Scanning electrons and light microscopy of the equine seminiferous tubule.

Changes within the equine seminiferous tubules during the cycle of the seminiferous epithelium were studied light and scanning electron microscopy (SEM). Once observed with SEM, tubules were sectioned and staged using light microscopy. As viewed by SEM, the weblike, spongy cytoplasm of germ cells or Sertoli cells in stages I and II extended over the entire height of the germinal epithelium. The cytoplasm of the basal portion of the germinal epithelium in stages III to VIII was similar to that in stages I and II. However, the cytoplasm which occupied the luminal third of the epithelium in stages III to VII was smooth appearance. The smooth-surfaced, periluminal cytoplasm diminished in stages VIII. Principal pieces of flagella from spermatids extended into the tubular lumina in all stages whereas the middle pieces extended into the lumen only in stage VIII. Later in stage VIII, the middle pieces, which were thickened with cytoplasm, were connected to the germinal epithelium by stalks. After spermiation, the diameter of the middle pieces was similar to that of ejaculated spermatozoa. Thus, the cytoplasm within the thickened middle pieces contributed to the formation of the cytoplasmic droplets.

Effect of centrifugation and seminal plasma on motility and fertility of stallion and bull spermatozoa.

The effect of centrifugation of diluted and undiluted semen on equine and bovine spermatozoan motility and fertility was examined, as was the effect of seminal plasma and dilution on stallion spermatozoa during incubation before and after freezing. Centrifugation at 370 g or 829 g was not detrimental (P greater than 0.05) to prefreeze or postfreeze motility if a final concentration of 10% seminal plasma was present. A reduction of seminal plasma from 10% to 2% significantly (P smaller than 0.05) reduced motility. A centrifugal force of 956 g significantly reduced prefreeze but not postfreeze motility of spermatozoa in undiluted semen, regardless of seminal plasma concentration. With a dried skim milk extender, prefreeze and postfreeze motility was greater in samples containing 20% seminal plasma. Motility was depressed by high and low concentrations of seminal plasma. The fertility of frozen or unfrozen stallion spermatozoa was not depressed (P greater than 0.05) by centrifugation at 310 g for 3.5 minutes. In contrast, the fertility of bull semen was significantly (P smaller than 0.05) lowered by centrifugation at 270 g for three minutes. Further, the fertility of centrifuged, diluted bovine semen was lower (P smaller than 0.05) than centrifuged, undiluted semen.

Gonadal and pituitary responsiveness of stallions is not down-regulated by prolonged pulsatile administration of GnRH.

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.

Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws.

Experiments were conducted to evaluate two extenders (egg-yolk Tris and egg-yolk lactose), varying concentrations of two cryopreservatives (glycerol and dimethyl sulfoxide), and rates for cooling to 5 degrees C, cooling from 5 to -100 degrees C, and warming for canine spermatozoa packaged in 0.5-ml French straws. At optimal concentrations of glycerol, egg-yolk Tris extender was superior to egg-yolk lactose in preserving spermatozoal motility. Addition of dimethyl sulfoxide, alone or in combination with glycerol in either extender, was not beneficial to spermatozoal survival after thawing. Canine spermatozoa withstood a range of cooling and equilibration times with no detrimental effect on spermatozoal motility prior to freezing. However, there were differences in spermatozoal motility immediately after thawing; these differences were variable, resulting in a cooling time by equilibration time interaction. Spermatozoal motility after thawing was best preserved by freezing in egg-yolk Tris extender containing 2-4% glycerol, using a moderate rate of cooling from 5 to -100 degrees C (-5 degrees C/min from 5 to -15 degrees C, then -20 degrees C/min from -15 to -100 degrees C). Three of 12 bitches inseminated intravaginally with semen frozen using this protocol became pregnant.

Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility 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).

Fertility of stallion semen processed, frozen and thawed by a new procedure.

The fertility of frozen-thawed and fresh semen from three stallions was compared in a trial using a randomized block design and 90 mares for 108 cycles. Semen was collected every third day, diluted to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium, and centrifuged. The cells were resuspended at 700 x 10(6) progressively motile sperm/1.0 ml of added lactose-EDTA-egg yolk extender containing 4% glycerol, packaged by placing 0.55 ml into polypropylene straws, and frozen. Semen was thawed by immersion in 75 degrees C water for 10 sec. All of the 43 ejaculates collected were frozen, but 21 were discarded because progressive sperm motility was <35% immediately after thawing or <40% after 30 min of incubation at 37 degrees C. semen from the same stallions was collected daily for inseminations with fresh semen. Semen containing 200 x 10(6) progressively motile sperm was added to 10 ml of heated skimmilk extender. Mares were inseminated daily starting on the third day of estrus or when a >/=4-cm follicle was detected, whichever came later, and continuing through the end of estrus or for nine days. Based on palpation per rectum on day 50 postovulation, the pregnancy rates from inseminations during one estrus were 50, 56 and 61% with frozen semen and 67, 67 and 61% with fresh semen (P>0.05) from the three stallions, respectively. Thus, mean pregnancy rate with frozen semen was 86% of the rate attained with fresh semen.

Extenders for preservation of canine and equine spermatozoa at 5 degrees C.

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.

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen.

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.

Use of two freezing extenders to cool stallion spermatozoa to 5 degrees C with and without seminal plasma.

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.

Motility and fertility of equine spermatozoa in a milk extender after 12 or 24 hours at 20 degrees C.

The effects of extender and storage at 20 degrees C on equine spermatozoa were evaluated in two experiments using embryo recovery as the end point. In both experiments, inseminations were every other day, starting on Day 2 or 3 of estrus or after a 35-mm follicle was detected, with 250 x 10(6) progressively motile cells (based on initial evaluation). In Experiment 1, semen from two stallions was used to compare the motility and fertility of spermatozoa maintained in a) heated skim milk extender at 37 degrees C with insemination in <1 h; b) E-Z Mixin extender at 37 degrees C with insemination in <1 h; and c) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 12 h at 20 degrees C. The percentage of motile spermatozoa was 34% after 12 h compared to 55% at 0 h (P < 0.05). However, the percentage of mares from which an embryo was recovered 6.5 d after ovulation was 62, 56, and 50% for Treatments A, B, and C (P > 0.05). In Experiment 2, semen from three stallions was used to compare the motility and fertility of spermatozoa in a) E-Z Mixin extender at 37 degrees C with insemination in <1 h or b) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 24 h at 20 degrees C. The percentage of motile spermatozoa was 17% after 24 h compared to 54% at 0 h (P < 0.05). There was no difference between treatments (P > 0.05) in the percentage of mares from which an embryo was recovered 6.0 d after ovulation (68 vs 62%) or among stallions. Thus, stallion semen extended in E-Z Mixin was held at 20 degrees C for 24 h without a marked decline in fertility.

Effect of antibiotics on motion characteristics of cooled stallion spermatozoa.

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.

Effect of seminal extenders containing egg yolk and glycerol on motion characteristics and fertility of stallion spermatozoa.

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.

Effects of linear cooling rate on motion characteristics of stallion spermatozoa.

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

Factors affecting motion characteristics of frozen-thawed stallion spermatozoa.

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.

Relationship of absolute numbers of Sertoli cells to testicular size and spermatogenesis in young beef bulls.

Testes were obtained from 34 Hereford or Angus bulls at about 1.5 yr of age and were used to investigate the relationship between the absolute number of Sertoli cells vs testicular size and daily spermatozoal production (DSP). Quantitative determination of DSP was based upon enumeration of elongated spermatids in testicular homogenates. The ratio of step 8 spermatids to Sertoli cells (S:SC) was established by direct counts of these cells in each of 20 round stage VIII seminiferous tubular cross sections for each bull. The number of Sertoli cells per paired testes was calculated as (total spermatids divided by S:SC)/.394, where total spermatids equalled the number of homogenization-resistant spermatids. The factor of .394 adjusted for the fact that the latter cells are present for only 39.4% of the spermatogenic cycle. All data were subjected to simple linear and second-order regression analyses. A positive linear relationship (P less than .005) was found between testicular weight (Y, in grams) and the absolute number of Sertoli cells per paired testes (X, in billions), which was characterized by the equation Y = 315.2 + 10.74X and a coefficient of correlation (r) of .56 (P less than .01). A similar relationship was observed between DSP (Y, in billions) and Sertoli cell numbers (X, in billions). This was characterized by the equation Y = 1.36 + .222X (P less than .005) and a coefficient of correlation of .70 (P less than .01). Daily sperm production was unrelated to the S:SC ratio (P greater than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of seminal additives and packaging systems on fertility of frozen bovine 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)

Restoration of reproductive capacity of stallions after suppression with exogenous testosterone.

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.

Effect of anabolic steroids on reproductive function of young stallions.

Thirty-two stallions were used to determine the effect of anabolic steroids on reproductive function. Stallions were assigned to one of the four treatments: 1) .23 ml sesame oil/kg of body weight (BW; control, C); 2) 4.4 mg boldenone undecylenate/kg BW (4E); 3) 1.1 mg boldenone undecylenate/kg BW (1E) and 4) 1.1 mg nandrolone decanoate/kg BW (D). Injections were given at 3-wk intervals for 15 wk. Semen was collected every other day for 3 wk before the first injection and at the same frequency during d 85 through 105 (d 0 = day of first injection). Libido was assessed on the basis of reaction time. Total scrotal width was determined every 2 wk. Serum was obtained at various intervals and analyzed for concentrations of luteinizing hormone (LH). Portions of testicular parenchyma were used to determine spermatid reserves and to permit quantitative histological evaluation of spermatogenesis. Gel, gel-free and total seminal volumes and pH were not affected (P greater than .05) by steroid treatment. However, spermatozoal motility, spermatozoal concentration and total sperm/ejaculate were severely lowered (P less than .05) by all anabolic steroid treatments. Total scrotal width for stallions in the D, 4E and 1E groups was less (P less than .05) than that of C stallions by wk 5. The weight of the testes of the D, 4E and 1E stallions averaged only 40.1, 44.9 and 61.6%, respectively, of that for the controls. Spermatozoal production was altered, as evidenced by smaller (P less than .05) numbers of spermatids/testis and primary spermatocytes for all treated groups than for the controls. Anabolic steroid treatment had no effect (P greater than .05) on erection time, time to first mount, ejaculation time or number of mounts/ejaculation. treatment with anabolic steroids resulted in a depression in concentration of LH in all treatment groups.

Sexual behavior, seminal pH and accessory sex gland weights in geldings administered testosterone and(or) estradiol-17 beta.

Sixteen stallions were castrated and 30 days later assigned to one of four treatments: (1) testosterone propionate (175 microgram/kg body weight), (2) 17 beta-estradiol-3-benzoate (44 micrograms/kg body weight), (3) a combination of both steroids or, (4) vehicle only. These dosage were administered every other day for 18 days. The dosages were then doubled and continued for 20 days. Concentrations of testosterone and estradiol in serum decreased rapidly after castration and stabilized within about 6 hours. Mean concentrations of testosterone and estradiol maintained by the steroids were 1.4 and 90 pg/ml, respectively, during treatment at the lower dosage, and 2.6 and 186 pg/ml during treatment at the higher dosage. Libido and the ability to ejaculate were gradually lost after castration. Testosterone restored both aspects of sexual behavior within 2 weeks. Estradiol effectively restored libido at the higher dosage, but was less effective in restoring the ability to ejaculate. The pH of gel-free semen increased after castration and was subsequently decreased by treatment with testosterone or the combination of both steroids. Estradiol had no effect on seminal pH. Weights of the seminal vesicles, ampullae and prostate were greater in geldings treated with testosterone or with both steroids than in estradiol-treated or control geldings. Since estradiol alone was able to restore libido in geldings, it is possible that the maintenance of libido in intact stallions involves either estradiol secreted directly by the testes or estradiol arising from aromatization in peripheral tissues. Treatment with estradiol at either concentration, did not appear to affect the size or function of sex glands in geldings.

Induction of abortion in mares with equimate: effect on secretion of progesterone, PMSG and reproductive performance.

Thirty-two light-horse mares were confirmed to be pregnant and assigned to one of four treatments: (1) injected with 250 micrograms of Equimate on day 70 and again on day 77 if abortion had not occurred; (2) injected with 250 micrograms of Equimate on day 70 and every 24 hr until abortion occurred (maximum four injections); (3) injected with 250 micrograms of Equimate on day 70 and every 12 hr until abortion (maximum eight injections); and (4) injected with 250 micrograms of Equimate once only on day 35 of gestation. Mares were observed four times daily for incidence of abortion or side effects. Estrual behavior was monitored daily and follicular activity either daily or every third or fourth day until estrus and ovulation. Samples of jugular blood were obtained at 0600 hr day -1 and every 6 hr until the first day of estrus after abortion, or for 2 weeks after abortion if estrus had not occurred, or for 1 week after treatment for mares that had not aborted. A single injection of Equimate terminated pregnancy in all but one mare injected on day 35, but none of the mares given an injection on day 70 and 77 aborted. Multiple injections of Equimate beginning on day 70 of gestation terminated pregnancy in all mares. Fewer (P less than .05) injections were required for abortion of mares injected daily. During the immediate week post-treatment, concentrations of progesterone decreased (P less than .05) in all mares injected with Equimate, but overall concentrations were greater (P less than .05) for mares injected once on day 70 than for those in the other three groups. Equimate did not affect secretion of PMSG in the day 70 group of mares. Estrus and ovulation after abortion were delayed (P less than 0.05) for mares injected daily or twice daily beginning on day 70 compared to those injected once on day 35. Thus, rebreeding of these mares during the same breeding season in which they were aborted would be difficult.