D.J. Jasko

Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa.

Experiments were conducted to determine temperatures between 24 and 4 degrees C at which stallion spermatozoa are most susceptible to cold shock damage. Semen was diluted to 25x10(6) spermatozoa/ml in a milk-based extender. Aliquots of extended semen were then cooled in programmable semen coolers. Semen was evaluated by computerized semen analysis initially and after 6, 12, 24, 36 and 48 hours of cooling. In Experiment 1A, semen was cooled rapidly (-0.7 degrees C/minute) from 24 degrees C to either 22, 20, 18 or 16 degrees C; then it was cooled slowly (-0.05 degrees C/minute) to a storage temperature of 4 degrees C. In Experiment 1B, rapid cooling proceeded from 24 degrees C to either 22, 19, 16, or 13 degrees C, and then slow cooling occurred to 4 degrees C. Initiating slow cooling at 22 or 20 degrees C resulted in higher (P<0.05) total and progressive motility over the first 24 hours of cooling than initiating slow cooling at 16 degrees C. Initiation of slow cooling at 22 or 19 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of cooled storage than initiation of slow cooling at 16 or 13 degrees C. In Experiment 2A, semen was cooled rapidly from 24 to 19 degrees C, and then cooled slowly to either 13, 10, 7 or 4 degrees C, at which point rapid cooling was resumed to 4 degrees C. Resuming the fast rate of cooling at 7 degrees C resulted in higher (P<0.05) total and progressive motility at 36 and 48 hours of cooled storage than resuming fast cooling at 10 or 13 degrees C. In Experiment 2B, slow cooling proceeded to either 10, 8, 6 or 4 degrees C before fast cooling resumed to 4 degrees C. There was no significant difference (P>0.05) at most storage times in total or progressive motility for spermatozoa when fast cooling was resumed at 8, 6 or 4 degrees C. In Experiment 3, cooling units were programmed to cool rapidly from 24 to 19 degrees C, then cool slowly from 19 to 8 degrees C, and then resume rapid cooling to storage temperatures of either 6, 4, 2 or 0 degrees C. Storage at 6 or 4 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of storage than 0 or 2 degrees C.

Effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen

Abstract Centrifugation of stallion semen to remove seminal plasma was hypothesized to be potentially beneficial in maintaining spermatozoal motility during cooled storage of extended semen. This hypothesis was tested using a series of experiments that evaluated spermatozoal motion characteristics, as determined by computerized semen analysis, of extended, cooled stallion semen. In the first experiment, aliquots of semen were extended to 50 × 10 6 sperm/ml with a nonfat, dried skim milk solids-glucose semen extender and subsequently were cooled slowly to 5°C and stored for 24 hours. Those aliquots utilizing high dilution ratios of extender (>1:2.1, volume of semen: volume of extender) maintained higher spermatozoal motility and mean velocity than aliquots using a lower dilution ratio. In the second experiment, semen was extended to 50 × 10 6 sperm/ml and centrifuged at 500 × g for 18 minutes. The sperm pellet was resuspended without the removal of extended seminal plasma. An immediate and significant (P 6 sperm/ml and centrifuged at 500 × g for 18 minutes, with the subsequent removal of extended seminal plasma and resuspension of the sperm pellet in fresh extender. A significant (P 6 sperm/ml.

Effect of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa

Abstract This study on extended, cooled spermatozoa was designed to determine 1) the concentration of seminal plasma required for the preservation of sperm motility, 2) whether the addition of egg yolk to a milk based semen extender improved the maintenance of sperm motility, and 3) whether there was a beneficial interaction of seminal plasma and egg yolk in the maintenance of sperm motility. For all experiments, semen was centrifuged for the removal or reduction of seminal plasma and spermatozoa were resuspended in a nonfat, dried skim milk - glucose extender. In Experiment 1, removal of all seminal plasma from spermatozoa and suspension in the extender resulted in a significant (P

METHODS FOR COLLECTING AND MATURING EQUINE OOCYTES IN VITRO

Abstract The objective of our study was to develop an effective method for collecting and maturing equine oocytes. In Experiments 1 and 2, oocytes were collected from excised ovaries obtained via colpotomy. In Experiment 3, oocytes were collected from ovaries obtained after slaughter. Follicles were aspirated and flushed with various treatments to recover the oocytes, which were then cultured and stained to observe the stage of meiosis. In Experiment 1, the aspiration treatments consisted of 0.5 ml of modified Dulbeccos PBS with 0, 100 or 500 lU/ml hyaluronidase. There was no increase (P>0.05) in oocyte recovery with the addition of hyaluronidase. The oocytes were cultured in either TCM-199 or Hams F-10 medium containing 0.5 ug/ml FSH, 1 ug/ml LH, 1 ug/ml estradiol 17β, 250 uM Na-pyruvate and 10% estrual mare serum for 0, 24, 36 or 48 h. Maturation rates were higher (P 0.05) in the percentage of maturation of oocytes between the 2 media at 48 h of culture. In Experiment 2, a single aspiration was performed with no flushing medium (dry aspiration) in 0.5 ml of PBS or in PBS with 1000 IU/ml hyaluronidase. The oocytes were then cultured in TCM-199 for 24, 30 or 36 h. There was an increase (P 0.05).

Effect of insemination volume and concentration of spermatozoa on embryo recovery in mares

Abstract Two experiments were conducted to determine 1) if an insemination volume of 50 ml yielded a lower embryo recovery rate than an insemination volume of 10 ml, when both volumes contained a total of 250 million progressively motile spermatozoa, and 2) if an insemination volume of 50 ml yielded a lower embryo recovery rate than an insemination volume of 10 ml, when both volumes contained 25 million motile spermatozoa/ml of extended semen. Mares exhibiting estrus for 2 or more days and having a follicle ≥ 35 mm were inseminated every other day throughout estrus with extended semen. For the first experiment, insemination volumes (10 or 50 ml) of extended semen contained 250 million progressively motile spermatozoa. There was a significant (P

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 <or=-0.1 degrees C/minute and preferably at -0.05 degrees C/min from 20 to 5 degrees C to maintain maximum sperm survival at 5 degrees C.

Fertilization rates in superovulated and spontaneously ovulating mares

Embryo recovery per ovulation has been shown to be lower in superovulated mares than in untreated controls. The objectives of this study were to 1) determine whether follicles stimulated with superovulatory treatment ovulate or luteinize without ovulation, 2) determine fertilization rates of oocytes in oviducts of superovulated and control mares, and 3) evaluate viability of early stage embryos from superovulated and control mares when cultured in equine oviductal cell-conditioned medium. Cyclic mares were randomly assigned to 1 of 2 groups (n=14 per group) on the day of ovulation (Day 0): Group 1 received 40 mg of equine pituitary extract (EPE; i.m.) daily beginning on Day 5 after ovulation; mares assigned to Group 2 served as untreated controls. All mares were given 10 mg PGF(2alpha) on Day 5 and Day 6, and 3,300 IU of human chorionic gonadotropin (hCG) were administered intravenously once mares developed 2 follicles >/=35 mm in diameter (Group 1) or 1 follicle >/=35 mm in diameter (Group 2). Mares in estrus were inseminated daily with 1 x 10(9) progressively motile spermatozoa once a >/=35 mm follicle was obtained. Two days after the last ovulation the ovaries and oviducts were removed. Ovaries were examined for ovulatory tracts to confirm ovulation, while the oviducts were trimmed and flushed with Dulbeccos PBS + 10% FCS to recover fertilized oocytes. All fertilized oocytes (embryos) recovered were cultured in vitro for 5 d using TCM-199 conditioned with equine oviductal cells. Ninety-two percent of the CLs from EPE mares resulted from ovulations compared with 94% for mares in the control group (P>0.05). The percentages of ovulations resulting in embryos were 57.1 and 62.5% for EPE-treated and control mares, respectively (P>0.05). Eighty-eight (Group 1) and 91% (Group 2) of the freshly ovulated oocytes recovered were fertilized (P>0.05). After 5 d of culture, 46.4 and 40.0% of the embryos from EPE-treated and control mares developed to the morula or early blastocyst stage (P>0.05). In summary, the CLs formed in superovulated mares were from ovulations not luteinizations. Although embryo recovery was less than expected, fertilization rates and embryo development were similar (P>0.05) between superovulated and control mares.

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.

Comparison of pregnancy rates from transfer of fresh versus cooled, transported equine embryos

Donor mares of mixed, light-horse breeds, maintained at Colorado State University, provided 104 embryos for immediate transfer (fresh embryos). One hundred and thirty-six additional embryos were collected on various breeding farms in the United States and were shipped to Colorado State University via commercial airlines (cooled embryos). Embryos were harvested 7 d after ovulation, graded, and either transferred into a mare immediately (<1 h) or placed in Hams F-10 medium plus 10% fetal calf serum in an atmosphere of 5% CO2, 5% O2, 90% N2 and packaged in a passive cooling unit (Equitainer) for shipment to our laboratory. All embryos were measured and graded just prior to surgical transfer via flank incision into synchronized mares. Recipients had ovulated 1 or 2 d before (+1, +2), on the same day as (0), or 1, 2 or 3 d after (-1, -2, -3) the donor mare. Pregnancy of recipients was determined by ultrasonography on 12, 35, and 50 d after ovulation of the donor. Pregnancy rates at 12, 35, and 50 d were similar for fresh (74, 64, 61%) and cooled embryos (80, 67, 66%), respectively. Overall, embryo size affected (P<0.05) pregnancy rates at 12, 35 and 50 d. Embryos of Grade 1 (excellent) or 2 resulted in more pregnancies than those of Grade 3 or 4 (poor) embryos. Embryonic losses between 12 and 35 d or between 35 and 50 d were not altered (P>0.05) by treatment (fresh or cooled) nor by age of the donor mare (P>0.05), but embryonic losses between 12 and 35 d were greater (P<0.06) for embryos stored for >12 h (25%) versus those stored for <12 h (10%). The duration needed for shipment (<12 h or >12 h) of cooled embryos did not alter pregnancy rates at 12 d (P>0.05). Age of donor mare had no effect (P>0.05) upon pregnancy rates of cooled or fresh embryos transferred nor on embryo quality. In summary, equine embryos can be cooled to 5 degrees C and maintained in storage for up to 24 h without decreased fertility, compared with those of embryos transferred in <1 hour.

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.