 h, respectively. There was a detrimental interaction between egg yolk and seminal plasma. Extenders in Experiment 3 were Colorado extender (CO3), CO3 + 4% egg yolk (EY), CO3 + 4% glycerol (GL), and CO3 + 4% egg yolk + 4% glycerol (EY + GL). Spermatozoa in CO3 + EY had the lowest (P < 0.05) PMOT at 24 and 48 h. CO3 did not protect spermatozoa cooled in the presence of seminal plasma. Therefore, in Experiment 4 we tested CO3 with seminal plasma present (control) and semen centrifuged and pellet resuspended in CO3 (CO3c), CO3 + EY (EYc), CO3 + GL (GLc) and CO3 + EY + GL (EY + GLc). Spermatozoa in CO3 had the lowest (P < 0.05) MOT and PMOT at all time periods, which suggested a detrimental interaction of this extender with seminal plasma.

The control of bacteria in semen of stallions has been most effective with the use of seminal extenders containing suitable concentrations of antibiotics. However, the detrimental effect of antibiotics on sperm motility may be greater in stored, cooled semen due to the prolonged exposure to the antibiotic. Therefore, a study was conducted to determine the effect of various antibiotics on sperm motion characteristics following short term exposure and during cooled storage of semen. Reagent grade amikacin sulfate, ticarcillin disodium, gentamicin sulfate and polymixin B sulfate were added to a nonfat, dried, skim milk - glucose seminal extender at concentrations of 1000 or 2000 mug or IU/ml. Aliquots of raw semen were diluted with extender-antibiotic combinations to a concentration of 25 x 10(6) spermatozoa/ml. An aliquot was also diluted with extender without antibiotic. Aliquots were incubated at 23 degrees C for 1 h. In addition, portions of the aliquots were cooled from 23 to 5 degrees C and stored for 48 h. During 1 h of incubation of extended semen at 23 degrees C, there was a significant (P<0.05) reduction in the percentage of progressively motile spermatozoa for samples containing gentamicin sulfate. After 24 h of storage at 5 degrees C, 2000 mug/ml of gentamicin and levels equal to and greater than 1000 IU/ml of polymixin B in seminal extender resulted in significant (P<0.05) reductions in the percentages of motile and progressively motile spermatozoa. After 48 h of cooled storage, a level of 1000 mug/ml of gentamicin sulfate. resulted in significant (P<0.05) reductions in the percentages of motile and progressively motile spermatozoa. Levels equal to or greater than 1000 IU/ml of polymixin B sulfate also resulted in a significant (P<0.05) reduction in mean curvilinear velocity. Levels up to 2000 mug/ml of amikacin sulfate and ticarcillin disodium had no significant effect on sperm motion characteristics during short-term incubation at 23 degrees C or storage for 24 h at 5 degrees C. Overall, the addition of antibiotics to extender did not significantly (P>0.05) improve motion characteristics of spermatozoa over control samples. However, levels of gentamicin sulfate greater than 1000 mug/ml and of polymixin B sulfate equal to or greater than 1000 IU/ml should be avoided in seminal extenders used for cooled semen.

Three experiments were designed to analyze the effects of cooling rate on survival of stallion spermatozoa in a milk-based extender, at 0 to 96 hours after reaching the desired temperature. The samples were warmed to 37 degrees C and were evaluated by computer-assisted analysis of sperm motility. In Experiment 1, rate of cooling between 37 and 20 degrees C was evaluated. Sperm motion was not affected by cooling at plunge, -0.42 or -0.28 degrees C/minute. However, storage of spermatozoa at 5 degrees C after slow cooling below 20 degrees C was superior to storage at 20 degrees C. In Experiment 2, 3 cooling rates from 37 degrees to 5 degrees C were evaluated. Cooling at either -0.05 or -0.7 degrees C/minute was superior (P<0.05) to plunging spermatozoa to 5 degrees C. Cooling at -0.05 degrees C/minute rather than -0.7 degrees C/minute maximized the percentage of motile spermatozoa and their curvilinear velocity. In Experiment 3, cooling rates from 20 to 5 degrees C were evaluated, with all samples cooled at -0.7 degrees C/minute from 37 to 20 degrees C. Sperm motion was similar (P>0.05) after cooling below 20 degrees C at -0.012, -0.05 or -0.10 degrees C/minute, and the 2 slower rates were superior (P<0.05) to cooling at -0.3 degrees C/minute. It was concluded that stallion spermatozoa can be cooled rapidly from 37 to 20 degrees C, but should be cooled at

Gonadotrophin-releasing hormone (GnRH) was administered subcutaneously to reproductively normal stallions, either in a pulsatile manner (10 micrograms GnRH/2 h; n = 6) or as a continuous infusion (10 micrograms GnRH/2 h; n = 6), and in a pulsatile manner to 9 reproductively abnormal stallions, from February to July, 1988. Hormonal secretion patterns, testicular parameters and semen characteristics were monitored before and during treatment. In general, pulsatile GnRH caused a significant increase (P less than 0.05) in luteinizing hormone (LH) concentrations in the peripheral blood of normal stallions. LH levels also appeared to increase in abnormal stallions but the rise was not significant (P greater than 0.05). Stallions given GnRH by continuous infusion and the untreated control stallions did not show an increase in LH concentrations during the treatment period. None of the treatments resulted in significant increases in peripheral blood concentrations of testosterone, although individual stallions that showed an increase in LH secretion appeared to show some increase in testosterone secretion rate. In general, and for individual stallions, none of the treatments resulted in increased total scrotal width, total number of spermatozoa per ejaculate or the percentage of progressively motile spermatozoa in the ejaculate. It was concluded that although pulsatile administration of GnRH may increase the secretion rate of LH and, consequently, testosterone, this adjustment does not increase testicular size or output and motility of spermatozoa.

Spermatozoa from four regions of the epididymis and from ejaculated semen were evaluated for their resistance to cold shock, progressive motility, and structural stability. Spermatozoa were incubated at 38 C and their percentage of eosinophilia was compared with that of spermatozoa cooled to 0 C in 2 minutes, 10 C in 12 minutes, or 4 C in 22 minutes. Spermatozoa motility was estimated visually under phase-contrast microscopy and was recorded by cinematography. Structural stability of spermatozoa incubated in 0.05 M sodium borate buffer, 0.035 M sodium dodecyl sulfate (SDS), 0.002 M dithiothreitol (DTT), or SDS+DDT was evaluated by phase-contrast and electron microscopy. The percentage of eosinophilic spermatozoa did not differ among regions of the epididymis and was not increased by cold shock. Cooling ejaculated spermatozoa to 0 C in 2 minutes increased (P < 0.01) the occurrence of eosinophilia (32% vs 73%). Spermatozoa released from the caput or proximal corpus epididymidis into 0.154 M NaCl were not progressively motile; only 4% of the spermatozoa from the distal corpus were motile Cauda epididymal and ejaculated spermatozoa exhibited similar motility (41% vs 47%). Stability of chromatin was greater in spermatozoa from the distal corpus than those from the caput epididymis. Chromatin of spermatozoa from the distal corpus was resistant to 7.5 minutes of SDS+DTT treatment, whereas virtually all spermatozoal nuclei from the caput were decondensed by SDS alone. Tail organelles of spermatozoa acquired stability between the proximal corpus and the cauda epididymidis. All tail organelles of spermatozoa from the proximal corpus were dissolved by 7.5 minutes of SDS treatment, whereas tail organelles of distal corpus epididymal spermatozoa had dissolved after 7.5 minutes of SDS+DTT treatment. Stability of tail organelles in cauda epididymal and ejaculated spermatozoa was similar. Seemingly, equine spermatozoa are infertile until they enter the cauda epididymidis.

Fifty-four normally cycling, non-lactating mares were given 2 injections (i.m.) of PGF-2 alpha (10 mg) 14 days apart without regard to stage of the oestrous cycle. At 19 days after the first PGF-2 alpha treatment, a single i.m. injection of either hCG (3300 i.u.) or a GnRH-analogue (500 micrograms) was administered. Each mare was inseminated with 100 X 10(6) motile spermatozoa at one of the following frequencies: once only on Day 20; every other day during oestrus or at least on Days 19 and 21; or daily during oestrus or at least on Days 19, 20, 21 and 22. Eighteen control mares received saline injections on Days 0 and 14, and were inseminated either on the 4th day of oestrus or every other day or daily beginning on the 2nd day of oestrus. More (P greater than 0.05) PGF-2 alpha treated mares displayed their 1st day of oestrus on Days 14 to 20 than control mares (80.6 versus 27.8%). During cycle 1, fewer (P greater than 0.05) treated mares became pregnant compared to controls; 38.9, 25.0 and 66.7% for PGF-2 alpha + hCG, PGF-2 alpha + GnRH-A and control mares, respectively. After three cycles, the pregnancy rates for mares inseminated every other day or daily were higher (P less than 0.05) than mares inseminated only once during oestrus (88.9 and 88.2 versus 64.7%).

Two experiments were conducted to evaluate the effects of six extenders and three glycerol levels on the motility of sperm stored at 5 degrees C. Using a split-ejaculated design, semen from 10 dogs and 12 stallions was extended with egg-yolk-tris (EYT), egg-yolk-bicarbonate (EGB), Beltsville F-3 (BF-3), Cornell University (CUE), caprogen (CAP) and heated skim milk (SM) extenders. After cooling to 5 degrees C, additional extender containing 0% to 12% glycerol was added to provide a final concentration of 0%, 3% or 6% glycerol. Regardless of glycerol level, a higher (P<0.05) percentage of canine sperm retained their potential for progressive motility in CAP extender than in EYT, SM, CUE, EGB or BF-3 extenders. The SM extender was the best (P<0.05) for maintaining motility of equine sperm. The inclusion of 6% glycerol depressed (P<0.05) motility of canine sperm, but there was no effect (P>0.05) of glycerol concentration on the percentage of motile equine sperm. For both species, the interaction of glycerol level and extender was nonsignificant. CAP may be useful for storage of canine sperm at 5 degrees C and SM may be satisfactory for storage of equine sperm.

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).

Semen from each of six bulls was used to study the effects of percent glycerol and rate of freezing on spermatozoa extended in egg yolk-sodium citrate extender and packaged in .25-ml Continental straws. Treatments were arranged factorially and included 5, 6, 7, 8, 9, 10, or 11% (volume/volume) glycerol; freezing rates (time from 5 to -130 C) of 1, 2, 3.5, 7, 12, or 20 min; and post-thaw incubation times of 0 and 2 h at 38 C. Post-thaw motility was similar for spermatozoa frozen in 2 to 20 min, and all means were greater than for spermatozoa frozen in 1 min. Post-thaw motility of spermatozoa frozen in 7% glycerol did not differ from that with 5 to 9% but was superior to that with 10 or 11% glycerol. Although glycerol and freezing rate interacted, 7% glycerol provided survival equal to or greater than that with the other percents of glycerol, regardless of freezing rate.

The relationship between post-thaw motility and the percentage of sperm with intact acrosomes was evaluated in semen from 26 yearling Hereford or Angus bulls. A total of 409 samples was used, which consisted of 208 pooled first and second ejaculates, split and frozen in ampules and straws. All bulls passed a breeding soundness exam several weeks prior to the experiment. However, bulls were not subjected to any further selection for seminal quality, and all were of unknown fertility. In addition, all ejaculates from each bull were frozen without regard to seminal quality. Motility was assessed immediately after thawing, whereas acrosomal integrity was evaluated after 2 h of post-thaw incubation at 38 degrees C. The simple coefficient of correlation among bulls between motility and percentage of sperm with intact acrosomes was only .33. When variation in seminal quality among bulls was removed statistically, the corresponding coefficient of correlation within bulls was only .23. Under conditions of this experiment, post-thaw motility and acrosomal integrity constituted distinct, separate features of spermatozoal integrity that varied independently of each other.

Two studies were conducted to determine the relationship between LH and progesterone and between PMSG and progesterone during pregnancy in mares. In the first, samples of jugular blood were collected daily from 7 mares from the first day of oestrus until Day 28 of pregnancy, and in the second, samples were collected weekly from 14 mares from Day 35 of gestation until parturition. In an attempt to prolong secretion of progesterone from accessory corpora lutea, 7 of these 14 mares were injected with increasing doses (2--10 mg) of diethylstilboestrol (DES) between Days 84 and 142 of gestation. The remaining 7 mares received injections of vehicle. Concentrations of LH, PMSG and progesterone in serum were determined by radioimmunoassay. From the onset of oestrus until Day 4 of gestation, serum concentrations of LH and progesterone were negatively correlated (r = 0.67, P less than 0.01), but from Days 5 to 28 a positive correlation (r = 0.80, P less than 0.01) was noted. Likewise, serum concentrations of PMSG and progesterone were highly correlated between Days 35 and 196 in mares injected with DES (r = 0.72, P less than 0.01) and the vehicle (r = 0.75, P less than 0.01). Injections of DES did not influence serum concentrations of LH, PMSG or progesterone, or affect the length of gestation. It was concluded that DES does not influence the maintenance of pregnancy in the mare.

The influence of percent egg yolk during cooling from 37 to 5 C on ability of sperm to withstand subsequent freezing was studied with semen from each of eight bulls. Treatments were arranged factorially and included .5, 1, 2, 4, 8, 16, and 32% egg yolk, cooling times of .5, 1, 2, and 4 h, and freezing in ampules and straws. Percent egg yolk did not influence motility or percent intact acrosomes when semen was frozen in ampules. For sperm in straws, both post-thaw percentages of motile sperm and intact acrosomes were similar for sperm cooled with .5, 1, 2, 4, or 8% egg yolk and greater than with 16 or 32% egg yolk. Optimal egg yolk during cooling was not dependent upon rate of cooling, regardless of how semen was packaged for freezing. The influence of percent egg yolk during freezing was investigated in a second study. Treatments included eight bulls; 1, 2, 4, 8, 16, and 32% egg yolk; freezing times from +5 to -130 C of 3.5, 20, and 40 min; and freezing in ampules or straws. Optimal egg yolk during freezing in either ampules or straws was independent of the rate of freezing. For sperm frozen in ampules, differences in motility or the percent intact acrosomes due to egg yolk were small. However, the use of only 1 or 2% egg yolk depressed motility whereas 16 or 32% egg yolk decreased the percentage of intact acrosomes. Thus, extremely high or low egg yolk should be avoided. For sperm in straws, survival was optimal when the extended semen contained 4 to 32% egg yolk during freezing.

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

Data from 257 normally cyclic mares used in trials over a 10-year period were examined to establish the relationship between the number of inseminations per cycle, duration of oestrus and pregnancy rate. Mares that became pregnant were inseminated more (P less than 0.05) frequently per cycle than mares that did not become pregnant (3.4 versus 2.8). First-cycle pregnancy rates of 22.2, 34.0, 38.6, 52.5, 58.3 and 52.2% were attained when mares were inseminated 1, 2, 3, 4, 5 or 6 or more times per cycle, respectively. The duration of oestrus in mares that became pregnancy was longer (P less than 0.01) than in those that did not conceive (7.9 versus 6.4 days). Studies of 64 mares in the transitional season showed that first-cycle pregnancy rates for mares in which oestrus lasted less than 10, greater than or equal to 10, greater than or equal to 20, greater than or equal to 30 and greater than 40 days were 56.2, 76.2, 76.5, 77.3 and 88.9%, respectively. Overall pregnancy rates (after 3 cycles) were lower (P less than 0.05) for mares that had an initial oestrus of less than 10 days. Mares were inseminated every other day of oestrus with 100 X 10(6) progressively motile spermatozoa. First-cycle pregnancy rates were 64.3, 75.0 and 75.0% for mares inseminated 1-4, 5-10 and greater than or equal to 12 times per cycle, respectively. Fewer (P less than 0.05) mares became pregnant after 3 cycles when inseminated 1-4 times per cycle than did mares inseminated greater than or equal to 12 times per cycle (51.3 versus 75.0%). Numerous inseminations per cycle of mares with extended oestrus were not detrimental to fertility.

Extragonadal reserves totalled 89 X 10(9) spermatozoa for 5--16-year-old sexually rested stallions and 60 X 10(9) for 2--4-year-olds. Regardless of age, the cauda epididymidis contained 62% of the total reserves and the vas deferens, including the ampulla, contained 7% of the total reserves of spermatozoa. The caput plus corpus epididymidis from 5--16-year-old stallions (N = 41) contained 14.9 X 10(9) spermatozoa per side as compared (P less than 0.01) to 8.5 X 10(9) for 2--4-year olds (N = 30). Frequency of ejaculation did not influence the number of spermatozoa found in caput plus corpus epididymidis. Across all ages, the number of spermatozoa potentially available for ejaculation from the cauda epididymidis and vas deferens on each side totalled 54 X 10(9). Collection of 5 successive ejaculates from sexually rested stallions removed 40--60% of the available spermatozoa while ejaculation every 2nd day reduced (P less than 0.05) the number of spermatozoa potentially available for ejaculation by 27%. Nevertheless, sufficient spermatozoa are produced daily (6.4 and 4.2 X 10(9) for 5--16-and 2--4-year-olds) to permit use of an average stallion once or twice daily, during spring and summer, providing sexual behaviour is adequate.

Preimplantation embryos from mice and cattle were exposed to bluetongue virus in vitro to determine whether the virus would replicate in these early embryos and, if so, what pathologic consequences would ensue. A high proportion of zona pellucida-free, 2-cell embryos and morulae from mice, and morulae from cattle became infected. The infection was rapidly cytopathic in embryos from both species. Indirect immunofluorescence was used to demonstrate accumulation of virus antigen in the blastomeres of these embryos. The zona pellucida of both murine and bovine embryos provided effective protection from virus present in culture fluid.

Twelve stallions that had been given 0, 50 or 200 micrograms testosterone propionate (TP)/kg body weight every other day for 88 days were examined for the effects of androgen withdrawal on spermatozoal production, seminal quality and libido. Although the lower dosage did not affect most of the traits studied, the higher dosage severely reduced scrotal width, spermatozoal production, the number of sperm per ejaculate, the percentage of progressively motile spermatozoa and the percentage of normal spermatozoa. These adverse effects were found to be largely reversible. By 90 days after the cessation of treatment, scrotal width, testicular weight and spermatozoal production were similar for treated and control stallions. Although the number of sperm per ejaculate remained lower for stallions given 200 micrograms TP than for controls during the recovery period, the number of sperm in the extragonadal ducts was similar for all groups after 90 days of recovery. Spermatozoal motility and morphological characteristics were normal for all three groups by the end of the recovery period. Libido was not affected by TP treatment or withdrawal.

Fertility of semen frozen in .25-ml Continental straws or 1.0-ml ampules was compared in two field trials. Semen from four bulls was used to inseminate 574 cows over 38 days in Trial 1. Rates of pregnancy for cows inseminated with semen in straws or ampules were 54 and 32% for first-service inseminations and 70 and 50% at the end of the breeding period. Semen from one bull was used to inseminate 204 cows over 44 days in Trial 2. Rates of pregnancy from single inseminations averaged 58 and 44%, while corresponding values at the end of the breeding period were 83 and 67% for semen in straws and ampules.

Two experiments established the relationships among total scrotal width (TSW), daily spermatozoal production (DSP), daily spermatozoal output (DSO) and extragonadal spermatozoal reserves (EGR) of dogs ejaculated daily. In Experiment 1, 11 dogs (14 to 36 kg) were ejaculated daily for two 10-day periods and in Experiment 2, seven dogs (15 to 47 kg) were ejaculated daily for two 20-day periods. Approximately six daily ejaculations were required for stabilizing DSO. After the second period of daily ejaculations, dogs were castrated or killed and the testes were weighed and DSP and EGR were determined. In Experiments 1 and 2, DSP averaged 11.7 +/- 0.5 and 16.7 +/- 1.4 X 10(6) per gram of testicular parenchyma, respectively, or 369 and 594 X 10(6) per dog. DSP per gram of testicular parenchyma was not significantly correlated with parenchymal weight for the same testis in either experiment (r = 0.04 and 0.26). Mean (+/- SEM) EGR of dogs in Experiment 1 were 4024 +/- 368 X 10(6) and 4791 +/- 767 X 10(6) in Experiment 2. Approximately 63% of the EGR were contained in the caput and corpus epididymidis, 36% in the cauda epididymidis and 1% in the ductus deferens. DSO averaged 79 and 82% of DSP for the 2 groups of dogs. Based on combined data for all 18 dogs, TSW was correlated with testicular weight (r = 0.95; P less than 0.01), DSP (r = 0.82; P less than 0.01) and DSO (r = 0.75; P less than 0.01). Thus, measurement of TSW is a useful predictor of a dog's ability to produce and ejaculate spermatozoa.

The following recommendations and conclusions are based upon results of fertility and laboratory studies, and general trends from field investigations. Fertility results due to the addition of enzymes have been variable and contradictory. Flushing of ampules with dry, gaseous nitrogen prior to filling has become a routine practice in processing semen to be frozen. For control of Vibrio fetus and Leptospira pomona, 2,000 micrograms of streptomycin and 1,000 u polymyxin B sulfate should be added per milliliter of raw semen immediately after collection. The extender for initial dilution should contain the same concentration of antibiotics used for raw semen plus 500 u penicillin. The glycerol portion of the extender should contain 500 u penicillin per milliliter. The effect of addition of sugars on fertility has been highly variable. The primary beneficial effect is probably due to their cryoprotective properties. A myriad of concoctions have been added to bovine semen and the results have been highly variable with respect to both motility and fertility. Results of subsequent experiments have rarely proven that addition of exotic compounds or mixtures has been of value. Higher mean fertility was obtained with semen in straws in 14 of 21 comparisons with ampules. The differences in favor of straws ranged from 1.1 to 18.9; while the range in favor of ampules was .1 to 4.4 percentage points. Fertility obtained with pellets has ranged from minus 12.8 to plus 11.9 percentage points in nonreturn rate (NR), compared to the corresponding NR with semen in ampules. Fertility of semen in ampules was higher in five of eight studies. Fertility of pelleted semen has ranged from minus 9.5 to plus 6.0 percentage points compared with straws. Fertility was higher for semen in pellets in only one of five investigations. Pellets should not be used until the potential for pathogenic contamination and exchange of spermatozoa among pellets is eliminated. There is a potential for higher fertility with semen in straws as compared to other packaging systems, but the issue of liquid nitrogen (LN) entry and possible contamination of semen should be further investigated. In general, fertility obtained with semen frozen in the .25 ml straw has been equal to or higher than semen in larger packages. However, they cannot be unequivocally recommended due to other considerations. From laboratory studies, it appears that greater spermatozoan survival is obtained when semen frozen in straws is thawed in water at 35 C or above.(ABSTRACT TRUNCATED AT 400 WORDS)

Factors affecting testicular measurements in situ and the relationships among the measurements and various reproductive characteristics were studied using data from 48 stallions. Mean values during the breeding season are provided for scrotal width, widths and lengths of individual testes, combined weight of testicular parenchyma, daily sperm production and daily sperm output. Testicular measurements were highly repeatable from day to day and for repeated measurements on a given day; technician provided the largest source of variation in the measurements of a given stallion. Age significantly affected all testicular measurements; testicular size for 2- to 3-year-old stallions did not differ (P greater than 0.05) from that for 4- to 6-year-olds, but was smaller (P less than 0.05) than testicular size of stallions greater than or equal to 7 years old. Scrotal width was correlated (P less than 0.01) with daily sperm production (r = 0.75) and daily sperm output (r = 0.55) and was generally the most repeatable measurement.

Experimental and clinical observations were made to treat abnormal sexual behaviour. The most common cause of abnormality was mismanagement of the animal; over-use and rough treatment at service and too-frequent ejaculation during winter had a detrimental effect on the behaviour of young stallions. Pain due to injury incurred at copulation or when associated with mounting attempts was also a common cause of impotence. Most impotent stallions responded well to re-training and recovery can be achieved without pharmacological treatment in most cases.

Five ejaculates were collected at hourly intervals from 32 sexually rested stallions. Gel volume, total seminal volume, sperm concentration and spermatozoa per ejaculate declined (P less than 0.01) from the first to the second or third ejaculate. Gel-free seminal volume or percentage of motile spermatozoa did not vary (P less than 0.05) among ejaculates. Ejaculates from 2- to 3-year-old stallions contained less volume and fewer spermatozoa than those from 9- to 16-year-old stallions. Regardless of the stallion's age the first, first 2, first 3 and first 4 ejaculates represented 50, 74, 86 and 93% of the total numbers of spermatozoa contained in the 5 ejaculates. In addition, the number of spermatozoa that might be obtained in 5 ejaculates can be predicted by multiplying the total number of spermatozoa in two successive ejaculates by 1.35 +/- 0.03. Although some fifth ejaculates contained as few as 0.02 X 10(9) motile spermatozoa, the mean number of motile spermatozoa in fifth ejaculates from sexually rested stallions was 0.52 X 10(9) spermatozoa; presumably enough to impregnate a mare during natural service.

Two experiments were conducted to determine the effects of HCG on duration of oestrus, dioestrus, the length of the oestrous cycle, the time of ovulation and fertility in non-lactating mares. In the first experiment, the injection of HCG was repeated for three successive cycles. Mares injected with 2000 i.u. HCG on Day 2 of oestrus during their first cycle had a shorter oestrus and ovulated sooner than untreated control mares, but in the third cycle, treated mares had a longer oestrus and ovulated longer after the onset of oestrus than controls. In the second experiment, one intramuscular injection of 3300 i.u. HCG was given 24 hr before the first insemination in the first cycle. Oestrus was shortened by 3-1 days, but there was no difference in pregnancy rate between treated and control mares (61-1 versus 63-9%). During the two cycles following injection, the conception rate in the treated mares was higher. The number of inseminations/cycle to effect fertility was significantly less over three cycles in the treated mares.

Sixteen bulls ranging in age from 2 to 11 years were experimentally inoculated with bluetongue virus to investigate the frequency and duration of seminal shedding of the virus. All bulls developed typical viremia, lasting 21 to 58 days, and they seroconverted 2 to 3 weeks after inoculation. Virus isolation was attempted from a total of 232 ejaculates, 163 (70%) of which were collected during the period of viremia. Bluetongue virus was not isolated from any of the ejaculates collected from 11 of the 16 bulls. Virus was isolated from 9 of 52 ejaculates collected from the other 5 bulls during the period of viremia. In no instance was virus isolated from semen without concurrent isolation from blood.

The relationship between daily sperm productions (DSP) and daily sperm output (DSO) was determined in eleven stallions. The DSO was determined by collecting single ejaculates, with an artificial vagina, at 24-hr intervals. The stallions were killed 24 hr after the last collection and the DSP was determined by quantitative testicular histology. The mean DSP was 8-0 X 10(9) (S.E. +/- 0-4 X 10(9), and the mean DSO was 7-0 X 10(9) (S.E. +/- 0-4 X 10(9)). It was estimated that 87% of the spermatozoa produced by the testes were harvested. The correlation between DSP and DSO was 0-80 (P less than 0-01), and between DSP and testis weight was 0-77 (P less than 0-01).