Effect of group thawing on post-thaw viability of bovine spermatozoa packaged in .5-milliliter French straws

The objective of this study was to determine the effects of thawing groups of 2, 5, 10, 15, or 20 .5-ml French straws on post-thaw spermatozoal viability. Thermostatically controlled and nonthermostatically controlled thawing baths were compared. Using a split-plot design, semen from 10 bulls was extended in egg yolk citrate, frozen, and then thawed (in the respective groups) at 36 degrees C in two types of thawing baths. Motility and percentage of intact acrosomes were determined immediately after thawing (0 h) and again after 4 h of incubation at the respective temperature of each thawing bath. Neither percentage of intact acrosomes nor motility was influenced by the number of straws thawed at 0 h (P greater than .05). Thawing bath had no effect (P greater than .05) on motility or percentage of intact acrosomes at 0 h. Bull variation was significant in both the 0- and 4-h evaluations. After 4 h of incubation, there was a significant (P less than .05) straw number x thawing bath interaction. When 15 or 20 straws were thawed in the thermostatically controlled bath there was a reduction (P less than .05) in motility and percentage of intact acrosomes. However, in the nonthermostatically controlled bath there was no reduction in motility and percentage of intact acrosomes as the size of straw group increased. Our results indicate that, when using a nonthermostatically controlled thawing bath, semen packaged in .5-ml straws can be thawed in groups of 20 without an effect on post-thaw sperm viability.     

Effects of Egg Yolk Source on the Cryopreservation of Stallion Spermatozoa

Three experiments were conducted to determine whether replacement of chicken egg yolk, as a component of freezing extenders, with egg yolk from other avian species would improve the post-thaw motility and percentage of intact acrosomes of stallion spermatozoa. In the first experiment, substitution of chicken egg yolk with chukar egg yolk, as a component of the lactose-ethylenediaminetetraacetic acid extender, improved (P ≤ .05) the post-thaw motility of stallion spermatozoa. These results were not replicated in (IMV Technologies, Maple Grove, MN, USA) a more expansive study comparing 2%, 4%, 6%, or 8% egg yolk combined with INRA 96 when a “slow freeze” method was used, or the same substitution at levels ranging from 13% to 22% when egg yolk was combined with lactose-ethylenediaminetetraacetic acid for diluents used for a “fast freeze” method of cryopreservation. In the third study, egg yolks from regular and high omega-3 chicken eggs as well as from turkey, chukar, and mallard duck eggs were analyzed for lipid content and fatty acid profile. The yolk from the turkey eggs was higher (1,300 mg/100 g) and that from mallard ducks was lower (560 mg/100 g) in cholesterol as compared with the two types of chicken eggs and chukar egg yolk (range, 1,046-1,094 mg/100 g). In addition, the high omega-3 eggs did test higher for fatty acids (4.51 g/100 g) than other types of eggs (range, 0.28-0.73 g/100 g). Substitution of chicken egg yolk with turkey, but not duck, egg yolk resulted in higher post-thaw total motility (P ≤ .05) for spermatozoa obtained from two of the three stallions used in the third experiment.     

Effect of prostaglandin F2 alpha, phenylephrine and ergonovine on uterine contractions in the ewe

Two experiments were conducted to determine the effect of prostaglandin F2 alpha (PGF2 alpha), phenylephrine and ergonovine on uterine contractions. In the first experiment, ewes were bilaterally ovariectomized, and a strain gauge force transducer was sutured to the serosa of one uterine horn. Each ewe was treated sc with 2 micrograms of estradiol-17 beta daily to prevent regression of the uterus. Beginning at least 5 d after ovariectomy, four dose levels of PGF2 alpha, phenylephrine and methoxamine were given by im injection and ergonovine was given by im or iv injection. Phenylephrine, methoxamine and ergonovine are alpha-adrenoceptor agonists. Uterine activity was recorded by physiograph for 30 min before and 90 min after treatment. Tracing were analyzed for 20-min periods before treatment and 4 to 24 min and 50 to 70 min after treatment. In Exp. 2, transducers were attached to uteri of intact ewes at d 10 to 12 of an estrous cycle. During subsequent estrus, one or two dose levels of PGF2 alpha, phenylephrine and ergonovine were given by im injection and uterine activity recorded. In Exp. 1, PGF2 alpha and phenylephrine increased (P less than .05 or .01) the number of amplitude of contractions at both 4 to 24 and 50 to 70 min. Ergonovine given im increased the number of contractions. In intact estrous ewes, PGF2 alpha increased the number and amplitude of contractions at 4 to 24 min, phenylephrine increased the number and amplitude at both 4 to 24 and 50 to 70 min, and ergonovine increased the number slightly but significantly at 4 to 24 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improvement by ergonovine of sperm transport, fertilization and pregnancy rates in ewes in natural or prostaglandin-induced estrus

Eight experiments were conducted with 451 ewes to test effects of ergonovine, prostaglandin F2 alpha (PGF2 alpha) and phenylephrine on sperm transport and fertility. In most experiments, ewes were mated at estrus and necropsied 2 or 3 h later. Sperm were flushed from the oviducts, uterus and anterior, middle and posterior thirds of the cervix and counted. Various doses of PGF2 alpha or phenylephrine given im at mating caused no significant increase in sperm numbers in any segment of the tract 2 h later. Three different dose levels of ergonovine were given im to ewes in natural estrus 1 h after mating and ewes were necropsied 3 h after mating. Doses of .2 and 1.0 mg were ineffective, but .5 mg increased sperm numbers about 10-fold in the oviducts and uterus. When given im at the time of artificial insemination, .6 mg of ergonovine increased the fertilization rate at 3 d from 5/25 in control ewes to 12/25 (P less than .05). In three experiments with ewes in PGF2 alpha-induced estrus, .6 mg of ergonovine increased sperm numbers in the cervix and uterus at 3 h after mating and in the uterus and oviducts at 23 h, near ovulation. Other ewes were artificially inseminated in the external cervical os and one-half of the ewes were given .6 mg of ergonovine im; ewes not returning to estrus were laparotomized at 22 to 26 d and embryos removed. After insemination during natural estrus with .2 ml of semen, pregnancy rates were 14/25 for control ewes and 15/25 for ergonovine-treated ewes; after insemination during natural estrus with .1 ml of semen, 6/35 and 18/35 (P less than .005); after insemination during PGF2 alpha-induced estrus with .2 ml of semen, 7/60 and 12/60. Fertilization and pregnancy rates combined were 32/145 (22%) for all control ewes and 57/145 (39%) for ergonovine-treated ewes (P less than .005).     

Acrosomal ultrastructure of stallion spermatozoa cryopreserved with ethylene glycol using two packaging systems

The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E10% and E/G, respectively. It was observed that the percentage of motile spermatozoa was significantly smaller (P<0.05) when semen was processed with E10%. A decrease in the acrosome integrity was observed in frozen thawed spermatozoa from all treated groups. It was observed that 28.0, 22.5, 25.5 and 22.5% of the sperm cells had a normal acrosome following freezing with G5%, E5%, E10% and E/G, respectively. Undulation of the outer acrosomal membrane, acrosomal swelling and loss of acrosomal content density and homogeneity were the most evident ultrastructural alterations observed. In Experiment 2, the post-thaw motility was higher (P<0.05) for sperm frozen in 0.5 ml straws than in 4.0 ml straws, regardless of the cryoprotector used. The ultrastructural evaluation showed 26.7 and 16.0% of intact acrosomes for sperm frozen in 0.5 ml and 4.0 ml straws, respectively. We concluded that ethylene glycol has similar cryoprotective properties to glycerol and that utilisation of 0.5 ml straws improved the ability of horse sperm cells to withstand damage after the cryopreservation process.     

Studies on the Effect of Supplementing Boar Semen Cryopreservation Media with Different Avian Egg Yolk Types on in Vitro Post-thaw Sperm Quality

Fertility after insemination of cryopreserved boar semen is currently below that of fresh semen. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using an adapted Westendorf method in which the chicken egg yolk was replaced by either duck or quail egg yolk. The different composition of the yolk types, particularly the amount of cholesterol, fatty acids and phospholipids, were thought to potentially afford a greater level of protection to sperm against damage during freezing and thawing. Sperm frozen in medium containing chicken egg yolk displayed higher motility immediately after thawing, but there was no difference in the motility of sperm frozen with different types of egg yolk 3 or 6 h after thawing and maintenance at 37 degrees C. Sperm frozen in media containing chicken or duck egg yolk had a higher proportion of intact acrosomes immediately after thawing than sperm frozen in medium containing quail egg yolk, but 6 h after thawing and maintenance at 37 degrees C the sperm that had been frozen in medium containing chicken egg yolk had a higher proportion of intact acrosomes than the sperm frozen in media containing duck or quail egg yolk. Analysis of the composition of the different yolk types showed that the basic components of the yolks were similar, but the ratios of fatty acids and phospholipid classes differed. Duck egg yolk had more monounsaturated fatty acids (MUFA) than chicken egg yolk, which had more MUFA than quail egg yolk. Duck egg yolk contained more phosphotidylinositol (PI) than chicken or quail egg yolks and quail egg yolk contained more phosphotidylserine than either chicken or duck egg yolks. The differences in post-thaw motility and acrosome integrity of boar sperm when frozen in media containing the different types of egg yolk may be due to the variation in composition.     

Effects of Hyaluronan Supplementation on Cryopreserved Equine Spermatozoa Hyaluronan and Cryopreserved Equine Spermatozoa

Addition of hyaluronan, a nonsulfated glycosaminoglycan, to fresh and frozen thawed human semen results in substantial retention of motility over time. Hyaluronan also has been reported to preserve postthaw viability and maintain membrane stability of boar spermatozoa. Therefore, experiments were designed to investigate the use of a commercially available hyaluronan (Map-5, Bioniche Animal Health, Inc., Athens, GA) in freezing extender for cryopreservation of equine spermatozoa. In experiment 1, aliquots from ejaculates were supplemented before freezing with one of four levels of hyaluronan: 100 μg/mL, 200 μg/mL, 400 μg/mL, and 1000 μg/mL along with an untreated control. No differences in sperm motility, assessed by computer-assisted sperm motility analysis (CASA), were found for any treatment at times 0, 30, or 60 minutes postthaw. Decreases in motility were noted in the highest hyaluronan group (1,000 μg/mL) after 90 and 120 minutes of incubation. Sperm viability, as assessed using SYBR-14/propidium iodide staining, was decreased (P < .05) when treated with 1,000 μg/mL compared with the control (37.1% and 46.1%, respectively). Motility parameters tended to remain elevated in those ejaculates treated with 200 μg/mL at various time points. Experiment 2, therefore, further investigated the effects of hyaluronan at 200 μg/mL on motility parameters and acrosome integrity and zona pellucida binding. Total (TM) and progressive (PM) motility of treated sperm immediately after thawing and at 60 minutes post-thaw were higher compared with control (P < .05). A tendency (P < .1) to maintain TM at 90 and 120 minutes post-thaw also was noted. No differences were noted for the mean number of spermatozoa bound to bovine oocytes for control or treated sperm (22 ± 14 vs 25 ± 17, respectively). Acrosome integrity also was unchanged between the two groups based on fluorescein isothiocyanate (FITC)−peanut agglutinin (PNA)/propidium iodide staining. All samples contained <1% live acrosome-damaged spermatozoa. In the final experiment, the effects of hyaluronan supplementation post-thaw was investigated using hyaluronan concentrations of 100, 200, and 400 μg/mL. Motility parameters studied over an 8-hour period at 37°C yielded no consistent differences. In conclusion, addition of hyaluronan at a concentration of 200 μg/mL before freezing increased spermatozoal post-thaw motility. High concentration of hyaluronan (1,000 μg/mL) appeared to be detrimental to post-thaw motility. Effects of hyaluronan on fertility are beyond the scope of this study and have yet to be determined.     

Pregnancy rates of mares inseminated with semen cooled for 18 hours and then frozen

The ability to ship cooled stallion sperm for subsequent freezing at a facility specializing in cryopreservation would be beneficial to the equine industry. Stallion sperm has been centrifuged, cooled to 5 degrees C for 12 h, and frozen without a detrimental effect on motility in a previous study; however, no fertility data were available. Experiment 1 compared the post-thaw motility of sperm cooled for 18 h at 15 or 5 degrees C at either 400 or 200 x 10(6) sperm/mL and then frozen. Storage temperature, sperm concentration, or the interaction of temperature and concentration had no effect on total (TM) and progressive motility (PM) after cooling. Post-thaw TM and PM were higher for control than (P < 0.05) for treated samples. There was no difference in post-thaw TM and PM due to temperature or concentration. Experiment 2 further evaluated procedures for cooling before freezing. Ejaculates were either cooled to 5 degrees C for 18 h and centrifuged, centrifuged at room temperature and then cooled to 5 degrees C for 18 h before freezing, or centrifuged and frozen immediately (control). There was no difference among treatments on post-thaw TM or PM. In Exp. 3, mares were inseminated with semen that had been extended in skim milk-egg yolk without glycerol, centrifuged, resuspended at 200 x 10(6) sperm/mL, cooled to 5 degrees C for 18 h, and then frozen or not cooled for 18 h before freezing (control). Pregnancy rates did not differ for mares receiving semen cooled and then frozen (21 of 30, 70%) or semen frozen directly without prior cooling (16 of 30, 53%). In summary, a procedure was developed for cooling stallion sperm for 18 h before freezing without a resultant decrease in fertility.     

Effects of addition of sodium lauryl sulfate on frozen-thawed canine spermatozoa

The addition of Orvus ES paste (OEP) to extender may be essential for preparing frozen dog semen. The major ingredient of OEP is sodium lauryl sulfate (SLS). In this study, we compared the effect of SLS on frozen dog semen with that of OEP. There were no significant differences between the 2-mg/ml SLS group and OEP group concerning sperm motility, viability and the percentage of viable sperm with intact acrosomes after freeze-thawing. These results suggest that the effectiveness of frozen dog semen extender containing 2 mg/ml of SLS is similar effective to that demonstrated for OEP.     

Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws

Experiments were conducted to study the effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa after cryopreservation in straws. Semen (split ejaculate) in maxi-straws (6 mm o.d.) was frozen using a programmable freezing chamber. Three methods for in vitro sperm evaluation were used: motility (MOT), acrosome integrity (NAR) and flow cytometric analysis of sperm treated with carboxyfluorescein diacetate and propidium iodide to assess sperm plasma membrane integrity (PMI). No interactions were found among the three variables evaluated. Length of prefreeze exposure to glycerol, ranging from .5 min to 75 min, had no effect on post-thaw sperm viability. Exposure of sperm to a glycerol-containing extender medium at 5 degrees C gave improved post-thaw viability over that exposed at 0 degree C (P less than .05). Glycerol at a concentration of 3 or 4% resulted in maximum post-thaw MOT. Acrosome integrity values were greatest for 2 and 3% glycerol, whereas PMI was greatest when glycerol concentration was 4 to 6%. The primary cryoprotective effect of glycerol on boar semen may be extracellular. It is concluded that 3 or 4% glycerol gives maximum viability of frozen-thawed spermatozoa when the present methods are employed.     

Serum concentrations of cefepime (BMY-28142), a broad-spectrum cephalosporin, in dogs.

Serum concentrations of cefepime (BMY-28142) were determined for four dosing regimes, 10 mg/kg or 20 mg/kg, given as single subcutaneous (SC) or intramuscular injections (IM) to dogs. Serial serum samples were analyzed for the presence of cefepime by high-performance liquid chromatography. In experiment 1, the overall mean (+/- SEM) serum concentration (for a 12-hour period) after a dose of 20 mg/kg for SC and IM routes (4.9 +/- 0.74 micrograms/ml and 5.5 +/- 0.63 micrograms/ml, respectively) was twice that for the 10 mg/kg dose given either SC or IM (2.2 +/- 0.31 micrograms/ml and 2.8 +/- 0.47 micrograms/ml, respectively). There was no significant difference (p greater than 0.05) in mean serum concentrations for SC and IM routes of administration at the same dosage. In subsequent experiments, 5 doses of cefepime (20 mg/kg) were administered IM at 12-hour (experiment 2) or 24-hour (experiment 3) intervals. The mean (+/- SEM) peak serum concentration was 12.1 +/- 1.59 micrograms/ml, 2 hours after the 2nd injection in experiment 2. In experiment 3, the mean (+/- SEM) peak serum concentration was 10.9 +/- 1.34 micrograms/ml, 4 hours after the 1st injection. Mean trough concentrations in experiment 2 were greater than or equal to 0.5 microgram/ml and less than or equal to 0.5 in experiment 3. Multiple IM doses produced transient edema at the injection site and mild lameness in all dogs. Cefepime was highly active against single canine isolates of Staphylococcus intermedius, Pseudomonas aeruginosa and Escherichia coli, with minimum inhibitory concentrations of 0.125 microgram/ml, 1 microgram/ml and 0.3 microgram/ml, respectively.     

The Effect of 2-Hydroxypropyl-β-Cyclodextrin on Post-Thaw Parameters of Cryopreserved Jack and Stallion Semen

Cyclodextrins improve post-thaw viability and motility of semen as well as mediate cholesterol efflux and subsequent acrosome reaction in spermatozoa from several species. The objectives of this study were: (a) to assess the effect of prefreeze addition of 60 mM hydroxypropyl-β-cyclodextrin (β-CD) on post-thaw viability and motility of jack and stallion semen cryopreserved in ethylene glycol-based freezing extenders containing 5% or 20% (v/v) egg yolk (LEY and HEY, respectively), and (b) to evaluate the ability of 1 μM calcium ionophore A23187 and/or 60 mM β-CD to induce acrosome reaction in thawed jack and stallion spermatozoa. Post-thaw motility of spermatozoa cryopreserved in HEY was higher (P < .05) for jack but lower (P < .05) for stallion spermatozoa when compared with LEY. Jack and stallion spermatozoa both exhibited higher (P < .05) motility when cryopreserved in 60 mM β-CD than without β-CD. Curvilinear velocity was faster (P < .05) for jack and stallion spermatozoa cryopreserved in LEY than in HEY. A treatment × time interaction affected (P < .05) the proportion of spermatozoa that underwent acrosome reaction. Post-thaw incubation of jack and stallion spermatozoa with β-CD for 90 minutes induced acrosome reaction in 85% and 22% of viable sperm cells, respectively; however, only 32% of jack and 8% of stallion spermatozoa incubated with calcium ionophore underwent acrosome reaction. This study is the first to evaluate the effect of β-CD (not loaded with cholesterol) on jack semen cryopreservation, and results reveal that β-CD may be a useful tool to enhance semen cryopreservation and to induce post-thaw acrosome reaction in jack spermatozoa.     

Microencapsulation of bovine spermatozoa

Two experiments were conducted to examine the efficacy of microencapsulation of bovine spermatozoa for use in artificial insemination. In Exp. 1, sperm were encapsulated at three different concentrations (45, 90 and 180 X 10(6) sperm/ml) in either .75- or 1.5-mm (diameter) microcapsules and incubated in vitro for 24 h at 37 C. Unencapsulated samples of each concentration served as controls. Capsule contents were evaluated for percentage of sperm motility and intact acrosomes at 2, 12 and 24 h of incubation. Capsule fragility was evaluated after 24 h incubation. Viability of spermatozoa was not influenced by sperm concentration or capsule size, and compared with controls, cellular injury after encapsulation was not apparent. Fragility of capsules was unaffected by capsule size; however, as the sperm concentration increased, integrity of the capsules decreased (P less than .05). In Exp. 2, using frozen-thawed semen, the effect of egg yolk content, presence of glycerol and viability of spermatozoa on the success of microencapsulation was measured. The extender was 2.9% sodium citrate with glycerol (7% v/v) and either 0, 5, 10 or 15% egg yolk (v/v). Uniformity of capsules in size and shape was evaluated subjectively. Capsule integrity and uniformity were unaffected by glycerol, sperm viability or egg yolk level up to 10% v/v; however, encapsulation of spermatozoa in 15%-yolk buffer increased the heterogeneity in capsule size and shape. Viability of encapsulated spermatozoa was maximal for extenders containing 10 or 15% yolk v/v. Reduced viability for the 5% yolk extender was due to pre-encapsulation injury associated with freezing. Microencapsulation procedures are compatible with sperm viability and can be adapted to an acceptable extender system used in artificial insemination.     

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility

Objective   We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility.
Design   A randomised 2 × 3 block design was used.
Procedure   Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen.
Results   There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% ( P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF ( P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol ( P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%.
Conclusion   In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.     

Cryopreservation of boar semen. II: Effect of cooling rate and duration of freezing point plateau on boar semen frozen in mini- and maxi-straws and plastic bags.

The post-thaw motility and the acrosome integrity of semen from 4 boars frozen with a programmable freezing machine, in mini (0.25 ml) and maxi (5 ml) plastic straws and in 10 x 5 cm Teflon FEP-plastic bags (0.12 mm thick, 5 ml), were compared. The freezing of the semen was monitored by way of thermo-couples placed in the straws and the bags. Three freezing programmes were used, namely A: from +5 degrees C, at a rate of 3 degrees C/min, to -6 degrees C, held for 1 min at -6 degrees C, and followed by a cooling rate of 20 degrees C/min to -100 degrees C; B: a similar curve except that there was no holding time at -6 degrees C and that the cooling rate was 30 degrees C/min, and C: from +5 degrees C to -100 degrees C, with a cooling rate of 35 degrees C/min, followed by storage in liquid N2. Despite the freezing curve assayed, both the mini-straws and the bags depicted much shorter freezing point plateaus as compared to the maxi-straws. Post-thaw sperm motility as well as the amount of normal apical ridges were equally significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. Significant differences in these post-thawing parameters were obtained between the freezing curves used. The stepwise freezing procedure A appeared as the best alternative for boar semen, considering this in vitro evaluation.     

Optimization of ethylene glycol concentrations, freezing rates and holding times in liquid nitrogen vapor for cryopreservation of rhesus macaque (Macaca mulatta) sperm

Ethylene glycol (EG) has been speculated to be the most appropriate penetrating cryoprotectant for cryopreservation of rhesus macaque sperm due to its higher permeability coefficient. The present study aimed to determine the optimal EG concentration, freezing rate and holding time in liquid nitrogen (LN(2)) vapor for rhesus sperm cryopreservation. Among six tested EG concentrations (0, 0.18, 0.35, 0.7, 1.4 and 2.1 M), 0.7 M EG showed the most effective cryoprotection (P<0.05). Sperm frozen with 0.7 M EG at -183°C/min showed higher post-thaw motility than sperm frozen at -10, -67 or -435°C/min (P<0.05). Sperm frozen in LN(2) vapor at -183°C/min with 0.7 M EG and a holding time of 10 min showed higher post-thaw motility compared with a holding time of 5 or 15 min (P<0.05). The function of sperm cryopreserved at the optimized EG concentration, freezing rate and holding time was further evaluated by in vitro fertilization. Of the 36 oocytes collected from gonadotropin-stimulated rhesus macaques, 61.1% were fertilized, and 61.1, 44.4 and 36.1% of the oocytes developed to 2 cells, morulae and blastocysts, respectively. Our findings provide an alternative penetrating cryoprotectant and optimal protocol for genetic preservation purposes in this important species.     

Cryopreservation of Cat Semen in Straws: Comparison of Five Different Freezing Rates

The first pregnancies in domestic cats were obtained using semen frozen in pellets ( Platz et al. 1978 ). Other freezing methods, vials ( Lengwinant and Blottner 1994 ) or straws ( Pope et al. 1991 ; Hay and Goodrowe 1993 ), have also been used. Pelleted freezing has often been the standard method ( Howard 1986 ). Opinions about the freezing method are discordant; the best method for Pope et al. (1991) was using straws; in fact, the post‐thaw motility and the percentage of normal acrosomes were of 44 ± 4 and 62 ± 3%, respectively, with straws and 11 ± 3 and 26 ± 4%, respectively, with pellets. According to Wood et al. (1993) , there are no differences between the two methods, with a motility of 66.2% and a percentage of normal acrosomes of 28.6% for the pellet method and a motility of 67.0% and a percentage of normal acrosomes of 27.4% for the straw container. However, these two authors used two different freezing protocols. A high concentration of glycerol (i.e. 8%, vol/vol) damages cat semen ( Zambelli 1994 ; Nelson et al. 1999 ), because of his toxicity to spermatozoa ( Graham 1996 ); while a concentration of 4% is suggested ( Zambelli 1994 ). Fast green FCF Bengal pink staining is often used to evaluate the acrosomal morphology ( Wood et al. 1993 ; Zambelli et al. 1993 ). As there are no studies on the influence of freezing rate on motility and on acrosomal morphology, the aim of this study was to test five freezing rates in order to verify which is the best for the cryopreservation of cat semen in straws.     

Control of Campylobacter fetus in artificially contaminated bovine semen by incubation with antibiotics before freezing

Fresh, diluted semen containing 1.55 X 10(6) cfu/ml of Campylobacter fetus subsp. venerealis was incubated with 500 iu of penicillin, 500 micrograms of streptomycin, 160 micrograms of lincomycin and 300 micrograms of spectinomycin per ml at 35 degrees C for 0, 1, 2, 5, 10, 20 or 40 minutes. The semen was cooled to 5 degrees C, packaged in 0.25 ml French straws and then frozen in liquid nitrogen for 2 weeks. Immediately after thawing and removal of the antibiotics by centrifugation semen samples from each of the seven treatment groups were cultured as for C. fetus. Semen samples were also examined by in-vitro tests for sperm motility prior to and post-freezing. Incubation with the antibiotics for 5, 10, 20 or 40 min prior to freezing reduced the numbers of C. fetus in the semen to non-detectable levels in 38%, 69%, 88% and 100% of samples respectively. The incubated semen showed no significant reduction of sperm motility although fertility trials have not been done.     

Concentrations of gentamicin in serum, milk, urine, endometrium, and skeletal muscle of cows after repeated intrauterine injections

Healthy mature cows (n = 6) were injected intrauterinally (IU) with gentamicin (50 ml of a 5% injectable solution) daily for 3 consecutive days. Venous blood and milk samples were collected at postinjection (initial) hours (PIH) 1, 3, 6, 9, 12, 24, 28, 31, 34, 37, 48, 51, 54, 57, 60, and 71, and endometrial biopsies were performed at PIH 6, 25, 48, 73, 95, and 119. Skeletal muscle biopsy samples were taken at PIH 25 and 73, and urine was collected every 1 or 2 hours during 12 consecutive hours after the first IU injection. Serum, milk, urine, and tissue concentrations of gentamicin were measured by radioimmunoassay. The highest mean serum concentration of gentamicin occurred during the 3 hours after each injection (2.49 +/- 1.46, 6.60 +/- 5.47, and 4.98 +/- 2.70 micrograms/ml). The mean peak concentration of gentamicin in milk occurred 3 to 6 hours after each injection. Mean peak urine concentration of gentamicin (256.8 +/- 127.9 micrograms/ml) was measured at PIH 6. The mean percentage of the first dose of gentamicin excreted in the urine within 12 hours was 14.78 +/- 3.56. The highest concentration of gentamicin in endometrial tissue (639.16 +/- 307.22 micrograms/g) was measured at PIH 6, decreasing to 9.64 +/- 3.55 micrograms/g before the next IU dose. Gentamicin was still detectable in endometrial tissue (0.86 +/- 0.43 microgram/g) 71 hours after the 3rd (last) IU injection.     

Post-thaw viability of european bison (Bison bonasus) semen frozen with extenders containing egg yolk or lipids of plant origin and examined with a heterologous in vitro fertilization assay.

Basic characteristics of European bison (Bison bonasus) semen were described and the efficacies of two extenders-Triladyl, containing egg yolk, and a synthetic extender, containing soybean lipids-were tested for semen cryopreservation. Seven ejaculates were collected by electroejaculation from a 10-yr-old, European bison bull. Each ejaculate was diluted at 37 degrees C to a final concentration of 200 x 10(6) sperm/ml with Triladyl or the synthetic extender. Extended semen samples were frozen according to a standard bull semen freezing protocol. After 2 wk of storage, one straw from each extender and ejaculate was thawed, and postthaw quality was evaluated by individual sperm motility and movement rate, numbers of sperm morphologic abnormalities and intact acrosomes, functional integrity of the sperm membranes determined by hypoosmotic swelling test (HOST), viability (live-dead, eosin-nigrosin stain), and a heterologous in vitro sperm penetration assay (SPA). A total of 600 in vitro-matured bovine oocytes were inseminated with 1 X 10(6) spermatozoa of Holstein semen frozen-thawed in Triladyl (control) or of European bison semen frozen in Triladyl or the synthetic extender. Nuclear status of the oocytes was determined after 18 h of sperm-oocyte coincubation. Extender had no effect on any evaluated parameters of semen after dilution and cooling (4 hr at 5 degrees C) or in postthaw individual motility, quality of movement, and sperm morphology. However, significantly (P < 0.05) higher numbers of spermatozoa with intact acrosomes, intact membranes (HOST), and viable sperm (P < 0.01) were in semen frozen in Triladyl than in the synthetic extender. Mean values for heterologous SPA for bull (control) and for bison semen frozen in the synthetic extender were very much alike-63.3+/-10.6% and 63.1 +/- 15.9%, respectively; bison semen frozen in Triladyl was lower, 43.0+/-24.2% but not significantly different. Cumulative results from a variety of viability assays of diluted/cooled and frozen-thawed semen, including the heterologous SPA, suggest that European bison semen can be successfully frozen in both extenders tested in this study.