The addition of ticarcillin-clavulanic acid to INRA 96 extender for stallion semen cooling
Reasons for performing study
A commonly used commercial extender (i.e. INRA 96) contains antimicrobials that may have limited effectiveness. Therefore, addition of ticarcillin-clavulanic acid to this extender is a widespread procedure in the equine breeding industry in the United States. However, such practice has not been critically evaluated.
To evaluate the addition of ticarcillin-clavulanic acid to INRA 96 and different extender and antimicrobial storage conditions on sperm function and antimicrobial effectiveness.
Gel-free semen (42 ejaculates from 14 mature Quarter Horse stallions) was extended with INRA 96 and stored for 24 h in an Equitainer II. The effects of added ticarcillin-clavulanic acid and different extender storage procedures on sperm motion characteristics (by computer-assisted analysis), sperm membrane integrity (by fluorescence-based measurement) and suppression of bacterial growth (by aerobic and anaerobic culture methods) were evaluated using analysis-of-variance and Chi-square statistical methods. The P value for significance was set at <0.05.
Freezing and thawing of modified or unmodified extender prior to use for stallion semen resulted in reduced sperm quality post cooling for 24 h, as evidenced by a significant reduction in sperm motility (i.e. total and progressive) and sperm membrane integrity. Addition of ticarcillin-clavulanic acid to extender resulted in higher sperm velocity when the reconstituted antimicrobial was subjected to cooled storage, as compared with frozen storage, prior to use. Only 28 of 42 ejaculates (67%) yielded presence of bacteria in neat semen but addition of ticarcillin-clavulanic acid to INRA 96 was not different than INRA 96 alone for inhibiting growth of bacteria (98 vs. 94%, respectively).
Addition of ticarcillin-clavulanic acid (1 mg/ml) to INRA 96 did not adversely affect sperm quality in extended semen after cooled storage. Extender freezing and thawing prior to use had detrimental effects on sperm quality.
These data suggest that INRA 96 should not be frozen and thawed prior to use. Addition of ticarcillin-clavulanic acid to INRA 96 did not impair sperm quality. All extender treatments effectively controlled the bacterial growth compared with neat semen.
The surfaces of the stallion's penis and prepuce are colonised by a variety of commensal bacteria that contaminate the mare's reproductive tract at breeding time [1-4]. Bacteria and metabolites of bacteria are known to have a detrimental effect on sperm motility upon cooled storage [5, 6]. As bacterial contamination of ejaculates obtained using an artificial vagina is common, semen collected using this method is customarily mixed with extender containing antimicrobial(s) to control bacterial growth, prior to insemination or when semen is processed for cooled transport or cryopreservation.
Milk-based extenders are commonly used in the United States  and Europe  to transport cooled stallion semen. Different studies have revealed that extenders containing only defined milk proteins (i.e. native phosphocaseinate) as a source of protein resulted in better semen quality [9, 10] and possibly fertility  when compared with skim milk-based extenders. INRA 961, a commercial milk fraction-based extender, has become more popular in the equine breeding industry in North America. This extender is commercialised as a premixed liquid formulation. Desirable features of this extender are minimal microscopic debris that could impair computerised semen evaluation and a chemically defined composition that allows the extender to be manufactured in a standardised manner, when compared with extenders containing products directly derived from animals such as skim milk and egg yolk. These latter products are highly variable in composition and consequently can yield variable results.
The types of antimicrobials used in commercial equine semen extenders are highly variable. Antimicrobials used for this purpose include amikacin gentamicin, streptomycin, potassium penicillin, sodium penicillin, pipericillin, ticarcillin, ticarcillin-clavulanic acid, polymixin B, ceftiofur, amphotericin B and antimicrobial combinations such as penicillin-streptomycin or potassium penicillin-amikacin [4, 12-15]. The effects of different antimicrobials in equine semen extenders have been studied through the evaluation of the seminal parameters (i.e. motility features and sperm membrane integrity) and bacterial growth upon storage [4, 12-16]. The INRA 96 extender contains gentamicin (0.105 mg/ml), penicillin (0.038 mg/ml) and amphotericin B (0.315 μg/ml; A. DeMirjyn, personal communication) but the concentrations of gentamicin and penicillin are lower than those used in studies conducted by others [4, 12, 15]. A recent study  showed that INRA 96 was unable to inhibit the growth of Taylorella equigenitalis when these bacteria were added to stallion extended semen. Conversely, addition of ticarcillin-clavulanic acid to the extender at concentrations of 1.0 or 1.5 mg/ml inhibited growth of Taylorella equigenitalis . In another study, addition of ticarcillin-clavulanic acid (1 mg/ml) to skim milk-based extender eliminated bacterial growth in stallion semen after extension and cooled storage for 24 h .
The aims of this study were to evaluate the effects of ticarcillin-clavulanic acid, when added to INRA 96 extender, on sperm motion characteristics, sperm membrane integrity and effectiveness at inhibiting bacterial growth after cooled storage of extended stallion semen. In addition, the effects of storage methods for reconstituted antimicrobial and for extender were tested using the same experimental endpoints.
Materials and methods
Stallions, semen collection and processing
Fourteen actively breeding, mature Quarter Horse stallions (mean age 10.6 years, range 5–20 years) were used. Ten stallions were housed in indoor stalls (7 × 7 m) bedded with wood shavings and stalls were cleaned several times daily. Four stallions were housed in partially covered dirt paddocks (7 × 17 m) cleaned once daily. Stallions housed in stalls were allowed exercise on a mechanical walker for 20 min once daily but were not permitted paddock exercise. Stallions housed in dirt paddocks were not exercised on the mechanical walker.
During November 2011, semen was collected from each stallion 3 times (every other day) to reduce extragonadal sperm reserves; thereafter, semen was collected 3 times at one week intervals using an artificial vagina (Missouri-Style AV [C26716N])2 equipped with an inline nylon micromesh filter (disposable nylon mesh gel filter)3 to permit collection of gel-free semen. Prior to semen collection, each stallion was exposed to a mare in oestrus to stimulate sexual arousal. Once erect, the penis was rinsed thoroughly with water and cotton immediately prior to each semen collection. The back of a mounting dummy was wrapped with disposable form-fitting plastic (Good Wrappers S244)4 to prevent potential cross contamination among stallions. After semen collection, the gel-free portion was transported to an adjacent laboratory and placed in an incubator (37°C) before processing and evaluation.
The gel-free semen volume was measured using a graduated cylinder and sperm concentration was measured using a fluorescence-based instrument (NucleoCounter SP-100)5, as described previously . The semen was extended to a final sperm concentration of approximately 30 × 106/ml. Aliquots of extended semen (1 ml) were maintained in polypropylene tubes (Cryogenic vials 1.2 ml)6 prior to storage or analysis.
The effects of semen processing technique on sperm motion characteristics, sperm membrane integrity and bacterial growth in extended semen were determined. A culture of neat semen was performed. Subsequently, aliquots of neat semen were extended with warmed (37°C) INRA 96 that was previously modified: Group 1 (control - commercial INRA 96); Group 2 (ticarcillin-clavulanic acid powder (Timentin,)8 added to INRA 96); Group 3 (ticarcillin-clavulanic acid powder added to INRA 96 and then stored at 4°C for one day); Group 4 (ticarcillin-clavulanic acid powder added to INRA 96 and then stored at 4°C for 7 days); Group 5 (ticarcillin-clavulanic acid powder reconstituted in sterile deionised water [200 mg/ml] and then added to INRA 96); Group 6 (ticarcillin-clavulanic acid powder reconstituted in sterile deionised water and then the stock solution frozen at -20°C for one day before being thawed in a water bath [37°C] and added to INRA 96); Group 7 (ticarcillin-clavulanic acid powder reconstituted in sterile deionised water and then the stock solution frozen at -20°C for 7 days before being thawed in a water bath [37°C] and added to INRA 96); Group 8 (ticarcillin-clavulanic acid powder reconstituted in sterile deionised water and then the stock solution frozen at -20°C for 7 days before being thawed at room temperature for 4 h and added to INRA 96); Group 9 (ticarcillin-clavulanic acid powder reconstituted in sterile deionised water and then the stock solution stored for one day at 4°C before being added to INRA 96); Group 10 (ticarcillin-clavulanic acid powder reconstituted in sterile deionised water and then the stock solution stored for 7 days at 4°C before being added to INRA 96); Group 11 (INRA 96 frozen at -20°C for 7 days before being thawed in a water bath [37°C]) and Group 12 (ticarcillin-clavulanic acid powder added to INRA 96 and then frozen at -20°C for 7 days before being thawed in a water bath [37°C]). It is worth noting that ticarcillin-clavulanic acid was added to INRA 96 at a final concentration of 1 mg/ml in treatments containing this antimicrobial.
Seminal parameters (i.e. total and progressive motility, mean curvilinear velocity and sperm membrane integrity) and bacteriological growth were analysed within 30 min after semen collection for Groups 1 and 2. In addition, aliquots of extended semen from all groups were maintained in an Equitainer II9 for 24 h. After this storage period, culture swabs (BBL CultureSwab Plus)7 of aliquots were obtained and samples warmed in an incubator (37°C) for 15 min prior to analysis of sperm motion characteristics and sperm membrane integrity.
Sperm motion characteristics
Sperm motion characteristics were analysed with a computer-assisted sperm motion analysis (HTM-IVOS, version 12.2 L)10 (CASMA) programme according to the recommendations published elsewhere . Briefly, warmed (37°C) analysis chambers (fixed height of 20 μm) affixed to microscope slides (Leja Standard Count 2 Chamber slides)11 were slowly loaded with a 6 μl volume of extended semen. The slides were then placed on a stage (37°C) and inserted into the CASMA instrument10 for evaluation. A total of 10 microscopic fields and a minimum of 500 sperm were examined per sample. Preset values for the CASMA were as those reported previously . Experimental endpoints included total motility, progressive motility and mean curvilinear velocity (μm/sec).
Sperm membrane integrity
The percentage of sperm with an intact plasma membrane was determined using an automated cell counter (NucleoCounter SP-100)5, as reported previously . Total sperm concentration was determined according to manufacturer's recommendations. Briefly, semen samples were first permeabilised by mixing semen with a detergent reagent (Reagent S-100)12. The sample was then transferred to a disposable cassette laced with propidium iodide. Placement of the cassette into the automated cell counter resulted in automatic transfer of contents into the measurement chamber. Green light excited DNA with intercalated propidium iodide and the emitted signal registered by a compact fluorescence microscope integrated within the instrument. The concentration of membrane-damaged sperm was determined by substituting phosphate buffered saline (Gibco Dulbecco's phosphate-buffered saline 1X)13 for detergent reagent. This protocol was designed to allow uptake of propidium iodide only in intrinsically membrane-damaged sperm. Dilution factors (i.e. semen and reagent volume) were prepared according to the manufacturer's recommendations. The total sperm concentration was assessed immediately prior to determining the membrane-damaged sperm concentration for each replicate. The percentage of membrane-intact sperm was calculated as: (concentration of total sperm - concentration of membrane-damaged sperm/concentration of total sperm), according to the manufacturer's recommendations.
Culture swabs (BBL CultureSwab Plus)7 were submerged into neat or extended semen and then placed in Aimes charcoal medium (BBL CultureSwab Plus)7 for transport in an insulated container containing ice packs to a diagnostic laboratory (Texas Veterinary Medical Diagnostic Laboratory, Amarillo, Texas, USA) for inoculation of media. Culture swabs were plated on blood and tergitol (blood agar, MacConkey agar, Tergitol 7 agar and triple sugar iron agar)14 agars within 24 h of collection. Bacteria were streaked on agar by standard methods . For each specimen, one blood agar plate and one tergitol plate were incubated under aerobic conditions at 37°C for up to 48 h. One blood agar plate for each specimen was also incubated at 37°C for 48 h under anaerobic conditions, as described previously [20, 21]. Bacterial isolates were identified by secondary plating on MacConkey agar14 and triple sugar iron agar14 and by standard laboratory techniques [4, 20, 21].
Data were analysed using the statistical package SAS 9.215. Analysis of variance was used to evaluate the effects of treatments on experimental endpoints for semen quality. Samples were blocked within ejaculates, and ejaculates were blocked within stallion in the statistical model. Variables measured as percentages were normalised by transformation to angles corresponding to arc sine of the square root of percentage for variance analyses. Tabular data are presented as nontransformed values to facilitate interpretation. The Tukey's honestly significant difference (HSD) test was used to separate main effect means when treatment F ratios were significant (P<0.05). The effectiveness of treatment (extender with, or without, added ticarcillin-clavulanic acid) and exposure time to extender for inhibiting bacterial growth (dichotomous outcome) was determined by Chi-square test. The P value significance was set at <0.05. Results are presented as means ± s.e.
The results for seminal parameters and bacterial cultures are summarised in Tables 1 and 2, respectively. At time 0 h, no difference was detected for total motility, progressive motility, mean curvilinear velocity and sperm membrane integrity between Groups 1 and 2 (Table 1). After 24 h of cooled storage of extended semen, differences were detected among the experimental treatments (Table 1). Freezing and thawing of extender prior to use (Groups 11 and 12) yielded lower values for total motility, progressive motility and sperm membrane integrity compared with other groups. Although mean values for total motility were similar among the other groups, isolated subtle differences were detected in progressive motility and sperm membrane integrity among those groups. Mean curvilinear velocity of sperm was lower in Group 11 than in all other groups. Mean curvilinear velocity was higher in groups in which powdered ticarcillin-clavulanic acid was added to INRA 96 (i.e. Groups 2, 3 and 4) as compared with groups in which ticarcillin-clavulanic acid was reconstituted in water and frozen–thawed prior to being added to INRA 96 (i.e. Groups 6, 7 and 8).
Table 1. Mean ± s.e – sperm motion characteristics and membrane integrity immediately following extension (T0) and after cooled storage for 24 h (T24; n = 42 ejaculates from 14 Quarter Horse stallions)
|Group 1||72 ± 3a||76 ± 3a||60 ± 3ab||65 ± 3a||201 ± 5ab||215 ± 5a||71 ± 3b||71 ± 3a|
|Group 2||73 ± 3a||77 ± 3a||59 ± 3ab||65 ± 3a||205 ± 5a||213 ± 5a||73 ± 3ab||73 ± 3a|
|Group 3|| ||72 ± 3a|| ||59 ± 3b|| ||204 ± 5a|| ||74 ± 3a|
|Group 4|| ||73 ± 3a|| ||59 ± 3b|| ||203 ± 5a|| ||73 ± 3a|
|Group 5|| ||72 ± 3a|| ||60 ± 3ab|| ||200 ± 5ab|| ||73 ± 3a|
|Group 6|| ||72 ± 3a|| ||61 ± 3ab|| ||194 ± 5bc|| ||73 ± 3ab|
|Group 7|| ||72 ± 3a|| ||60 ± 3ab|| ||194 ± 5bcd|| ||73 ± 3a|
|Group 8|| ||71 ± 3a|| ||62 ± 3ab|| ||186 ± 5de|| ||73 ± 3a|
|Group 9|| ||71 ± 3a|| ||61 ± 3ab|| ||188 ± 5cde|| ||73 ± 3a|
|Group 10|| ||71 ± 3a|| ||62 ± 3a|| ||182 ± 5e|| ||73 ± 3a|
|Group 11|| ||62 ± 3b|| ||52 ± 3c|| ||169 ± 5f|| ||65 ± 3c|
|Group 12|| ||64 ± 3b|| ||53 ± 3c|| ||180 ± 6e|| ||67 ± 3c|
Table 2. Bacteria isolated from neat semen and effectiveness of treatments for elimination of bacterial growth following 15 min at 37°C or 24 h in Equitainer (n = 28 ejaculates from 14 stallions)
|Neat semen||–||–||Bacillus sp., Enterobacter sp., Lactobacillus sp., Micrococcus sp., Streptococcus constellatus, Streptococcus intermedius, Peptostreptococcus micros, Pseudomonas sp. (not P. aeruginosa)|
|Group 1||24/28 (86%)a||27/28 (96%)a||Micrococcus sp., Peptostreptococcus micros, Streptococcus intermedius|
|Group 2||26/28 (93%)a||28/28 (100%)a||Micrococcus sp.|
|Group 3|| ||28/28 (100%)a||None|
|Group 4|| ||28/28 (100%)a||None|
|Group 5|| ||28/28 (100%)a||None|
|Group 6|| ||27/28 (96%)a||Streptococcus intermedius|
|Group 7|| ||28/28 (100%)a||None|
|Group 8|| ||27/28 (96%)a||Streptococcus intermedius|
|Group 9|| ||28/28 (100%)a||None|
|Group 10|| ||27/28 (96%)a||Micrococcus sp.|
|Group 11|| ||28/28 (100%)a||None|
|Group 12|| ||28/28 (100%)a||None|
Bacteria were isolated from neat semen in 28 of 42 ejaculates (67%). All bacteria isolated were considered to be commensal bacteria originating from the surface of the penis (Table 2). No potentially pathogenic bacteria (i.e. Pseudomonas aeruginosa or Klebsiella pneumonia) were isolated from neat semen.
Four of the 14 stallions had bacteria isolated from neat semen in all ejaculates collected. Three of these 4 stallions were housed in dirt paddocks. The mean number of mounts on a breeding dummy required for successful ejaculation over all stallions and semen-collection sessions was 1.2 (range 1–3). Ejaculation occurred during the first mount on 83% (35/42) of the semen-collection sessions. Bacteria were isolated from neat semen on 5 of 7 occasions (71%) when a stallion required more than one mount for ejaculation, but bacteria were also isolated from neat semen on 23 of 35 occasions (66%) when a stallion required only one mount for ejaculation.
No difference was detected between Groups 1 and 2 for effectiveness at eliminating bacterial growth in neat semen after 15 min of exposure of semen to extender. Similarly, no difference was detected among Groups 1–12 after exposure of semen to extender for 24 h. Over all treatments, addition of ticarcillin-clavulanic acid to INRA 96 was not different than INRA 96 alone for inhibiting growth of bacteria (303/308 [98%] vs. 79/84 [94%], respectively). Over all treatments, elimination of bacterial growth was better when extended semen was subjected to cooled storage for 24 h (332/336; 99%) as compared with 15 min exposure at 37°C (50/56; 89%).
Our data indicate that addition of ticarcillin-clavulanic acid to INRA 96 does not negatively impact on sperm quality (i.e. motility features and sperm membrane integrity) in cooled semen. Addition of powdered ticarcillin-clavulanic acid to INRA 96 resulted in increased curvilinear velocity after cooled storage, as compared with addition of ticarcillin-clavulanic acid that had previously been reconstituted in water, then frozen and thawed.
In this study, the bactericidal effectiveness of INRA 96 was not improved by the addition of ticarcillin-clavulanic acid. The selection of ticarcillin-clavulanic acid for this purpose was based on previous testing of this antimicrobial in milk-based semen extender for longevity of sperm motility and inhibition of bacterial growth . The dosage used in this study, 1 mg/ml, was also based on previous studies [4, 16]. While the current study yielded no potentially pathogenic bacteria (i.e. Pseudomonas aeruginosa or Klebsiella pneumonae) in the collected semen, bacteria such as these are found to colonise the penile and preputial skin of some stallions [1-3, 22]. The data sheet that accompanies ticarcillin-clavulanic acid is indicative of a broad antibacterial spectrum, including effectiveness against Pseudomonas aeruginosa and Klebsiella pneumonae. In addition, a previous study revealed that this antimicrobial also inhibited growth of Taylorella equigenitalis when added to INRA 96 at concentrations of 1.0 or 1.5 mg/ml . As the present study was conducted at a commercial equine breeding facility, we were unable to inoculate the semen samples with suspensions of potentially pathogenic bacteria. Of interest, others have reported that certain bacteria considered to be nonpathogenic (i.e. Streptococcus dysgalactiae subsp. equisimilis) can have a detrimental effect on semen quality after cooled storage .
Subjecting the extender to freezing and thawing prior to use negatively affected semen quality (i.e. motility features and sperm membrane integrity) post cooling for 24 h. The INRA 96 manufacturer indicates that any unused extender can be frozen and thawed one time prior to reuse but our findings do not support this recommendation. The freezing and thawing of extender prior to use was not confounded by the addition of ticarcillin-clavulanic acid, as this treatment (i.e. Group 12) resulted in similar values for total motility, progressive motility, and sperm membrane integrity as those identified for the frozen–thawed control group (i.e. Group 11) and curvilinear velocity was higher than for the frozen–thawed control group. Maintaining the extender containing ticarcillin-clavulanic powder at 4°C for 7 days (i.e. Group 4), resulted in similar seminal parameters (i.e. total motility, progressive motility, sperm membrane integrity and curvilinear velocity) to the control group (i.e. Group 1).
The addition of ticarcillin-clavulanic acid to INRA 96 extender is becoming more popular in the equine breeding industry in the United States. As such, we were interested in determining the effect of various methods for storing ticarcillin-clavulanic acid from an opened bottle prior to use. In this study, we found similar values for total motility, progressive motility, and sperm membrane integrity when the ticarcillin-clavulanic acid powder was added directly to extender or when ticarcillin-clavulanic acid powder was reconstituted in water prior to addition to extender. However, curvilinear velocity tended to be higher when the antimicrobial was added to the extender as a powder, as opposed to first being reconstituted in water and frozen–thawed prior to use. We also tested whether the method of thawing reconstituted and frozen ticarcillin-clavulanic acid affected semen quality when added to the extender INRA 96 by comparing Group 7 with Group 8. Thawing method did not affect semen quality (i.e. total sperm motility, progressive sperm motility, sperm membrane integrity and curvilinear velocity).
All of the stallions housed in dirt paddocks consistently had ejaculates contaminated with bacteria. This finding is consistent with a previous report that bedding type and cleaning frequency can have an impact on contamination of semen with bacteria . Others have also reported that semen-collection method can reduce  or eliminate  contamination of semen from bacteria residing on the penile and preputial skin.
Adding antimicrobials to different equine semen extenders warrants further studies, as highly inconsistent results have been reported. For instance, Aurich and Spergser  reported that gentamicin can negatively affect sperm function in extended semen during cooled storage and also that optimal concentrations should be tested for each extender type. Jasko et al.  concluded that a gentamicin concentration greater than 1 mg/ml in extender can be detrimental to sperm motility after cooled storage. Varner et al.  reported that extended cooled stallion semen in a skim milk extender containing either gentamicin or amikacin (1 mg/ml) had better sperm motility when compared with the control group (extender with no antimicrobials). The concentration of gentamicin in INRA 96 (0.105 mg/ml) is similar to that reported by Vaillancourt et al. who reported that gentamicin added to extended semen at a concentration of 0.1 mg/ml inhibited growth of inoculated Pseudomonas aeruginosa after 24 h of cooled storage .
Storage of extended semen at a cooled temperature for 24 h inhibited bacterial growth in semen more effectively than 15 min of semen exposure to extender. Storage temperature of extended semen is known to have an impact on bacterial growth patterns in extended semen. Vaillancourt et al.  reported no increase in bacterial number in extended semen containing no antimicrobial when a storage temperature of 4°C was maintained; however, preservation of semen in similar extender resulted in a 3- to 4-fold increase in bacteria after storage of semen at 20°C for 48 h. Others have reported that semen quality was better maintained at 15°C when the extender contained antimicrobial; however, storage of extended semen at 5°C did not require the addition of antimicrobial to maintain semen quality .
All the stallions used in this study produced semen that responded well to extension and cooling. It is possible that stallion semen with less tolerance to extension and cooling would respond differently to these experimental conditions. Brinsko et al. reported that centrifugation of extended semen and resuspension of sperm in extender containing a reduced amount of seminal plasma improved sperm motility for some stallions whose sperm did not withstand cooled storage by conventional methods .
In summary, addition of ticarcillin-clavulanic acid to INRA 96 did not impair sperm quality (i.e. motility features and sperm membrane integrity) of cooled stored stallion semen for 24 h. Freezing and thawing INRA 96 should be discouraged as our results revealed a corresponding reduction of semen quality upon cooled storage for 24 h. Further studies are warranted to evaluate the effects of ticarcillin-clavulanic acid added to INRA 96 involving fertility trials, different storage temperatures and possible inoculations with pathogenic bacteria such as Pseudomonas aeroginosa and Klebsiella pneumonia.
Authors' declaration of interests
No competing interests have been declared.
Source of funding
Burnett Ranches, LLC (6666 Ranch); Legends Premier Stallion Season Auction Fund; Texas A&M University.
The authors would like to gratefully acknowledge Sheila Teague and Rusty Raleigh for laboratory assistance. Semen extender in this study was donated by IMV Technologies, Maple Grove, Minnesota, USA.
C.J. Dean was involved in experimental design, data acquisition, and manuscript preparation. A.M. Hobgood was involved in data acquisition. G.P. Blodgett was involved in experimental design, data acquisition, and manuscript preparation. C.C. Love was involved in experimental design and manuscript preparation. T.L. Blanchard was involved in experimental design and manuscript preparation. D.D. Varner was involved in experimental design, statistical analysis, and manuscript preparation.
INRA 96, IMV Technologies, L'Aigle, France.
Nasco, Ft. Atkinson, Wisconsin, USA.
Animal Reproduction Systems, Chino, California, USA.
Uline, Pleasant Prairie, Wisconsin, USA.
Chemometec A/S, Allerød, Denmark.
Corning Life Sciences, Lowell, Massachusetts, USA.
Becton-Dickinson and Co., Sparks, Maryland, USA.
GlaxoSmithKline, Research Triangle Park, North Carolina, USA.
Hamilton Research Inc., South Hamilton, Massachusetts, USA.
Hamilton Thorne Biosciences, Beverly, Massachusetts, USA.
Leja Products B.V., Nieuw-Vennep, The Netherlands.
Chemometec A/S, Allerød, Denmark.
Invitrogen, Grand Island, New York, USA.
Remel Inc., Lenexa, Kansas, USA.
SAS 9.2, SAS Institute Inc., Cary, North Carolina, USA.