Field investigations of bacterial contaminants and their effects on extended porcine semen

Field investigations (n=23) were made over a 3-yr period at North American boar studs and farms in which the primary complaint was sperm agglutination in association with decreased sperm longevity of extended semen, and increased regular returns to estrus and/or vaginal discharges across parity. Microscopic examination of extended semen from these units revealed depressed gross motility (usually <30%), sperm agglutination, and sperm cell death occurring within 2 d of semen collection and processing regardless of the semen extender used. The extended semen exhibited a high number of induced acrosome abnormalities (>20%). Sample pH was acidic (5.7 to 6.4) in 93% of the submitted samples. Aerobic culture yielded a variety of bacteria from different genera. A single bacterial contaminant was obtained from 66% of the submitted samples (n=37 doses); 34% contained 2 or more different bacterial genera. The most frequently isolated contaminant bacteria from porcine extended semen were Alcaligenes xylosoxydans (n=3), Burkholderia cepacia (n=6), Enterobacter cloacae (n=6), Escherichia coli (n=6), Serratia marcescens (n=5), and Stenotrophomonas [Xanthomonas] maltophilia (n=6); these 6 bacteria accounted for 71% of all contaminated samples, and were spermicidal when re-inoculated and incubated in fresh, high quality extended semen. All contaminant bacteria were found to be resistant to the aminoglycoside gentamicin, a common preservative antibiotic used in commercial porcine semen extenders. Eleven genera were spermicidal in conjunction with an acidic environment, while 2 strains (E. coli, S. maltophilia) were spermicidal without this characteristic acidic environment. Bacteria originated from multiple sources at the stud/farm, and were of animal and nonanimal origin. A minimum contamination technique (MCT) protocol was developed to standardize hygiene and sanitation. This protocol focused on MCT's during boar preparation, semen collection, semen processing and laboratory sanitation. Implementation of the MCT, in addition to specific recommendations in stud management, resulted in the control of bacterial contamination in the extended semen.     

Cationic Synthetic Peptides: Assessment of Their Antimicrobial Potency in Liquid Preserved Boar Semen

Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs) received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional antibiotics in liquid boar sperm preservation. The antibacterial activity of two cyclic AMPs (c-WWW, c-WFW) and a helical magainin II amide analog (MK5E) was studied in vitro against two Gram-positive and eleven Gram-negative bacteria. Isolates included ATCC reference strains, multi-resistant E. coli and bacteria cultured from boar semen. Using broth microdilution, minimum inhibitory concentrations were determined for all AMPs. All AMPs revealed activity towards the majority of bacteria but not against Proteus spp. (all AMPs) and Staphylococcus aureus ATCC 29213 (MK5E). We could also demonstrate that c-WWW and c-WFW were effective against bacterial growth in liquid preserved boar semen in situ, especially when combined with a small amount of gentamicin. Our results suggest that albeit not offering a complete alternative to traditional antibiotics, the use of AMPs offers a promising solution to decrease the use of conventional antibiotics and thereby limit the selection of multi-resistant strains.     

Thermotemporal dynamics of contaminant bacteria and antimicrobials in extended porcine semen

Bacterial contamination of extended porcine semen has been associated with deleterious effects on both semen quality and sow fertility. Retrospective, prospective and in vitro studies were performed to delineate the prevalence and behavior of certain bacterial contaminants in extended semen, and antimicrobial pharmacodynamics in various semen diluents. Retrospective review of extended semen samples submitted from North American boar studs for microbiological screening at the University of Pennsylvania Reference Andrology Laboratory in 2005 and 2006 yielded bacteriospermia prevalence rates of 17% (144/832) and 26% (256/984), respectively. In a prospective study of regional boar studs, of 91 extended semen samples tested over 1-y, 29% were positive for bacteriospermia. Retrospective and prospective studies both showed that the preponderance of contaminant positive samples occurred during the fall months (P<0.05). To better understand behavior of select contaminant bacteria, generation intervals were determined for Serratia marcescens (SM) and Achromobacter xylosoxidans (AX) at 16, 22 and 37 degrees C. Generation times were temperature-dependent, with intervals decreasing two- to four-fold as incubation temperature increased. Growth patterns for SM, AX and Burkholderia cepacia were evaluated in various semen diluents. The different diluents exhibited constant or episodic patterns of growth within and among bacteria throughout the 5-d test period. Kill-time kinetics at 37 degrees C of several genera of bacteria in four semen diluents containing amoxicillin, gentamicin, tylosin, and lincomycin/spectinomycin (single drug or combination) ranged from 75 to over 360min, and was highly dependent (P<0.05) upon both type of bacteria and semen diluent.     

The fecundity of porcine semen stored for 2 to 6 days in Androhep and X-CELL extenders

Extending the raw ejaculate prior to artificial insemination (AI) is beneficial, in part, due to the increased number of females that are bred from an ejaculate, along with prolonged shelf life of the semen. The objective of this study was to examine the affects of storage time on the fecundity of porcine semen diluted in 2 semen extenders, Androhep and X-CELL. A completely randomized design with a factorial arrangement of treatments was utilized in which 429 high quality, gel-free ejaculates from 48 boars were used in a timed, double insemination of 1,431 first-service gilts. The gilts were divided into groups and inseminated with semen stored in Androhep or X-CELL for 2 to 3 d, 3 to 4 d, 4 to 5 d, or 5 to 6 d prior to use (day of collection = Day 0). Sperm age was identical, and both extenders were used concurrently each day of the trial. Farrowing rate and litter size data were recorded. Farrowing rates did not differ between extenders through Days 4 to 5 of storage. Gilts inseminated with Androhep diluted stored semen showed a decrease (P < 0.001) in farrowing rate compared with those inseminated with semen extended in X-CELL stored for 5 to 6 d. Mean litter sizes did not differ between extenders through Days 2 to 3 of storage. Compared with the X-CELL extended semen, gilts inseminated with Androhep extended semen produced smaller litters when semen was stored for 4 to 5 d (P < 0.05). Within the Androhep treatment, smaller mean litter sizes (P < 0.05) were evident when the semen was stored for 3 to 4 and 4 to 5 d. No differences were detected in litter size or farrowing rate for gilts bred with semen stored for 2 to 6 d in the X-CELL extender (P > 0.1). The results of this study indicate that extender type influences the fertility potential of fresh porcine semen stored for 2 to 6 d. For optimal fecundity in gilts, semen extended with Androhep extender should be used for AI within 3 d. The X-CELL extended semen can be used for up to 6 d without significant decrease in litter size or farrowing rate. These recommendations are dependent upon using high quality semen that is properly handled from collection through insemination.     

The effect of diode laser and light emitting diode on the bacterial contamination of semen medium for artificial insemination.

Animal artificial insemination (AI) suffers from bacterial contamination of semen media which results in decreased success of the process of artificial insemination. It is difficult to treat the semen extender medium to reduce the bacterial growth in the presence of semen using the conventional techniques of bacterial inhibition. In the present work, a new optical method developed for bacterial growth inhibition in semen containing extender medium using diode laser (DL) and commercial cheap light emitting diode (LED) is presented. Certain wavelengths and exposure times suitable for the process of artificial insemination are found to be optimum at reducing bacterial growth with a minimum significant effect on the semen motility and viability.     

Recent advances in cooled-semen technology

The majority of horse registries approve the use of artificial insemination, and horse breeding has widely taken benefit from the use of cooled-stored semen. New insights into cooled-semen technology open possibilities to reduce problems such as impaired semen quality after cooled-storage in individual stallions. The stallion itself has major impacts on quality and fertility of cooled-stored semen. Dietary supplementation of antioxidants and polyunsaturated fatty acids improves semen quality in a variety of species, but only few studies on this topic exist in the horse. Proper semen collection and handling is the main key to the maintenance of semen quality during cooled-storage. Semen collection should be achieved by minimal sexual stimulation with a single mount; this results in high sperm concentration, low content of seminal plasma and minimal contamination with bacteria. Milk-based semen extenders are most popular for semen processing and storage. The development of more defined extenders containing only the beneficial milk ingredients has made extender quality more constant and reliable. Semen is often centrifuged to decrease the seminal plasma content. Centrifugation results in a recovery rate of only 75% of spermatozoa in the semen pellet. Recovery rates after centrifugation may be improved with use of a "cushion technique" allowing higher centrifugation force and duration. However, this is not routinely used in cooled-semen technology. After slow-cooling, semen-storage and shipping is best performed at 5 degrees C, maintaining semen motility, membrane integrity and DNA integrity for up to 40 h after collection. Shipping containers created from Styrofoam boxes provide maintenance of semen quality at low cost.     

Equine spermatozoal motility and fertility associated with the incorporation of d-(+)-mannose into semen extender

Mannose is capable of decreasing bacterial attachment to the uterine mucosa in mares. Bacteria gain entry into the mare's uterus during breeding; therefore, a practical method to deliver mannose to the uterus is to incorporate it into semen extenders. The effect of mannose on spermatozoal motility and subsequent sperm fertilizing capability is unknown. The present study evaluated progressive spermatozoal motility in semen extender formulations incorporating mannose and assessed the fertility of mares inseminated with a mannose-containing semen extender. In Experiment 1, progressive spermatozoal motility in extender mixtures containing 0 mannose (control), 25, 37 or 49 mg/mL mannose was evaluated at 20 degrees C or 5 degrees C holding temperatures for 0, 12, 24 and 48 h post-dilution. Measures were repeated three times using five stallions of proven fertility. High concentrations of mannose in the extender affected progressive motility beyond the time and temperature effects noted in the controls. Extender containing only mannose sugar (49 mg/mL) displayed an immediate depression in progressive motility compared with controls (45.5% versus 62.9%, respectively; P<0.001). The 37 mg/mL mannose extender had a less dramatic decrease in motility (P<0.05) and only after storage at 5 degrees C for > or =12h (48.7% versus 58.0%, respectively). Extender with 25 mg/mL mannose performed no differently than the control formulation under all conditions. In Experiment 2, two groups of mares (n=11 each) were inseminated with 500 x 10(6) progressively motile spermatozoa extended in a traditional skim milk (control) extender or the 37 mg/mL mannose extender preparation. A single-cycle pregnancy rate of 72% was achieved by both groups. Present data suggest that a semen extender containing up to 37 mg/mL mannose could maintain motile spermatozoa for on-farm use and 25 mg/mL mannose concentrations preserved motility during long-term cooling. Likewise, sperm extended with up to 37 mg/mL of mannose had the same fertilizing capability as sperm in traditional extender mixtures.     

A comparison of two methods for the isolation of bovine herpesvirus 1 (BHV-1) from extended bovine semen

Two methods for the isolation of BHV-1 from extended bovine semen are described and their sensitivities compared. A method based on differential centrifugation is demonstrated to be superior to a standard method based on dilution when the semen extender used was milk; the two methods were equally effective when the semen extender used was egg yolk. The possible consequences of BHV-1 contamination of semen, and limitations of other isolation methods are briefly discussed.     

Fertility of ram semen frozen in Bioexcell and used for cervical artificial insemination

The current use of ingredients of animal origin, such as egg yolk, in semen extenders presents a risk of microbial contamination, and has led to the search for alternatives. Such an extender is commercially available for bull semen (Bioexcell), IMV, L'Aigle, France), and it has previously been tested in vitro for freezing ram semen, with satisfactory results. The aim of the present study was to compare the fertility results of ewes in Uruguay, after cervical insemination with ram semen that was frozen in Bioexcell versus semen frozen in a conventional milk-egg yolk extender (control). Semen from five Corriedale rams was frozen, using a split sample design, in either milk-egg yolk or Bioexcell extender, using a two-step extension method. The sperm parameters assessed after thawing were subjective motility, membrane integrity (SYBR-14/PI), and capacitation status (CTC). Thawed semen was inseminated intracervically once during spontaneous estrus in 970 Corriedale ewes that grazed in natural pastures, under extensive management conditions. Fertility was recorded as nonreturn rates at 21 days (NRR-21) and 36 days (NRR-36) after artificial insemination (AI), as well as pregnancy rate (PR-US, diagnosed ultrasonographically 50 days after AI of the last ewe). Subjective motility was slightly higher in Bioexcell than in the milk extender (47 vs. 46.5%; NS), as was membrane integrity (38 vs. 37.7%; NS) and the percentage of uncapacitated spermatozoa (28.5 vs. 26.3%; NS). There were no statistically significant differences in fertility rates found between Bioexcell and the control extender: NRR-21 (35.9 vs. 33.2%), NRR-36 (34.8 vs. 32.6%), and PR-US (28.4 vs. 27.2%). In conclusion, Bioexcell appears to be an alternative to the conventional milk-egg yolk extender for freezing ram semen, and provides similar fertility results after cervical AI under extensive management conditions. Thus, Bioexcell, containing no additives of animal origin, can offer a safer alternative when frozen semen is used for introducing new genetic material into a flock or a country.     

Influence of glucose and fructose in the extender during long-term storage of chilled canine semen

The use of chilled, extended semen in dog breeding is becoming increasingly popular as preparation and transportation is less expensive and regulations are often less complicated than for frozen semen. Sugar is one of the main constituents in semen extenders, and glucose and fructose are metabolized in separate pathways by freshly ejaculated dog sperm. In this study, glucose, fructose or an equal mixture of both were used in an egg-yolk-tris (EYT) extender at two different concentrations (10 and 70 mM). EYT extender without sugar supplementation, providing only the glucose (3-4 mM) originating from the egg-yolk, served as a control. The longevity of the chilled semen at 5 degrees C was 23 days: the quality of physical and functional characteristics decreasing with time. Glucose and fructose had a strong influence on motility and movement patterns of chilled canine semen. The beneficial effect of 70 mM sugar concentrations compared to 10 mM and the control was pronounced, and maintained sperm motility > or = 70% for 8 days of storage, compared to for 4 days in the control extender. Fructose maintained higher sperm motility than did glucose and the mixture. VAP values were higher in sugar-supplemented extenders (P < 0.05). Neither type nor concentration of the two sugars influenced sperm plasma membrane, acrosome integrity or the acrosome reaction following ionophore challenge (ARIC). Sugar consumption by dog sperm varied between the different periods of storage and with sugar concentrations provided in the extenders. Glucose consumption by dog sperm was greater than fructose consumption when both sugars were present in equal amounts, indicating that dog sperm used glucose in preference to fructose. In conclusion, the major influence of the two sugars on chilled semen was to support motility. EYT extender supplemented with fructose at a concentration of 70 mM was found to be the best of the tested extenders for long-term preservation of chilled canine semen.     

Activity of glutathione peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen during storage at 5 degrees C

Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 degrees C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the concentration of thiobarbituric acid reactive substances (TBARS) as an indicator of lipid peroxidation in native semen and after addition of extender, cooling and storage. Semen was collected from fertile Shetland stallions. In experiment 1, activity of antioxidative enzymes was determined immediately after semen collection and after 24 h storage at 5 degrees C. Enzyme activities were measured in native semen, semen diluted with semen extender, spermatozoa resuspended after centrifugation in extender and 0.9% NaCl as well as in undiluted and extender-diluted seminal plasma. In experiment 2, TBARS concentrations were analysed during storage of semen at 5 degrees C for 24 h. Semen storage for 24 h at 5 degrees C did not change activity of the examined enzymes. Antioxidative activity was significantly higher in extended than in native semen as well as in extended plasma than in undiluted plasma. In conclusion, the addition of semen extender increases the antioxidative activity in seminal plasma of stallions. Basal antioxidative activity in native semen as well as increased activity in extended semen are maintained over 24 h storage at 5 degrees C. TBARS content did not increase during semen storage. In conclusion, lipid peroxidation does not increase substantially during semen storage. The enzymatic antioxidative activity in semen apparently prevents ROS formation and is further increased by addition of semen extender.     

Effect of cryopreservation protocol on postthaw characteristics of stallion sperm

Three ejaculates from each of eight stallions were subjected to cryopreservation in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Semen was exposed to a fast prefreeze cooling rate (FAST; semen immediately subjected to cryopreservation) or a slow prefreeze cooling rate (SLOW; semen pre-cooled at a controlled rate for 80 min prior to cryopreservation). Postthaw semen was diluted in initial freezing medium (FM) or INRA 96 (IMV Technologies, L'Aigle, France) prior to analysis of 10 experimental end points: total motility (MOT; %), progressive motility (PMOT; %), curvilinear velocity (VCL; μm/s), linearity (LIN; %), intact acrosomal and plasma membranes (AIMI; %), intact acrosomal membranes (AI; %), intact plasma membranes (MI; %), and DNA quality. Eight of 10 experimental endpoints (MOT, PMOT, average-path velocity [VAP], mean straight-line velocity [VSL], LIN AIMI, AI, and MI) were affected by extender type, with egg yolk-based extender yielding higher values than milk/egg yolk-based extender (P < 0.05). Exposure of extended semen to a slow prefreeze cooling period resulted in increased values for six of eight endpoints (MOT, PMOT, VCL, AIMI, AI, and MI), as compared with a fast prefreeze cooling period (P < 0.05). As a postthaw diluent, INRA 96 yielded higher mean values than FM for MOT, PMOT, VCL, average-path velocity, and mean straight-line velocity (P < 0.05). Treatment group FM yielded slightly higher values than INRA 96 for LIN and MI (P < 0.05). In conclusion, a slow prefreeze cooling rate was superior to a fast prefreeze cooling rate, regardless of freezing extender used, and INRA 96 served as a satisfactory postthaw diluent prior to semen analysis.     

Advances in cooled semen technology.

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen.Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed.These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.     

In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen

Semen diluents containing egg yolk as a cryoprotectant may pose hygienic risks and are difficult to standardize. Although a new generation of semen diluents free of animal ingredients is available, egg yolk-containing extenders are still widely used for cryopreserving semen. We compared the effects of using different extenders on bovine sperm function in vitro and on fertility in vivo. A soy lecithin extender (SL; AndroMed) and an egg yolk-containing (TRIS-EY) extender were tested. No differences (P>0.05) were detected between the two extenders for sperm-zona pellucida binding capacity (HZI=115+/-13). Assessment of the inducibility of the acrosome reaction with progesterone showed no differences (P>0.05) between extenders for live acrosome-reacted sperm (15+/-2.36 and 14.42+/-2.02%, respectively, for SL and TRIS-EY). However, post-thaw sperm motility was significantly lower (P<0.05) when semen was extended in the TRIS-EY diluent. Field trials revealed that nonreturn rates of SL-extended semen showed significantly higher insemination success (P<0.0001) compared with the nonreturn rates for the TRIS-EY extender (70.45 and 67.85%, respectively). We suggest that consistent with quality standards that should be required for cryoprotectant media and because of the superior quality of the egg yolk-free extender, a defined soybean lecithin-containing diluter might be the better choice as a semen extender in the future.     

Comparison of ticarcillin and piperacillin in Kenney's semen extender

Ticarcillin and piperacillin were compared to determine their effect on sperm motility and bacterial growth of equine semen samples diluted in Kenney's glucose skim milk semen extender. Each ejaculate (n=11) was divided into three portions and glucose skim milk semen extender solution was added. The control semen extender solution contained extended semen and no antibiotic, whereas ticarcillin and piperacillin solutions contained extended semen plus 1.0mg/mL of ticarcillin or piperacillin, respectively. An aliquot was removed (1h after collection) to evaluate sperm motility and microbial concentration. All three solutions were stored at 4 degrees C and aliquots were obtained at 24 and 48 h to determine sperm motility and microbial concentration. Mean percentages of motile and progressively motile sperm did not differ significantly among control and antibiotic-containing solutions after storage. Control-extended semen samples from ejaculates of stallions (n=11) were contaminated with aerobic gram-positive and gram-negative bacteria. In solutions that contained either antibiotic, growth of these microbes was inhibited after 1, 24, and 48 h at 4 degrees C. Semen samples from stallions (n=5) were extended with Kenney's glucose skim milk extender containing no antibiotic, ticarcillin or piperacillin and then inoculated with approximately 5 x 10(2)CFU/mL Klebsiella pneumoniae or Pseudomonas aeruginosa; there was no significant difference between antibiotics in the inhibition of microbial growth. In conclusion, piperacillin was an appropriate alternative to ticarcillin in extenders for equine semen.     

Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity

This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.     

Comparison of two dilution rates on canine semen quality after cryopreservation in a coconut water extender

The objective of the present study was to evaluate the effect of sperm dilution (one part semen:one part extender or at 200 x 10(6) spermatozoa/mL) using a coconut water extender on the post-thaw sperm quality. Twelve ejaculates were collected from six dogs. Semen was divided into two aliquots, one for dilution one part semen:one part extender (group 1) and another for a concentration of 200 x 10(6) spermatozoa/mL (group 2). Semen was initially extended at 37 degrees C at a proportion of one part semen:half part extender (1:1/2) for group 1 (A-fraction). For group 2, the volume for a concentration of 200 x 10(6) spermatozoa/mL was calculated and a half of this volume was used for the initial dilution (A-fraction, 37 degrees C). Coconut water extender containing 20% egg yolk was used for this initial dilution in both groups. After dilution, the semen was cooled for 40 min in a thermal box (15 degrees C) and for 30 min in a refrigerator. The other half of the extender (B-fraction) containing egg yolk and glycerol (12%) was added to semen in both groups. Subsequently, the final concentration of glycerol in the extender was 6%. Ejaculates were frozen in 0.25 mL straws 5 cm above the surface of liquid nitrogen and stored at -196 degrees C. After 1 week, straws were thawed at 37 degrees C for 1 min and the microscopic criteria were evaluated. The dilution method had no influence on sperm motility, vigor and normal spermatozoa (71.4 compared with 67.7%). There was no effect of dog, ejaculate within male on post-thaw semen quality. Moreover, there was not a male x treatment interaction. Both treatments were efficient in preserving sperm quality.     

In vivo fertility of bull semen following cryopreservation with an LDL (low density lipoprotein) extender: preliminary results of artificial inseminations

A semen extender made with low density lipoproteins (LDL) has been used instead of a standard extender that is already available on the market for the cryopreservation of bovine semen. However, in order to extend its use to artificial insemination centres, in vivo fertility studies were required. Semen was taken from three bulls and frozen-thawed in two extenders: the LDL extender and a standard Tris-egg-yolk (20%) extender used by AI centres. The quality of the semen was assessed prior to artificial insemination: motility was assessed using an image analyser (Computer Assisted Semen Analysis (Hamilton Thorne)), and the integrity of the plasma membrane was assessed using the hypo-osmotic test (HOS test). For the first time, gestations were obtained following the artificial insemination of cows in the field (n=193) with semen that had been frozen-thawed in the LDL extender. No significant difference (p>0.05) was detected between the success rates of AI between the semen that had been frozen-thawed in the LDL extender (59.2%) and the control extender, Tris-20% egg yolk (65.3%). In conclusion, the in vivo fertility of semen that has been frozen-thawed in the LDL extender is maintained since gestations are obtained following AI.     

Influence of antibiotics on bacterial load and sperm parameters during short-term preservation of collared peccary semen

Studies on semen and sperm cells are critical to develop assisted reproductive technologies for the conservation of the collared peccary. The objective of the study was to compare the effect of different antibiotics on the bacterial load and sperm quality during short-term storage of peccary semen. Fresh semen samples from 10 males were extended in Tris-egg yolk or Tris-Aloe vera supplemented with streptomycin-penicillin (SP; 1 mg/mL - 1000 IU/mL or 2 mg/mL - 2000 IU/mL) or gentamicin (30 µg/mL or 70 µg/mL) before storage at 5°C. Bacterial load and sperm motility, membrane integrity and function, mitochondrial activity, and morphology, were evaluated at different time points for 36 h. The SP and gentamicin treatments concentration inhibited (p < 0.05) bacterial growth for 36 h regardless of the extender. Compared to the other treatments, Tris-egg yolk plus 70 µg/mL gentamicin maintained the sperm parameters for longer, including total motility (41.9 ± 6.1%) at 24 h, and membrane integrity (58.3 ± 2.1%) at 36 h. In contrast, the highest SP concentration in both extenders impaired sperm membrane integrity at 36 h (p < 0.05). For the liquid storage of collared peccary semen, it therefore is recommended to use Tris extender supplemented with egg yolk and gentamicin (70 µg/mL).     

Effect of extender supplementation with various antimicrobial agents on viability of Brucella ovis and Actinobacillus seminis in cryopreserved ovine semen

The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 10⁶ spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P < 0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis.