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Factors associated with failure in breeding soundness examination of Western USA rams
Preventive Veterinary Medicine 105 (2012) 118–126
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Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Factors associated with failure in breeding soundness examination of Western USA rams David C. Van Metre a,∗ , Sangeeta Rao a , Cleon V. Kimberling b , Paul S. Morley a a b
Animal Population Health Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1678, USA Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1678, USA
a r t i c l e
i n f o
Article history: Received 12 July 2011 Received in revised form 31 January 2012 Accepted 3 February 2012 Keywords: Sheep-reproduction Sheep-microbiological diseases Fertility Brucella spp.
a b s t r a c t Breeding-soundness examination (BSE) and eradication of Brucella ovis infection in rams are critical components of flock-health programs. The aims of this retrospective, cross-sectional study were to describe the results of BSE in a large sample of rams in the Western USA and to determine the association between BSE outcome and the semen collection method (penis manually extended vs. retained in the preputial cavity), ram body-condition score (BCS), the presence of ulcerative posthitis, and the size of the flock of origin. We evaluated the first BSE in a given year for rams from Colorado, Wyoming, and Utah, USA, from 2000 through 2007. Breeding-soundness examination consisted of physical examination, scrotal circumference and BCS measurement, semen collection by electroejaculation, and microscopic examination of semen motility, morphology, and leukocyte concentration. We assigned a reason for failure to each failed BSE and used multivariable logistic and Poisson regressions to measure associations between ram and flock variables and the risk or reason for failure on BSE. A non-random, owner-selected subset of rams was tested for antibodies to B. ovis by serum indirect ELISA (iELISA). The Rogan–Gladen corrected B. ovis seroprevalence was measured. Of the 14,667 BSEs performed on 11,804 rams, 29.0% were classified as “failed;” the most common reason for failure was substandard semen parameters (43.8%). Breedingsoundness examinations were more likely to have been categorized as failure for inflammatory causes when performed on rams from medium-sized flocks (OR 1.6; 95% CI 1.1, 2.3) and large flocks (OR 1.4; 95% CI 1.0, 1.9) (P = 0.02), suggesting that larger flocks are at higher risk of contagious diseases. The adjusted seroprevalence of B. ovis antibodies among tested rams in this study was 10.0%. Of 233 rams seropositive to B. ovis, 125 (53.6%) were subclinical, a finding that supports the importance of this test in ram BSE. We found that emaciation in rams was associated with an increased risk of BSE failure from substandard semen parameters (P < 0.001), but ulcerative posthitis and the semen collection method were not (P = 0.09 and 0.34, respectively). However, collection of semen with the penis retained in the preputial cavity resulted in greater odds of leukospermia relative to semen collection with the penis extended (OR 4.1; 95% CI 2.9, 5.9; P < 0.001), presumably from contamination of the semen sample with preputial leukocytes. For ram BSE, therefore, semen collection with the penis manually extended from the sheath is recommended to limit leukocyte contamination of the sample. © 2012 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author. Tel.: +1 970 297 1299; fax: +1 970 297 1275. E-mail address: [email protected] (D.C. Van Metre). 0167-5877/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2012.02.002
Breeding-soundness examination (BSE) of rams is a clinical evaluation of the potential for a ram to impregnate a
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defined number of healthy ewes by natural service during a breeding season (Hulet et al., 1965; Ott and Memnon, 1980; Kimberling and Parsons, 2007). The use of rams deemed satisfactory by BSE is associated with an increase in the pregnancy risk in the ewe flock and the size of the lamb crop (Stobart et al., 1992; Kimberling and Butler, 1986; Kimberling and Schweitzer, 1989). Use of fertile rams can increase the proportion of lambs born early in the lambing season and therefore indirectly result in increased weaning weights (Stobart et al., 1992). In addition to BSE, testing rams by culture (Bulgin, 1990a) or serology (Gall et al., 2003) for contagious infection with Brucella ovis is an important component of the ram-fertility evaluation (Ott and Memnon, 1980; Kimberling and Parsons, 2007). Clinical infection with B. ovis typically manifests as epididymitis, with pain, swelling, and induration (evident on palpation) of the epididymis (Biberstein et al., 1962, 1964; Webb et al., 1980). Infection with B. ovis causes reduced fertility or infertility by induction of leukospermia, reduced sperm motility, and/or an increase in morphologically abnormal sperm evident on microscopic examination of semen (Cameron, 1976; Webb et al., 1980; Kimberling et al., 1986). Importantly, however, B. ovis infection can also be subclinical, resulting in infected rams appearing normal on BSE (Cameron, 1976; Webb et al., 1980; Kimberling et al., 1986; Bulgin, 1990a; Dargatz et al., 1990). Transmission of B. ovis occurs primarily through venereal spread (Buddle, 1955; Hartley et al., 1955; Biberstein et al., 1964), although spread through homosexual activity (Hartley et al., 1955) and oral contact with urine from infected rams (Bulgin and Anderson, 1983) are possible. Detrimental economic effects of this disease include a reduction in the number of lambs born alive, increase in the number of non-pregnant ewes, increased lamb death loss, and prolongation of the subsequent lambing season (McGowan and Devine, 1960; Swift and Weyerts, 1970; Bagley et al., 1984). Previous studies have characterized BSE results in rams of various ages (Wiemer and Ruttle, 1987; Ruttle and Southward, 1988), breeds (Ley et al., 1990), and status relative to B. ovis infection (Cameron, 1976; Kott et al., 1988) or relative to the presence or absence of undifferentiated epididymitis (Bagley et al., 1984). In some of these studies, associations were detected between certain physical characteristics of the rams and parameters of the BSE (Wiemer and Ruttle, 1987), outcome of the BSE (Wiemer and Ruttle, 1987; Ruttle and Southward, 1988), conception rates (Mickelson et al., 1981a), and number of lambs born (McGowan and Devine, 1960; Mickelson et al., 1981a). While particular ram and flock factors have been presumed to be associated with the outcome of BSE, objective data supporting these associations are lacking; these factors include the size of the flock of origin, the semen collection method (with the penis retained in the preputial cavity or manually extended from the sheath), ram body condition score (BCS), and the presence of ulcerative posthitis, a bacterial dermatitis of the preputial orifice. Our aims were to describe the BSE classifications of a large number of Western USA rams and the associations between both BSE failure and reason for failure and the semen-collection method, presence of ulcerative posthitis, ram BCS, and the size of the flock of origin.
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2. Materials and methods 2.1. Case-selection criteria This was a retrospective, cross-sectional study. We searched the case records of the James L. Voss Veterinary Teaching Hospital, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, to identify rams that had been evaluated by BSE on the farm or ranch in Colorado, Wyoming, and Utah from 1 January 2000 through 31 December 2007. All rams eligible for inclusion in the study had at least one BSE performed in the study period. Some rams were examined more than once in a given year; however, we could not determine from the medical records whether a medical or dietary intervention had preceded the subsequent BSE(s) in a given year. Therefore, to reduce confounding caused by such interventions, we included only the first BSE in a given year for each ram. For a BSE to be eligible for inclusion in the study, the medical record had to include the identification of the owner and the ram, the date of BSE, and BSE data to support the classification of the ram as shown in Table 1. Rams were identified by name or by ear tag. During review of the records, if two rams that had identical ear tag identification or names were found to have been tested in a particular flock on a given day, we compared the two rams’ breed, age, and test results to determine whether the rams were different individuals with the same identification or the same ram had been examined twice on that day. If we considered the rams to be different individuals, then we assigned a unique identification number to each ram. If, on the other hand, we considered the ram to have been examined twice on that day, the results of the subsequent BSE(s) for that day were excluded. Information obtained from medical records consisted of owner identification and address, the date of BSE, animal identification, age, breed, BCS, scrotal circumference, and whether or not palpable lesions of epididymitis were present, abnormalities were detected on physical examination, ulcerative posthitis was present, and semen collection was performed. For rams from which semen collection was attempted, information obtained from the medical record included whether or not ejaculation occurred, the percentage of spermatozoa with progressive motility, the percentage of spermatozoa with normal morphology, whether or not leukospermia was present, and the density of white blood cells (WBCs) in the semen sample when viewed at 100× magnification (absent/present at 1–9 WBCs/field/present at ≥10 WBCs/field) (Kimberling et al., 1986). The semen-collection method was designated as either standard collection (with the penis manually extended from the sheath) or preputial collection (with the penis retained in the preputial cavity). A veterinarian supervised the BSE and a veterinarian or animal-health technician performed all microscopic examinations of semen samples; however, the individual conducting each component of the BSE was not recorded in the medical record. We categorized ram breeds into the major breeds represented in the study population: Suffolk, Columbia, Hampshire, and Rambouillet. Black-faced rams that were reported by the owner to be a cross among black-face
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Table 1 Criteria used to classify rams for breeding potential, 14,667 BSEs of 11,804 Western USA rams, 2000–2007. Parameters
Categories of classification on BSE
Body condition Physical abnormalitiesa Epididymitis Ulcerative posthitis
Semen collected by electroejaculation Progressive sperm motility (%) Normal sperm morphology (%) Leukospermia: # white blood cells in semen/100× fieldb Brucella ovis indirect ELISA Scrotal circumference (cm): Rams aged 6–13 mo Rams aged ≥14 moc
Questionable potential breeder (fail)
Satisfactory potential breeder (pass)
Excellent potential breeder (pass)
2.4 Absent Absent Healed or no lesions
3 Absent Absent Healed or no lesions
Yes 10–30 30–50 0–9
Yes 31–69 51–79 0–9
Yes 70–100 ≥80 0–9
Positive
Indeterminate
Negative
Negative
<26 <29
27–29 30–32
30–35 33–39
≥36 ≥40
Unsatisfactory potential breeder (fail) 1 (emaciated), 5 (obese) Present Present Active and severe, and/or narrowing or distortion of preputial orifice; and/or internal form present No 0 ≤29 ≥10
Absent Absent Active and mild to moderate
a Physical problems included febrile illness (rectal temperature >104 ◦ F [40 ◦ C]), lameness, blindness, conformational defects, severe wear or absence of lower incisor teeth, prognathism, brachygnathism, abdominal or inguinal hernias, abnormal testicular shape or consistency (unilateral or bilateral), abnormalities in the spermatic cord (unilateral or bilateral), penile deformities, dermatitis, injury, or deformity of the scrotum, substandard scrotal circumference, and/or an injury or infectious disease (other than epididymitis or ulcerative posthitis) judged to preclude the ram from being capable of breeding ewes. Injuries and infectious diseases were treated by the attending veterinarian upon request by the owner. b Rams with preputial collection were not classified on the basis of WBCs in semen, owing to the likelihood of preputial contamination of the sample with WBCs. c Rams with dentition of 1-yr-olds for which the date of birth was not available were judged on the basis of 6- to 14-mo-old rams.
breeds, or for which breed data were unavailable at the time of BSE, were classified as the “Black Face” breed. Similarly, white-faced rams that were either reported by the owner to be a cross among white-face breeds, or for which breed data were unavailable at the time of BSE, were classified as the “White Face” breed. All other breeds were categorized as the “Other” breed. We determined the age of each ram by dental examination at the time of its first BSE, and we categorized rams as lambs (<1 yr old), yearlings (1 yr old), adult rams (2–5 yr), and aged (≥6 yr old) (similar to categorization in Wiemer and Ruttle, 1987).
2.2. Flock size determination We considered all rams owned by a given owner to be a flock. For flocks evaluated by BSE for >1 yr in the study, we expressed flock size as the average number of rams examined for that owner per year over the course of the study. We categorized flock size as small (≤20 rams), medium (21–50 rams), and large (≥51 rams). In this region, flocks with ≤20 rams are typically confined to small pastures or corrals during the breeding season, while flocks with ≥51 rams are usually managed on open rangelands (often state- or federally-owned forests or grasslands) wherein sheep from different flocks can intermingle. In extensive range settings, comingling of rams from different flocks facilitates transmission of B. ovis among rams (Bagley et al., 1985). Flocks with 21–50 rams might be kept in either fenced pastures or open rangelands.
2.3. Physical examination and semen collection When possible, feed was withheld from rams for 12–24 h prior to BSE (to facilitate manual extension of the penis from the sheath during semen collection). Rams were restrained for BSE in a standing position or in lateral recumbency on a modified calf table. A BCS of 1 (emaciated), 2 (thin), 3 (normal), 4 (well-conditioned) or 5 (obese) was assigned by palpation of the dorsal lumbar area (Scott, 2001). A cursory physical examination was conducted. The scrotum and prepuce were examined visually and palpated. The testes, epididymides, and spermatic cords were palpated. Scrotal circumference was measured with scrotal measuring tape (Scrotal Measuring Tape, Nasco International, Inc., Atkinson, WI, USA) and expressed in cm. No sedatives or anesthetics were administered for semen collection. A hand-held, battery-powered, rectally inserted electroejaculation probe (Lane Ram Ejaculator, Lane Manufacturing, Inc., Denver, CO, USA) was used as described by Kimberling and Parsons (2007). During all BSEs, manual extension of the penis was attempted prior to initiation of electroejaculation. If manual extension was successful, the penis was visually examined, then secured with cotton gauze with the glans directed into a warm, sterile, 16-ml polypropylene tube (Kimberling and Parsons, 2007). If samples of semen were collected with the penis extended, we designated those as “standard collections.” If extension of the penis could not be achieved, the semen sample was collected as it exited the preputial orifice; we designated such samples as “preputial collections.” Immediately after semen collection, a drop of semen was secured with a warm glass Pasteur pipette, placed
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on a warm, clean microscope slide, diluted in approximately 0.1 ml of warm Lactated Ringer’s solution, covered with a cover slip, and examined microscopically with a phase-contrast microscope at 100×. The proportion of spermatozoa that showed progressive motility were estimated and recorded as percent motility. Samples with leukospermia were categorized on the basis of the number of WBCs per visual field at 100× magnification as shown in Table 1 (Kimberling et al., 1986). However, we did not classify rams with preputial collections on the basis of leukospermia because these semen samples were commonly contaminated with WBCs and other debris from the preputial cavity. For evaluation of sperm morphology, a drop of semen and a drop of eosin–nigrosin stain (Live Dead Semen Stain, Jorgensen Laboratories, Loveland, CO, USA) were mixed together, smeared, allowed to dry, and examined under phase contrast with oil immersion at 1000×. One hundred cells were examined, and the proportion of morphologically normal spermatozoa was measured and recorded as % normal morphology (Kimberling and Parsons, 2007).
2.4. Serologic testing for antibodies to B. ovis We evaluated a non-random, owner-selected subset of rams in this study population to quantify serum antibodies to B. ovis using an iELISA (Gall et al., 2003). Serologic testing was conducted at the Colorado State University Veterinary Diagnostic Laboratory. Considering the complement fixation test as the reference method (WOAH, 2011), the sensitivity of iELISA was previously estimated to be 96.3% and the specificity was 99.6% (Gall et al., 2003). From 1 January 2000 to 9 May 2006, the antigen utilized for the iELISA was B. ovis Complement Fixation Antigen (National Veterinary Services Laboratory, Ames, IA); the spectrophotographic optical density (OD) was read at 450 nm. Because supplies of the previous antigen became depleted, from 16 May 2006 until the end of the study, the antigen utilized for the iELISA was changed to B. ovis ELISA Antigen, reagent code 9-ELA (National Veterinary Services Laboratory, Ames, IA); the spectrophotographic OD was read at 590 nm. Throughout the study period, the same 3 reference sera were used to standardize iELISA results: “high positive” sera (high serum titer of antibodies to B. ovis), “low positive” sera (lower serum titer), and negative (no detectable antibodies). These reference sera were obtained from rams with semen culture-confirmed status for B. ovis infection. Rams were classified on the as negative, indeterminate, or positive for B. ovis infection by comparison of ram sera OD values to OD cutoff values obtained from these reference sera. Cutoff values for classification of samples varied over the course of the study; however, classification was considered internally consistent over the study period because test results were standardized using these same reference sera and internal validation procedures revealed similar classification of banked ram serum samples as test protocols were changed (B. Hannafious, personal communication, 2012). For an estimate of true seroprevalence, we only considered rams with positive results on iELISA to be considered seropositive.
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The true seroprevalence was calculated using the formula of Rogan and Gladen (1978): True prevalence =
Apparent prevalence + Specificity − 1 Sensitivity + Specificity − 1
2.5. Classification of the ram Based on criteria derived from Sharkey et al. (2001), BSE data were compiled upon completion of the exam, and the ram was classified as displaying the characteristics of an excellent, satisfactory, questionable, or unsatisfactory potential breeder, as shown in Table 1. The lowest rating among all the BSE parameters determined the ram’s overall BSE rating (Kasimanickam et al., 2006). For analysis, classification of the ram as either an “excellent” and “satisfactory” potential breeder resulted in classification of that examination as a “passed BSE;” otherwise, it was classified as a “failed BSE.” We classified rams that were seropositive to B. ovis as “unsatisfactory potential breeders” and rams with an indeterminate result on B. ovis indirect ELISA as “questionable potential breeders.” In some instances, semen collection was performed on rams with physical abnormalities. In such cases, the ram was classified as a “questionable” or “unsatisfactory” potential breeder (depending on the severity of the physical problem and its potential for spontaneous resolution or resolution with treatment, as judged by the attending veterinarian). Similarly, semen collection was performed on most rams with ulcerative posthitis, and classification of the ram was based on the characteristics of the lesion as described in Table 1. 2.6. Determination of reason for failure Any given BSE could have been classified as a “failed BSE” on the basis of more than one parameter; therefore, to identify one abnormal parameter that resulted in failure on each BSE, we applied a hierarchical algorithm to each failed examination. The reasons for failure were categorized in the following order, based on the sequence of data acquisition during the BSE: (1) Owner cull: The ram possessed a phenotypic characteristic considered “unfavorable” by the owner, who decided to cull the animal. (2) Inflammatory causes: Enlargement, pain and/or induration in one or both epididymides or ≥10 WBCs visible per 100× microscopic field and/or positive or indeterminate on serologic testing for B. ovis. (3) Emaciation: BCS = 1. (4) Obesity: BCS = 5. (5) Physical abnormalities as defined in Table 1. (6) Ulcerative posthitis. (7) Failure to collect (electroejaculation was attempted but semen could not be obtained). (8) Substandard semen parameters: Semen was obtained by electroejaculation, but microscopic examination revealed substandard motility and/or morphologic abnormalities as defined in Table 1.
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2.7. Categorization of year and season for BSEs The total number of years during which each ram in the study was tested at least once was expressed as 1 yr or ≥2 yr. Each BSE was assigned to its corresponding season based on the month in which it was performed: January–June, July–September, and October–December. Michelson et al. (1981b) found that scrotal circumference and the proportion of morphologically normal sperm in rams were highest in the fall months and gradually decreased to the lowest values in February. Our intent was to capture this nadir of ram fertility in the middle of a 3-month period; however, because only four BSEs were performed during January–March, the first quarter of the year was combined with the second. 2.8. Data analysis Analyses were performed using commercially available statistical software (SAS version 9.2, Cary, NC, USA). We calculated descriptive statistics at the level of the individual ram for breed category, age category at the time of first examination, the total number of years tested, and whether ever tested positive for B. ovis infection. We used chi-square to analyze the relationship between flock size category and whether rams were examined in only 1 yr or ≥2 yr. Most inferential analyses performed at the level of the BSEs used pass/fail categorization as the primary outcome variable in logistic and Poisson regressions (PROC GENMOD, SAS version 9.2, Cary, NC); we estimated odds ratios (OR) with associated 95% confidence intervals (CIs) and risks of failure (respectively). We used GEE models to control for repeated measures within ram, nested within flock. Exposures (independent variables) in these analyses included age category, breed category, flock-size category, year of examination, season, and collection method. In univariable analyses, we found that the first four of these were all significantly (P < 0.001) associated with several different outcomes; therefore, we included these variables in all multivariable models to control for potential confounding associated with them. Factors that were potentially associated with BSE failure specifically attributed to substandard semen parameters were investigated within the subset of failed BSEs; these factors were body-condition category, ulcerative posthitis, and collection type. We also investigated (using multivariable logistic regression) whether breed or age categories for rams were associated semen collection method. We also included ram-age and flock-size categories in the model investigating inflammatory causes within the subset of failed BSEs. We used multivariable logistic regression to evaluate the effect of collection type on the odds of leukospermia being present, as well as to evaluate the effect of age and flock size on the odds of failure from inflammatory causes. Model diagnostics were used to evaluate the validity of all logistic and Poisson regression models. This included evaluations for collinearity of explanatory variables (by examining the variance inflation factor, simple correlations and cross-tabulations for explanatory variables), influential observations, and goodness-of-fit statistics (Dohoo
Table 2 Semen-collection method, classification, and reasons for failure, 14,667 BSEs of 11,804 Western USA Rams, 2000–2007. BSEs N Semen-collection method Standarda Preputialb
%
13,910 212
98.5c 1.5
Classification Excellent (pass) Satisfactory (pass) Questionable (fail) Unsatisfactory (fail)
3029 7386 1584 2668
20.7 50.4 10.8 18.1
Reason for failure Owner cull Inflammatory causes Emaciation Obesity Physical abnormalities Ulcerative posthitis Failure to collect Substandard semen parameters
6 852 605 48 658 196 24 1863
0.1 20.0 14.2 1.1 15.5 4.6 0.6 43.8
a
Semen collected with the penis manually extended from the sheath. Semen collected with the penis retained in the preputial cavity. c Expressed as a percentage of the 14,122 BSEs in which semen collection was performed. b
et al., 2009). Statistical significance of independent variables was evaluated using two-tailed Type III P-values obtained from the GEE models, with a critical alpha of 0.05. All first-order interaction terms were evaluated singly (in turn) for all independent variables included in final models.
3. Results A total of 14,674 BSEs from 11,811 rams met the eligibility criteria for inclusion in the study. Seven BSEs from 7 rams were excluded because the reason for classification could not be determined from the medical record (resulting in the inclusion in the study of 14,667 BSEs from 11,804 rams). Of the 11,804 rams in the 8-yr study, 9818 rams (83.2%) were examined in only 1 yr, 1355 rams (11.5%) were examined in only 2 yr, 445 rams (3.8%) were examined in only 3 yr, and 186 rams (1.5%) were examined in ≥4 yr. A larger proportion of rams from large flocks (1703/8638, 19.7%) were examined in ≥2 yr, compared to rams from medium-sized flocks (203/2036, 10.0%) and rams from small flocks (80/1130, 7.1%) (P < 0.001). Ninety-five rams underwent a second BSE on the same day as the first BSE in a given year. A total of 211 rams had ≥2 BSEs in a given year. We could not determine from the medical records the reason for these subsequent BSEs. Descriptive statistics for semen collection method, BSE classification, and reasons for BSE failure are shown in Table 2. In Table 3, we show the variables significantly associated with risk of failure on BSE (for any cause), as determined by multivariable Poisson regression. Semencollection method and season of BSE were not significant and were not retained. In Table 4, we show the variables associated with the odds of failure for inflammatory causes, as determined by multivariable logistic regression.
D.C. Van Metre et al. / Preventive Veterinary Medicine 105 (2012) 118–126 Table 3 Results of multivariable Poisson regression evaluating the risk of failure on BSE, 14,667 BSEs of 11,804 Western USA Rams, 2000–2007a . Variable b
Age (yr)
Flock size
Breed
Year
Category
% Failurec
(95% CI)
P-value
<1 1 2–5 ≥6
34.4a,d 28.7b 26.8c 36.7d
32.1, 36.8 26.9, 30.7 25.5, 28.1 32.5, 41.3
<0.001
Small (≤20) Medium (21–50) Large (≥51)
34.7a 29.7b 29.9b
31.9, 37.9 27.5, 32 28.6, 31.3
0.005
Suffolk Black Faced Columbia White Facee Rambouillet Hampshire Other
36.9a 34.9a,d 35.1a,f 30.8b,d,e,f 29.9b 28.7b,e 25.1c
34.9, 38.9 32.2, 37.8 31.7, 38.9 27.1, 35 27.7, 32.3 26.2, 31.3 22.7, 27.7
<0.001
2000 2001 2002 2003 2004 2005 2006 2007
37.6a 33.8b,c 33.6b,c 38.7a 31.4c 34.8a,b 25.6d 26.4d
35, 40.4 31.2, 36.5 30.9, 36.4 35.9, 41.7 29, 34.1 32.2, 37.6 23.5, 28 24.2, 28.9
<0.001
a Correlation in responses associated with inclusion of >1 BSE for some rams was controlled using generalized estimating equations. b Ages were missing for 36/14,667 (0.2%) of BSEs. c For each variable, estimates of risk of failure with different letters differ at a significance level of ≤5%. d Black-faced breeds other than Suffolk or Hampshire. e White-faced breeds other than Columbia or Rambouillet.
We determined that there was no difference among breed categories or in ram-age category in the proportion of semen collections that were standard collections (P = 0.07 and 0.76, respectively). Preputial collection, however, was associated with greater odds for leukospermia to be detected on microscopic examination of semen, relative to samples obtained by standard collection (OR 4.1; 95% CI 2.9, 5.9; P < 0.001). In Table 5, we show the association between semen collection method, ulcerative posthitis,
Table 4 Multivariable logistic regression evaluating the odds of failure for inflammatory causesa , 4252 BSEs classified as “failed” of 3989 Western USA Rams, 2000–2007b , c . Variable
Category
OR (95% CI)
P-value
Age (yr)
<1 1 2–5 ≥6
Reference 1.6 (1.2, 2.1) 1.3 (1.0, 1.7) 0.8 (0.5, 1.2)
<0.001
Flock size
Small (≤20) Medium (21–50) Large (≥51)
Reference 1.6 (1.1, 2.3) 1.4 (1.0, 1.9)
0.02
a Failure from inflammatory causes includes BSEs failed due to the presence of palpable epididymitis, ≥10 WBCs per 100× field, and/or positive or indeterminate results for B. ovis serology. b Breed of ram and year of examination were also controlled as fixed effects in this regression model. c Correlation in responses associated with inclusion of >1 BSE for some rams was controlled using generalized estimating equations. ˇ0 = −1.48; 95% CI: −2.04 to −0.91.
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Table 5 Multivariable Poisson regression evaluating the risk of failure on BSE due to substandard semen motility or morphology, 14,667 BSEs of 11,804 Western USA rams, 2000–2007a , b . Variable
Category
% Failurec
(95% CI)
P-value
Collection method
Standard Preputial
25.6 27.9
22.0, 29.0 21.9, 33.5
0.34
Ulcerative posthitisd
Present Absent
28.8 24.6
22.8, 34.4 21.0, 28.1
0.09
Body condition score
1 2 3 4 5
31.6a 27.1b 24.0c 24.9b,c 25.9a,b,c
26.8, 36.2 23.1, 31.0 20.1, 27.6 20.8, 28.8 14.2, 36.1
<0.001
a Breed and age categories of ram, flock size category, and year of examination were also controlled as fixed effects in this regression model. b Correlation in responses associated with inclusion of >1 BSE for some rams was controlled using generalized estimating equations. c For each variable, estimates of risk of failure with different letters differ at a significance level of ≤5%. d Semen was collected in 250/258 BSEs (96.9%) performed on rams with ulcerative posthitis.
and BCS and the risk of failure on BSE due to substandard semen parameters. A total of 2317 rams were tested at least once by iELISA for antibodies to B. ovis. Of these, 233 were positive; the Rogan–Gladen adjusted true prevalence in this subset of rams was 10.0% (Rogan and Gladen, 1978). Importantly, 125 of 233 rams (53.6%) that were seropositive for B. ovis were otherwise normal on BSE, with no abnormalities detected in these rams on physical examination or semen examination. 4. Discussion The most important findings of this study were measurement of the association between outcome of BSE (pass/fail) and variables relevant to ram BSE technique (semen-collection method), ram characteristics (BCS and ulcerative posthitis), and flock size. These variables have been deemed relevant to ovine BSE in the published literature but have not previously been objectively evaluated. To our knowledge, this is the largest study of BSEs on rams in the veterinary literature. The primary limitation of this study is that the study population is not necessarily representative of all rams in the Western USA. The state in which the owner resided was not analyzed because animals in many flocks traveled across state lines during multiple years of the study. Further, the persons who performed each component of the BSE were not listed in the medical records. The skill and experience of the individuals conducting the various procedures included in ovine BSE likely varied, as did skill and experience within a given individual across different years of the study; this variation in examiners (and also in climate, feed quality, etc.) could have affected BSE outcomes across years. Further, those performing semen microscopy were not blinded to the physical properties of the rams; it is possible that this lack of blinding could have influenced classification relative to semen characteristics.
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This study is important because it is the first to evaluate the association between semen collection method and the outcome of ram BSE objectively. Semen collection without manual extrusion of the penis from the preputial cavity has been described (Hindson and Winter, 2002; Mobini et al., 2002); this type of collection is often practiced because spontaneous penile extension during electroejaculation does not consistently occur in rams (Ball, 1976). Preputial contents can contain endogenous debris such sloughed epithelial cells and white blood cells, as well as exogenous debris (e.g. plant material). Manual extrusion of the penis from the sheath prior to semen collection has been recommended by others, in order to minimize contamination of the sample with preputial contents (Kimberling and Parsons, 2007; Scott, 2007; Parkinson, 2009). This recommendation was substantiated by our data: preputial collection was associated with greater odds of leukospermia. Accurate interpretation of leukospermia is essential during BSE of the ram. B. ovis could be isolated from ram semen samples that showed leukospermia and otherwise normal sperm motility and morphology (Kimberling et al., 1986). In addition, leukospermia is one of the earliest changes in the BSE following experimental B. ovis infection of rams (Webb et al., 1980). However, in our study, the semen collection method was not associated with the proportion of BSEs that failed for substandard semen parameters; this implies that collection method does not influence semen motility and morphology. Manual extension of the ram’s penis could not be performed for a relatively small proportion of BSEs (1.5%) in this study. Manual extension of the penis can be difficult to accomplish – particularly in larger rams or rams that have been recently fed – owing to pressure from the full rumen on the ventral body wall and penis. Notably, however, neither breed nor the age of the ram were associated with the semen collection method. Ulcerative posthitis is a bacterial infection of the prepuce and skin of the sheath of male small ruminants. It results from proliferation of Corynebacterium renale (a normal bacterial inhabitant of the prepuce) in response to the increased concentration of urea in the urine (which, in turn, results from ingestion of excessive amounts of protein in the diet) (Brook et al., 1966). The presence of ulcerative posthitis had no detectable effect on semen parameters. However, ulcerative posthitis might influence the ram’s willingness to breed (libido) because of pain when the lesion contacts the ewe during breeding. We therefore recommend that rams with such lesions be categorized as “questionable” or “unsatisfactory” potential breeders and treated with dietary protein modification—specifically, reduction in dietary protein intake (Brook et al., 1966) and antimicrobial therapy (with re-evaluation by a veterinarian prior to breeding). Although others have recommended rams to be in good body condition prior to the onset of the breeding season (Ott and Memnon, 1980; Kimberling and Parsons, 2007), the influence of extremes of BCS on semen parameters has not been described previously. In this study, emaciation was associated with a higher risk of failure because of substandard semen parameters. Thin rams might have had diseases that adversely affected semen parameters,
or there could be a direct effect between thin body condition and semen quality. Whether or not emaciated rams experience improvements in semen parameters with improvement in BCS remains to be determined. In contrast, no detectable difference in risk of failure from substandard semen parameters could be detected for obese rams even though obesity has been hypothesized to affect ram fertility adversely (Wise, 1983). However, we had relatively few obese rams (N = 48) and therefore perhaps lacked sufficient power to detect an adverse effect of obesity on semen parameters. Further, it is plausible that obesity in rams negatively affects sperm quality, libido, and/or thermoregulatory capacity during the breeding season, which might not be detected during BSE. The estimated risk of failure for BSEs conducted on rams from small flocks were higher than those of BSEs conducted on rams from large- and medium-sized flocks. However, failed BSEs that were performed on rams from large- and medium-sized flocks were at higher odds to be categorized as failure from inflammatory causes than were failed BSEs performed on rams from small flocks. We speculate that this difference in odds of failure due to inflammatory causes is related to the difficulty in establishing good ram flock biosecurity practices under the open-range conditions common to larger sheep flocks in the Western USA, as has been previously proposed by Bagley et al. (1985). Further, when purchasing new rams, operators from smaller flocks might exercise different purchasing practices or use different criteria to select source flocks for such rams relative to owners of larger flocks. We found that 1-yr-old and 2- to 5-yr-old rams had higher odds of failure from inflammatory causes relative to ram lambs and aged rams. Although B. ovis infection has been documented in virgin ram lambs (Bulgin, 1990b), it is considered to be a more common isolate from mature rams (Bulgin and Anderson, 1983), which might partly explain in part the increased risk of failure from inflammatory causes in the 1-yr-old and 2- to 5-yr-old rams of our study. Further, rams of these two age groups might be more sexually active during the breeding season than ram lambs or aged rams, thereby increasing their risk of infectious diseases involving venereal transmission. Ram lambs and aged rams showed the highest estimated risks of failure. Ram lambs could have been sexually immature at the time of BSE, whereas aged rams could have greater risk for chronic, debilitating diseases, physical injuries, dental disease, or age-related reduction in fertility. In a previous study, relative to younger rams, a significantly greater proportion of rams >6 yr old had testicular lesions; these older rams also showed significantly fewer motile sperm cells and significantly greater morphologically abnormal sperm than younger rams (Wiemer and Ruttle, 1987). The estimated risk of failure of BSE (from any cause) was lowest for the breeds included in the “other” category, followed by the Hampshire and Rambouillet breeds. Breedcategory differences in maturation rate and susceptibility to disease (as well as differences in flock management) might have influenced the disparity among breed categories in BSE failure in our study. In contrast, Ley et al. (1990) failed to detect a difference among 1 yr-old Dorset,
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Hampshire, and Suffolk breeds in failure percentage on a single BSE. We evaluated a subset of rams in this study population by testing for antibodies to B. ovis. Most rams that were seropositive were otherwise normal on BSE—findings that corroborate those of previous studies wherein infection with B. ovis was documented commonly to be subclinical (Cameron, 1976; Webb et al., 1980; Bagley et al., 1984; Kimberling et al., 1986; Kott et al., 1988; Bulgin, 1990a; Dargatz et al., 1990). In light of these findings, serologic testing for B. ovis should continue to be considered as an integral component of the BSE in rams. However, only a small subset of rams in this study was tested for B. ovis infection, and these animals were selected by the owners. Therefore, our serologic data are heavily biased and should not be interpreted as representative of the true population of rams in the Western USA. The proportion of BSEs in the present study that were categorized as passed (71.0%) is similar to that (73.8%) reported by Kimberling and Butler (1986) in a different Colorado study and to that (70.7%) reported by Wiemer and Ruttle (1987) in a New Mexico, USA study. The pass percentage for BSEs in our study is also similar to that reported for rams in Virginia, USA (71.7%) by Ley et al. (1990). The most common reason for failure on BSE in our study was substandard semen parameters, which is similar to the findings of Ley et al. (1990). That those previous studies were performed >20 years ago suggests minimal change in the proportion of rams in the USA that are classified as passed on BSE over that time period, as well as minimal change in the primary reason for failure on BSE.
5. Conclusions Collection of semen with the penis retained in the preputial cavity was associated with higher odds of leukocytes in the semen sample relative to semen collection with the penis extended from the sheath. Because leukospermia can indicate contagious epididymitis caused by B. ovis, semen collection with the penis extended is recommended to prevent spurious contamination of the semen sample with leukocytes from the prepuce. We found that emaciation in rams was associated with an increased risk of BSE failure from substandard semen parameters, but ulcerative posthitis was not. Rams from medium- and large-sized flocks showed the highest odds of BSE failure from inflammatory causes–suggesting potential disparate risk for contagious venereal diseases among flocks of different sizes. As shown in previous studies, rams that are seropositive to B. ovis can be otherwise normal on BSE; therefore, to detect subclinically infected rams, serologic testing for B. ovis should be performed in conjunction with BSE.
Conflict of interest statement The authors of this manuscript have no conflicts of interest to report that are relevant to this work.
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Acknowledgements We thank Gerilyn Parsons, Anthony Knight, Jeruesha Nichols, Roger Ellis, and Jennifer Fraser for assistance with this study. References Bagley, C.V., Burrell, W.C., Esplin, G.M., Walters, J.L., 1984. Effect of epididymitis on semen quality of rams. J. Am. Vet. Med. Assoc. 185, 876–877. Bagley, C.V., Paskett, M.E., Matthews, N.J., Stenquist, N.J., 1985. Prevalence and causes of ram epididymitis in Utah. J. Am. Vet. Med. Assoc. 186, 798–801. Ball, L., 1976. Electroejaculation. In: Klemm, W.R. (Ed.), Applied Electronics for Veterinary Medicine and Animal Physiology. Charles C. Thomas, Springfield, IL, USA, pp. 394–441. Biberstein, E.L., McGowan, B., Robinson, E.A., Harrold, D.R., 1962. Epididymitis in rams: studies on immunity. Cornell Vet. 52, 214–227. Biberstein, E.L., McGowan, B., Olander, H., Kennedy, P.C., 1964. Epididymitis in rams: studies on pathogenesis. Cornell Vet. 54, 27–41. Brook, A.H., Southcott, W.H., Stacy, B.D., 1966. Etiology of ovine posthitis: relationship between ovine posthitis and a causal organism. Aust. Vet. J. 42, 9–12. Buddle, M.B., 1955. Observations on the transmission of Brucella infection in sheep. N. Zeal. Vet. J. 3, 10–19. Bulgin, M.S., 1990a. Brucella ovis excretion in semen of seronegative, clinically normal breeding rams. J. Am. Vet. Med. Assoc. 196, 313–315. Bulgin, M.S., 1990b. Brucella ovis epizootic in virgin ram lambs. J. Am. Vet. Med. Assoc. 196, 1120–1122. Bulgin, M.S., Anderson, B.C., 1983. Association of sexual experience with isolation of various bacteria in cases of ovine epididymitis. J. Am. Vet. Med. Assoc. 182, 372–374. Cameron, R.D.A., 1976. Characteristics of semen changes during Brucella ovis infection of rams. Vet. Rec. 99, 231–233. Dargatz, D.A., Smith, J.A., Knight, A.P., Farin, P.W., Kimberling, C.V., 1990. Antimicrobial therapy for rams with Brucella ovis infection of the urogenital tract. J. Am. Vet. Med. Assoc. 196, 605–610. Dohoo, I.A., Martin, S.W., Stryhn, H., 2009. Veterinary Epidemiological Research, 2nd ed. VER Inc., Charlottetown, Prince Edward Island, Canada. Gall, D., Nielsen, K., Vigliocco, A., Smith, P., Perez, B., Rojas, X., Robles, C., 2003. Evaluation of an indirect enzyme-linked immunoassay for presumptive serodiagnosis of Brucella ovis in sheep. Small Rumin. Res. 48, 173–179. Hartley, W.J., Jebson, J.L., Macfarlane, D., 1955. Some observations on natural transmission of ovine brucellosis. N. Zeal. Vet. J. 3, 5–10. Hindson, J.C., Winter, A.C., 2002. Manual of Sheep Diseases. Blackwell Science Ltd., Oxford, UK, pp. 42–52. Hulet, C.V., Foote, W.C., Blackwell, R.L., 1965. Relationship of semen quality and fertility in the ram to fecundity in the ewe. J. Reprod. Fertil. 9, 311–315. Kasimanickam, R., Pelzer, K.D., Kasimanickam, V., Swecker, W.S., Thatcher, C.D., 2006. Association of classical semen parameters, sperm DNA fragmentation index, lipid peroxidation and antioxidant enzymatic activity of semen in ram-lambs. Theriogenology 65, 1407–1421. Kimberling, C.V., Arnold, K.S., Schweitzer, D.J., Jones, R.L., VonByern, H., Lucas, M., 1986. Correlation of the presence of seminal white blood cells and the prevalence of separated spermatozoal heads with subclinical Brucella ovis infection in rams. J. Am. Vet. Med. Assoc. 189, 73–76. Kimberling, C., Butler, J., 1986. Colorado’s ram breeding soundness program. In: 5th Ann. Mtg. of the WRCC-46 for Ram Epididymitis, Albuquerque, NM, pp. 41–47. Kimberling, C.V., Schweitzer, D., 1989. Brucella ovis infection and its management in ovine reproduction. Agri. Pract. 10, 36–39. Kimberling, C.V., Parsons, G.A., 2007. Breeding soundness evaluation and surgical sterilization of the ram. In: Younquist, R.S., Threlfall, W.R. (Eds.), Current Therapy in Large Animal Theriogenology. , 2nd ed. Saunders Elsevier, St. Louis, MO, USA, pp. 620–628. Kott, R.W., Halver, G.C., Firehammer, B., Thomas, V.M., 1988. Relationships between Brucella ovis semen culture and various semen and serum parameters. Theriogenology 29, 961–970. Ley, W.B., Sprecher, J., Thatcher, C.D., Pelzer, K.D., Umberger, S.H., 1990. Use of the point score system for breeding soundness examination in yearling Dorset, Hampshire, and Suffolk rams. Theriogenology 34, 721–733.
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Scott, P.R., 2001. Health and production management in sheep flocks. In: Radostits, O.M. (Ed.), Herd Health: Food Animal Production Medicine. , 3rd ed. Philadelphia, PA, pp. 765–844. Scott, P.R., 2007. Sheep Medicine. In: London, 1st ed, pp. 33–82. Sharkey, S., Callan, R.J., Mortimer, R., Kimberling, C., 2001. Reproductive techniques in sheep. Vet. Clin. North Am. Food Anim. Pract. 17, 435–455. Stobart, R.H., Nix, K.J., Kimberling, C.V., Riley, M.L., 1992. Influence of ram epididymitis caused by Brucella ovis on conception rate and lambing percentage in range ewes. Sheep Res. J. 8, 66–68. Swift, B.L., Weyerts, P.R., 1970. Ram epididymitis: a study on infertility. Cornell Vet. 60, 204–214. Webb, R.F., Quinn, C.A., Cockram, F.A., Husband, A.J., 1980. Evaluation of procedures for diagnosis of Brucella ovis infection in rams. Aust. Vet. J. 56, 172–175. Wiemer, K.E., Ruttle, J.L., 1987. Semen characteristics, scrotal circumference, and bacterial isolates of fine wool range rams. Theriogenology 28, 625–637. Wise, C.M., 1983. Ram management for maximum reproductive performance in the range flock. Comp. Cont. Ed. Pract. Vet. 5, S649–S653. World Organization for Animal Health, 2011. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, http://www.oie.int/fileadmin/Home/eng/Health standards/tahm/ 2.07.09 OVINE EPID.pdf.
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Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Factors associated with failure in breeding soundness examination of Western USA rams David C. Van Metre a,∗ , Sangeeta Rao a , Cleon V. Kimberling b , Paul S. Morley a a b
Animal Population Health Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1678, USA Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1678, USA
a r t i c l e
i n f o
Article history: Received 12 July 2011 Received in revised form 31 January 2012 Accepted 3 February 2012 Keywords: Sheep-reproduction Sheep-microbiological diseases Fertility Brucella spp.
a b s t r a c t Breeding-soundness examination (BSE) and eradication of Brucella ovis infection in rams are critical components of flock-health programs. The aims of this retrospective, cross-sectional study were to describe the results of BSE in a large sample of rams in the Western USA and to determine the association between BSE outcome and the semen collection method (penis manually extended vs. retained in the preputial cavity), ram body-condition score (BCS), the presence of ulcerative posthitis, and the size of the flock of origin. We evaluated the first BSE in a given year for rams from Colorado, Wyoming, and Utah, USA, from 2000 through 2007. Breeding-soundness examination consisted of physical examination, scrotal circumference and BCS measurement, semen collection by electroejaculation, and microscopic examination of semen motility, morphology, and leukocyte concentration. We assigned a reason for failure to each failed BSE and used multivariable logistic and Poisson regressions to measure associations between ram and flock variables and the risk or reason for failure on BSE. A non-random, owner-selected subset of rams was tested for antibodies to B. ovis by serum indirect ELISA (iELISA). The Rogan–Gladen corrected B. ovis seroprevalence was measured. Of the 14,667 BSEs performed on 11,804 rams, 29.0% were classified as “failed;” the most common reason for failure was substandard semen parameters (43.8%). Breedingsoundness examinations were more likely to have been categorized as failure for inflammatory causes when performed on rams from medium-sized flocks (OR 1.6; 95% CI 1.1, 2.3) and large flocks (OR 1.4; 95% CI 1.0, 1.9) (P = 0.02), suggesting that larger flocks are at higher risk of contagious diseases. The adjusted seroprevalence of B. ovis antibodies among tested rams in this study was 10.0%. Of 233 rams seropositive to B. ovis, 125 (53.6%) were subclinical, a finding that supports the importance of this test in ram BSE. We found that emaciation in rams was associated with an increased risk of BSE failure from substandard semen parameters (P < 0.001), but ulcerative posthitis and the semen collection method were not (P = 0.09 and 0.34, respectively). However, collection of semen with the penis retained in the preputial cavity resulted in greater odds of leukospermia relative to semen collection with the penis extended (OR 4.1; 95% CI 2.9, 5.9; P < 0.001), presumably from contamination of the semen sample with preputial leukocytes. For ram BSE, therefore, semen collection with the penis manually extended from the sheath is recommended to limit leukocyte contamination of the sample. © 2012 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author. Tel.: +1 970 297 1299; fax: +1 970 297 1275. E-mail address: [email protected] (D.C. Van Metre). 0167-5877/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2012.02.002
Breeding-soundness examination (BSE) of rams is a clinical evaluation of the potential for a ram to impregnate a
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defined number of healthy ewes by natural service during a breeding season (Hulet et al., 1965; Ott and Memnon, 1980; Kimberling and Parsons, 2007). The use of rams deemed satisfactory by BSE is associated with an increase in the pregnancy risk in the ewe flock and the size of the lamb crop (Stobart et al., 1992; Kimberling and Butler, 1986; Kimberling and Schweitzer, 1989). Use of fertile rams can increase the proportion of lambs born early in the lambing season and therefore indirectly result in increased weaning weights (Stobart et al., 1992). In addition to BSE, testing rams by culture (Bulgin, 1990a) or serology (Gall et al., 2003) for contagious infection with Brucella ovis is an important component of the ram-fertility evaluation (Ott and Memnon, 1980; Kimberling and Parsons, 2007). Clinical infection with B. ovis typically manifests as epididymitis, with pain, swelling, and induration (evident on palpation) of the epididymis (Biberstein et al., 1962, 1964; Webb et al., 1980). Infection with B. ovis causes reduced fertility or infertility by induction of leukospermia, reduced sperm motility, and/or an increase in morphologically abnormal sperm evident on microscopic examination of semen (Cameron, 1976; Webb et al., 1980; Kimberling et al., 1986). Importantly, however, B. ovis infection can also be subclinical, resulting in infected rams appearing normal on BSE (Cameron, 1976; Webb et al., 1980; Kimberling et al., 1986; Bulgin, 1990a; Dargatz et al., 1990). Transmission of B. ovis occurs primarily through venereal spread (Buddle, 1955; Hartley et al., 1955; Biberstein et al., 1964), although spread through homosexual activity (Hartley et al., 1955) and oral contact with urine from infected rams (Bulgin and Anderson, 1983) are possible. Detrimental economic effects of this disease include a reduction in the number of lambs born alive, increase in the number of non-pregnant ewes, increased lamb death loss, and prolongation of the subsequent lambing season (McGowan and Devine, 1960; Swift and Weyerts, 1970; Bagley et al., 1984). Previous studies have characterized BSE results in rams of various ages (Wiemer and Ruttle, 1987; Ruttle and Southward, 1988), breeds (Ley et al., 1990), and status relative to B. ovis infection (Cameron, 1976; Kott et al., 1988) or relative to the presence or absence of undifferentiated epididymitis (Bagley et al., 1984). In some of these studies, associations were detected between certain physical characteristics of the rams and parameters of the BSE (Wiemer and Ruttle, 1987), outcome of the BSE (Wiemer and Ruttle, 1987; Ruttle and Southward, 1988), conception rates (Mickelson et al., 1981a), and number of lambs born (McGowan and Devine, 1960; Mickelson et al., 1981a). While particular ram and flock factors have been presumed to be associated with the outcome of BSE, objective data supporting these associations are lacking; these factors include the size of the flock of origin, the semen collection method (with the penis retained in the preputial cavity or manually extended from the sheath), ram body condition score (BCS), and the presence of ulcerative posthitis, a bacterial dermatitis of the preputial orifice. Our aims were to describe the BSE classifications of a large number of Western USA rams and the associations between both BSE failure and reason for failure and the semen-collection method, presence of ulcerative posthitis, ram BCS, and the size of the flock of origin.
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2. Materials and methods 2.1. Case-selection criteria This was a retrospective, cross-sectional study. We searched the case records of the James L. Voss Veterinary Teaching Hospital, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, to identify rams that had been evaluated by BSE on the farm or ranch in Colorado, Wyoming, and Utah from 1 January 2000 through 31 December 2007. All rams eligible for inclusion in the study had at least one BSE performed in the study period. Some rams were examined more than once in a given year; however, we could not determine from the medical records whether a medical or dietary intervention had preceded the subsequent BSE(s) in a given year. Therefore, to reduce confounding caused by such interventions, we included only the first BSE in a given year for each ram. For a BSE to be eligible for inclusion in the study, the medical record had to include the identification of the owner and the ram, the date of BSE, and BSE data to support the classification of the ram as shown in Table 1. Rams were identified by name or by ear tag. During review of the records, if two rams that had identical ear tag identification or names were found to have been tested in a particular flock on a given day, we compared the two rams’ breed, age, and test results to determine whether the rams were different individuals with the same identification or the same ram had been examined twice on that day. If we considered the rams to be different individuals, then we assigned a unique identification number to each ram. If, on the other hand, we considered the ram to have been examined twice on that day, the results of the subsequent BSE(s) for that day were excluded. Information obtained from medical records consisted of owner identification and address, the date of BSE, animal identification, age, breed, BCS, scrotal circumference, and whether or not palpable lesions of epididymitis were present, abnormalities were detected on physical examination, ulcerative posthitis was present, and semen collection was performed. For rams from which semen collection was attempted, information obtained from the medical record included whether or not ejaculation occurred, the percentage of spermatozoa with progressive motility, the percentage of spermatozoa with normal morphology, whether or not leukospermia was present, and the density of white blood cells (WBCs) in the semen sample when viewed at 100× magnification (absent/present at 1–9 WBCs/field/present at ≥10 WBCs/field) (Kimberling et al., 1986). The semen-collection method was designated as either standard collection (with the penis manually extended from the sheath) or preputial collection (with the penis retained in the preputial cavity). A veterinarian supervised the BSE and a veterinarian or animal-health technician performed all microscopic examinations of semen samples; however, the individual conducting each component of the BSE was not recorded in the medical record. We categorized ram breeds into the major breeds represented in the study population: Suffolk, Columbia, Hampshire, and Rambouillet. Black-faced rams that were reported by the owner to be a cross among black-face
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Table 1 Criteria used to classify rams for breeding potential, 14,667 BSEs of 11,804 Western USA rams, 2000–2007. Parameters
Categories of classification on BSE
Body condition Physical abnormalitiesa Epididymitis Ulcerative posthitis
Semen collected by electroejaculation Progressive sperm motility (%) Normal sperm morphology (%) Leukospermia: # white blood cells in semen/100× fieldb Brucella ovis indirect ELISA Scrotal circumference (cm): Rams aged 6–13 mo Rams aged ≥14 moc
Questionable potential breeder (fail)
Satisfactory potential breeder (pass)
Excellent potential breeder (pass)
2.4 Absent Absent Healed or no lesions
3 Absent Absent Healed or no lesions
Yes 10–30 30–50 0–9
Yes 31–69 51–79 0–9
Yes 70–100 ≥80 0–9
Positive
Indeterminate
Negative
Negative
<26 <29
27–29 30–32
30–35 33–39
≥36 ≥40
Unsatisfactory potential breeder (fail) 1 (emaciated), 5 (obese) Present Present Active and severe, and/or narrowing or distortion of preputial orifice; and/or internal form present No 0 ≤29 ≥10
Absent Absent Active and mild to moderate
a Physical problems included febrile illness (rectal temperature >104 ◦ F [40 ◦ C]), lameness, blindness, conformational defects, severe wear or absence of lower incisor teeth, prognathism, brachygnathism, abdominal or inguinal hernias, abnormal testicular shape or consistency (unilateral or bilateral), abnormalities in the spermatic cord (unilateral or bilateral), penile deformities, dermatitis, injury, or deformity of the scrotum, substandard scrotal circumference, and/or an injury or infectious disease (other than epididymitis or ulcerative posthitis) judged to preclude the ram from being capable of breeding ewes. Injuries and infectious diseases were treated by the attending veterinarian upon request by the owner. b Rams with preputial collection were not classified on the basis of WBCs in semen, owing to the likelihood of preputial contamination of the sample with WBCs. c Rams with dentition of 1-yr-olds for which the date of birth was not available were judged on the basis of 6- to 14-mo-old rams.
breeds, or for which breed data were unavailable at the time of BSE, were classified as the “Black Face” breed. Similarly, white-faced rams that were either reported by the owner to be a cross among white-face breeds, or for which breed data were unavailable at the time of BSE, were classified as the “White Face” breed. All other breeds were categorized as the “Other” breed. We determined the age of each ram by dental examination at the time of its first BSE, and we categorized rams as lambs (<1 yr old), yearlings (1 yr old), adult rams (2–5 yr), and aged (≥6 yr old) (similar to categorization in Wiemer and Ruttle, 1987).
2.2. Flock size determination We considered all rams owned by a given owner to be a flock. For flocks evaluated by BSE for >1 yr in the study, we expressed flock size as the average number of rams examined for that owner per year over the course of the study. We categorized flock size as small (≤20 rams), medium (21–50 rams), and large (≥51 rams). In this region, flocks with ≤20 rams are typically confined to small pastures or corrals during the breeding season, while flocks with ≥51 rams are usually managed on open rangelands (often state- or federally-owned forests or grasslands) wherein sheep from different flocks can intermingle. In extensive range settings, comingling of rams from different flocks facilitates transmission of B. ovis among rams (Bagley et al., 1985). Flocks with 21–50 rams might be kept in either fenced pastures or open rangelands.
2.3. Physical examination and semen collection When possible, feed was withheld from rams for 12–24 h prior to BSE (to facilitate manual extension of the penis from the sheath during semen collection). Rams were restrained for BSE in a standing position or in lateral recumbency on a modified calf table. A BCS of 1 (emaciated), 2 (thin), 3 (normal), 4 (well-conditioned) or 5 (obese) was assigned by palpation of the dorsal lumbar area (Scott, 2001). A cursory physical examination was conducted. The scrotum and prepuce were examined visually and palpated. The testes, epididymides, and spermatic cords were palpated. Scrotal circumference was measured with scrotal measuring tape (Scrotal Measuring Tape, Nasco International, Inc., Atkinson, WI, USA) and expressed in cm. No sedatives or anesthetics were administered for semen collection. A hand-held, battery-powered, rectally inserted electroejaculation probe (Lane Ram Ejaculator, Lane Manufacturing, Inc., Denver, CO, USA) was used as described by Kimberling and Parsons (2007). During all BSEs, manual extension of the penis was attempted prior to initiation of electroejaculation. If manual extension was successful, the penis was visually examined, then secured with cotton gauze with the glans directed into a warm, sterile, 16-ml polypropylene tube (Kimberling and Parsons, 2007). If samples of semen were collected with the penis extended, we designated those as “standard collections.” If extension of the penis could not be achieved, the semen sample was collected as it exited the preputial orifice; we designated such samples as “preputial collections.” Immediately after semen collection, a drop of semen was secured with a warm glass Pasteur pipette, placed
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on a warm, clean microscope slide, diluted in approximately 0.1 ml of warm Lactated Ringer’s solution, covered with a cover slip, and examined microscopically with a phase-contrast microscope at 100×. The proportion of spermatozoa that showed progressive motility were estimated and recorded as percent motility. Samples with leukospermia were categorized on the basis of the number of WBCs per visual field at 100× magnification as shown in Table 1 (Kimberling et al., 1986). However, we did not classify rams with preputial collections on the basis of leukospermia because these semen samples were commonly contaminated with WBCs and other debris from the preputial cavity. For evaluation of sperm morphology, a drop of semen and a drop of eosin–nigrosin stain (Live Dead Semen Stain, Jorgensen Laboratories, Loveland, CO, USA) were mixed together, smeared, allowed to dry, and examined under phase contrast with oil immersion at 1000×. One hundred cells were examined, and the proportion of morphologically normal spermatozoa was measured and recorded as % normal morphology (Kimberling and Parsons, 2007).
2.4. Serologic testing for antibodies to B. ovis We evaluated a non-random, owner-selected subset of rams in this study population to quantify serum antibodies to B. ovis using an iELISA (Gall et al., 2003). Serologic testing was conducted at the Colorado State University Veterinary Diagnostic Laboratory. Considering the complement fixation test as the reference method (WOAH, 2011), the sensitivity of iELISA was previously estimated to be 96.3% and the specificity was 99.6% (Gall et al., 2003). From 1 January 2000 to 9 May 2006, the antigen utilized for the iELISA was B. ovis Complement Fixation Antigen (National Veterinary Services Laboratory, Ames, IA); the spectrophotographic optical density (OD) was read at 450 nm. Because supplies of the previous antigen became depleted, from 16 May 2006 until the end of the study, the antigen utilized for the iELISA was changed to B. ovis ELISA Antigen, reagent code 9-ELA (National Veterinary Services Laboratory, Ames, IA); the spectrophotographic OD was read at 590 nm. Throughout the study period, the same 3 reference sera were used to standardize iELISA results: “high positive” sera (high serum titer of antibodies to B. ovis), “low positive” sera (lower serum titer), and negative (no detectable antibodies). These reference sera were obtained from rams with semen culture-confirmed status for B. ovis infection. Rams were classified on the as negative, indeterminate, or positive for B. ovis infection by comparison of ram sera OD values to OD cutoff values obtained from these reference sera. Cutoff values for classification of samples varied over the course of the study; however, classification was considered internally consistent over the study period because test results were standardized using these same reference sera and internal validation procedures revealed similar classification of banked ram serum samples as test protocols were changed (B. Hannafious, personal communication, 2012). For an estimate of true seroprevalence, we only considered rams with positive results on iELISA to be considered seropositive.
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The true seroprevalence was calculated using the formula of Rogan and Gladen (1978): True prevalence =
Apparent prevalence + Specificity − 1 Sensitivity + Specificity − 1
2.5. Classification of the ram Based on criteria derived from Sharkey et al. (2001), BSE data were compiled upon completion of the exam, and the ram was classified as displaying the characteristics of an excellent, satisfactory, questionable, or unsatisfactory potential breeder, as shown in Table 1. The lowest rating among all the BSE parameters determined the ram’s overall BSE rating (Kasimanickam et al., 2006). For analysis, classification of the ram as either an “excellent” and “satisfactory” potential breeder resulted in classification of that examination as a “passed BSE;” otherwise, it was classified as a “failed BSE.” We classified rams that were seropositive to B. ovis as “unsatisfactory potential breeders” and rams with an indeterminate result on B. ovis indirect ELISA as “questionable potential breeders.” In some instances, semen collection was performed on rams with physical abnormalities. In such cases, the ram was classified as a “questionable” or “unsatisfactory” potential breeder (depending on the severity of the physical problem and its potential for spontaneous resolution or resolution with treatment, as judged by the attending veterinarian). Similarly, semen collection was performed on most rams with ulcerative posthitis, and classification of the ram was based on the characteristics of the lesion as described in Table 1. 2.6. Determination of reason for failure Any given BSE could have been classified as a “failed BSE” on the basis of more than one parameter; therefore, to identify one abnormal parameter that resulted in failure on each BSE, we applied a hierarchical algorithm to each failed examination. The reasons for failure were categorized in the following order, based on the sequence of data acquisition during the BSE: (1) Owner cull: The ram possessed a phenotypic characteristic considered “unfavorable” by the owner, who decided to cull the animal. (2) Inflammatory causes: Enlargement, pain and/or induration in one or both epididymides or ≥10 WBCs visible per 100× microscopic field and/or positive or indeterminate on serologic testing for B. ovis. (3) Emaciation: BCS = 1. (4) Obesity: BCS = 5. (5) Physical abnormalities as defined in Table 1. (6) Ulcerative posthitis. (7) Failure to collect (electroejaculation was attempted but semen could not be obtained). (8) Substandard semen parameters: Semen was obtained by electroejaculation, but microscopic examination revealed substandard motility and/or morphologic abnormalities as defined in Table 1.
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2.7. Categorization of year and season for BSEs The total number of years during which each ram in the study was tested at least once was expressed as 1 yr or ≥2 yr. Each BSE was assigned to its corresponding season based on the month in which it was performed: January–June, July–September, and October–December. Michelson et al. (1981b) found that scrotal circumference and the proportion of morphologically normal sperm in rams were highest in the fall months and gradually decreased to the lowest values in February. Our intent was to capture this nadir of ram fertility in the middle of a 3-month period; however, because only four BSEs were performed during January–March, the first quarter of the year was combined with the second. 2.8. Data analysis Analyses were performed using commercially available statistical software (SAS version 9.2, Cary, NC, USA). We calculated descriptive statistics at the level of the individual ram for breed category, age category at the time of first examination, the total number of years tested, and whether ever tested positive for B. ovis infection. We used chi-square to analyze the relationship between flock size category and whether rams were examined in only 1 yr or ≥2 yr. Most inferential analyses performed at the level of the BSEs used pass/fail categorization as the primary outcome variable in logistic and Poisson regressions (PROC GENMOD, SAS version 9.2, Cary, NC); we estimated odds ratios (OR) with associated 95% confidence intervals (CIs) and risks of failure (respectively). We used GEE models to control for repeated measures within ram, nested within flock. Exposures (independent variables) in these analyses included age category, breed category, flock-size category, year of examination, season, and collection method. In univariable analyses, we found that the first four of these were all significantly (P < 0.001) associated with several different outcomes; therefore, we included these variables in all multivariable models to control for potential confounding associated with them. Factors that were potentially associated with BSE failure specifically attributed to substandard semen parameters were investigated within the subset of failed BSEs; these factors were body-condition category, ulcerative posthitis, and collection type. We also investigated (using multivariable logistic regression) whether breed or age categories for rams were associated semen collection method. We also included ram-age and flock-size categories in the model investigating inflammatory causes within the subset of failed BSEs. We used multivariable logistic regression to evaluate the effect of collection type on the odds of leukospermia being present, as well as to evaluate the effect of age and flock size on the odds of failure from inflammatory causes. Model diagnostics were used to evaluate the validity of all logistic and Poisson regression models. This included evaluations for collinearity of explanatory variables (by examining the variance inflation factor, simple correlations and cross-tabulations for explanatory variables), influential observations, and goodness-of-fit statistics (Dohoo
Table 2 Semen-collection method, classification, and reasons for failure, 14,667 BSEs of 11,804 Western USA Rams, 2000–2007. BSEs N Semen-collection method Standarda Preputialb
%
13,910 212
98.5c 1.5
Classification Excellent (pass) Satisfactory (pass) Questionable (fail) Unsatisfactory (fail)
3029 7386 1584 2668
20.7 50.4 10.8 18.1
Reason for failure Owner cull Inflammatory causes Emaciation Obesity Physical abnormalities Ulcerative posthitis Failure to collect Substandard semen parameters
6 852 605 48 658 196 24 1863
0.1 20.0 14.2 1.1 15.5 4.6 0.6 43.8
a
Semen collected with the penis manually extended from the sheath. Semen collected with the penis retained in the preputial cavity. c Expressed as a percentage of the 14,122 BSEs in which semen collection was performed. b
et al., 2009). Statistical significance of independent variables was evaluated using two-tailed Type III P-values obtained from the GEE models, with a critical alpha of 0.05. All first-order interaction terms were evaluated singly (in turn) for all independent variables included in final models.
3. Results A total of 14,674 BSEs from 11,811 rams met the eligibility criteria for inclusion in the study. Seven BSEs from 7 rams were excluded because the reason for classification could not be determined from the medical record (resulting in the inclusion in the study of 14,667 BSEs from 11,804 rams). Of the 11,804 rams in the 8-yr study, 9818 rams (83.2%) were examined in only 1 yr, 1355 rams (11.5%) were examined in only 2 yr, 445 rams (3.8%) were examined in only 3 yr, and 186 rams (1.5%) were examined in ≥4 yr. A larger proportion of rams from large flocks (1703/8638, 19.7%) were examined in ≥2 yr, compared to rams from medium-sized flocks (203/2036, 10.0%) and rams from small flocks (80/1130, 7.1%) (P < 0.001). Ninety-five rams underwent a second BSE on the same day as the first BSE in a given year. A total of 211 rams had ≥2 BSEs in a given year. We could not determine from the medical records the reason for these subsequent BSEs. Descriptive statistics for semen collection method, BSE classification, and reasons for BSE failure are shown in Table 2. In Table 3, we show the variables significantly associated with risk of failure on BSE (for any cause), as determined by multivariable Poisson regression. Semencollection method and season of BSE were not significant and were not retained. In Table 4, we show the variables associated with the odds of failure for inflammatory causes, as determined by multivariable logistic regression.
D.C. Van Metre et al. / Preventive Veterinary Medicine 105 (2012) 118–126 Table 3 Results of multivariable Poisson regression evaluating the risk of failure on BSE, 14,667 BSEs of 11,804 Western USA Rams, 2000–2007a . Variable b
Age (yr)
Flock size
Breed
Year
Category
% Failurec
(95% CI)
P-value
<1 1 2–5 ≥6
34.4a,d 28.7b 26.8c 36.7d
32.1, 36.8 26.9, 30.7 25.5, 28.1 32.5, 41.3
<0.001
Small (≤20) Medium (21–50) Large (≥51)
34.7a 29.7b 29.9b
31.9, 37.9 27.5, 32 28.6, 31.3
0.005
Suffolk Black Faced Columbia White Facee Rambouillet Hampshire Other
36.9a 34.9a,d 35.1a,f 30.8b,d,e,f 29.9b 28.7b,e 25.1c
34.9, 38.9 32.2, 37.8 31.7, 38.9 27.1, 35 27.7, 32.3 26.2, 31.3 22.7, 27.7
<0.001
2000 2001 2002 2003 2004 2005 2006 2007
37.6a 33.8b,c 33.6b,c 38.7a 31.4c 34.8a,b 25.6d 26.4d
35, 40.4 31.2, 36.5 30.9, 36.4 35.9, 41.7 29, 34.1 32.2, 37.6 23.5, 28 24.2, 28.9
<0.001
a Correlation in responses associated with inclusion of >1 BSE for some rams was controlled using generalized estimating equations. b Ages were missing for 36/14,667 (0.2%) of BSEs. c For each variable, estimates of risk of failure with different letters differ at a significance level of ≤5%. d Black-faced breeds other than Suffolk or Hampshire. e White-faced breeds other than Columbia or Rambouillet.
We determined that there was no difference among breed categories or in ram-age category in the proportion of semen collections that were standard collections (P = 0.07 and 0.76, respectively). Preputial collection, however, was associated with greater odds for leukospermia to be detected on microscopic examination of semen, relative to samples obtained by standard collection (OR 4.1; 95% CI 2.9, 5.9; P < 0.001). In Table 5, we show the association between semen collection method, ulcerative posthitis,
Table 4 Multivariable logistic regression evaluating the odds of failure for inflammatory causesa , 4252 BSEs classified as “failed” of 3989 Western USA Rams, 2000–2007b , c . Variable
Category
OR (95% CI)
P-value
Age (yr)
<1 1 2–5 ≥6
Reference 1.6 (1.2, 2.1) 1.3 (1.0, 1.7) 0.8 (0.5, 1.2)
<0.001
Flock size
Small (≤20) Medium (21–50) Large (≥51)
Reference 1.6 (1.1, 2.3) 1.4 (1.0, 1.9)
0.02
a Failure from inflammatory causes includes BSEs failed due to the presence of palpable epididymitis, ≥10 WBCs per 100× field, and/or positive or indeterminate results for B. ovis serology. b Breed of ram and year of examination were also controlled as fixed effects in this regression model. c Correlation in responses associated with inclusion of >1 BSE for some rams was controlled using generalized estimating equations. ˇ0 = −1.48; 95% CI: −2.04 to −0.91.
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Table 5 Multivariable Poisson regression evaluating the risk of failure on BSE due to substandard semen motility or morphology, 14,667 BSEs of 11,804 Western USA rams, 2000–2007a , b . Variable
Category
% Failurec
(95% CI)
P-value
Collection method
Standard Preputial
25.6 27.9
22.0, 29.0 21.9, 33.5
0.34
Ulcerative posthitisd
Present Absent
28.8 24.6
22.8, 34.4 21.0, 28.1
0.09
Body condition score
1 2 3 4 5
31.6a 27.1b 24.0c 24.9b,c 25.9a,b,c
26.8, 36.2 23.1, 31.0 20.1, 27.6 20.8, 28.8 14.2, 36.1
<0.001
a Breed and age categories of ram, flock size category, and year of examination were also controlled as fixed effects in this regression model. b Correlation in responses associated with inclusion of >1 BSE for some rams was controlled using generalized estimating equations. c For each variable, estimates of risk of failure with different letters differ at a significance level of ≤5%. d Semen was collected in 250/258 BSEs (96.9%) performed on rams with ulcerative posthitis.
and BCS and the risk of failure on BSE due to substandard semen parameters. A total of 2317 rams were tested at least once by iELISA for antibodies to B. ovis. Of these, 233 were positive; the Rogan–Gladen adjusted true prevalence in this subset of rams was 10.0% (Rogan and Gladen, 1978). Importantly, 125 of 233 rams (53.6%) that were seropositive for B. ovis were otherwise normal on BSE, with no abnormalities detected in these rams on physical examination or semen examination. 4. Discussion The most important findings of this study were measurement of the association between outcome of BSE (pass/fail) and variables relevant to ram BSE technique (semen-collection method), ram characteristics (BCS and ulcerative posthitis), and flock size. These variables have been deemed relevant to ovine BSE in the published literature but have not previously been objectively evaluated. To our knowledge, this is the largest study of BSEs on rams in the veterinary literature. The primary limitation of this study is that the study population is not necessarily representative of all rams in the Western USA. The state in which the owner resided was not analyzed because animals in many flocks traveled across state lines during multiple years of the study. Further, the persons who performed each component of the BSE were not listed in the medical records. The skill and experience of the individuals conducting the various procedures included in ovine BSE likely varied, as did skill and experience within a given individual across different years of the study; this variation in examiners (and also in climate, feed quality, etc.) could have affected BSE outcomes across years. Further, those performing semen microscopy were not blinded to the physical properties of the rams; it is possible that this lack of blinding could have influenced classification relative to semen characteristics.
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This study is important because it is the first to evaluate the association between semen collection method and the outcome of ram BSE objectively. Semen collection without manual extrusion of the penis from the preputial cavity has been described (Hindson and Winter, 2002; Mobini et al., 2002); this type of collection is often practiced because spontaneous penile extension during electroejaculation does not consistently occur in rams (Ball, 1976). Preputial contents can contain endogenous debris such sloughed epithelial cells and white blood cells, as well as exogenous debris (e.g. plant material). Manual extrusion of the penis from the sheath prior to semen collection has been recommended by others, in order to minimize contamination of the sample with preputial contents (Kimberling and Parsons, 2007; Scott, 2007; Parkinson, 2009). This recommendation was substantiated by our data: preputial collection was associated with greater odds of leukospermia. Accurate interpretation of leukospermia is essential during BSE of the ram. B. ovis could be isolated from ram semen samples that showed leukospermia and otherwise normal sperm motility and morphology (Kimberling et al., 1986). In addition, leukospermia is one of the earliest changes in the BSE following experimental B. ovis infection of rams (Webb et al., 1980). However, in our study, the semen collection method was not associated with the proportion of BSEs that failed for substandard semen parameters; this implies that collection method does not influence semen motility and morphology. Manual extension of the ram’s penis could not be performed for a relatively small proportion of BSEs (1.5%) in this study. Manual extension of the penis can be difficult to accomplish – particularly in larger rams or rams that have been recently fed – owing to pressure from the full rumen on the ventral body wall and penis. Notably, however, neither breed nor the age of the ram were associated with the semen collection method. Ulcerative posthitis is a bacterial infection of the prepuce and skin of the sheath of male small ruminants. It results from proliferation of Corynebacterium renale (a normal bacterial inhabitant of the prepuce) in response to the increased concentration of urea in the urine (which, in turn, results from ingestion of excessive amounts of protein in the diet) (Brook et al., 1966). The presence of ulcerative posthitis had no detectable effect on semen parameters. However, ulcerative posthitis might influence the ram’s willingness to breed (libido) because of pain when the lesion contacts the ewe during breeding. We therefore recommend that rams with such lesions be categorized as “questionable” or “unsatisfactory” potential breeders and treated with dietary protein modification—specifically, reduction in dietary protein intake (Brook et al., 1966) and antimicrobial therapy (with re-evaluation by a veterinarian prior to breeding). Although others have recommended rams to be in good body condition prior to the onset of the breeding season (Ott and Memnon, 1980; Kimberling and Parsons, 2007), the influence of extremes of BCS on semen parameters has not been described previously. In this study, emaciation was associated with a higher risk of failure because of substandard semen parameters. Thin rams might have had diseases that adversely affected semen parameters,
or there could be a direct effect between thin body condition and semen quality. Whether or not emaciated rams experience improvements in semen parameters with improvement in BCS remains to be determined. In contrast, no detectable difference in risk of failure from substandard semen parameters could be detected for obese rams even though obesity has been hypothesized to affect ram fertility adversely (Wise, 1983). However, we had relatively few obese rams (N = 48) and therefore perhaps lacked sufficient power to detect an adverse effect of obesity on semen parameters. Further, it is plausible that obesity in rams negatively affects sperm quality, libido, and/or thermoregulatory capacity during the breeding season, which might not be detected during BSE. The estimated risk of failure for BSEs conducted on rams from small flocks were higher than those of BSEs conducted on rams from large- and medium-sized flocks. However, failed BSEs that were performed on rams from large- and medium-sized flocks were at higher odds to be categorized as failure from inflammatory causes than were failed BSEs performed on rams from small flocks. We speculate that this difference in odds of failure due to inflammatory causes is related to the difficulty in establishing good ram flock biosecurity practices under the open-range conditions common to larger sheep flocks in the Western USA, as has been previously proposed by Bagley et al. (1985). Further, when purchasing new rams, operators from smaller flocks might exercise different purchasing practices or use different criteria to select source flocks for such rams relative to owners of larger flocks. We found that 1-yr-old and 2- to 5-yr-old rams had higher odds of failure from inflammatory causes relative to ram lambs and aged rams. Although B. ovis infection has been documented in virgin ram lambs (Bulgin, 1990b), it is considered to be a more common isolate from mature rams (Bulgin and Anderson, 1983), which might partly explain in part the increased risk of failure from inflammatory causes in the 1-yr-old and 2- to 5-yr-old rams of our study. Further, rams of these two age groups might be more sexually active during the breeding season than ram lambs or aged rams, thereby increasing their risk of infectious diseases involving venereal transmission. Ram lambs and aged rams showed the highest estimated risks of failure. Ram lambs could have been sexually immature at the time of BSE, whereas aged rams could have greater risk for chronic, debilitating diseases, physical injuries, dental disease, or age-related reduction in fertility. In a previous study, relative to younger rams, a significantly greater proportion of rams >6 yr old had testicular lesions; these older rams also showed significantly fewer motile sperm cells and significantly greater morphologically abnormal sperm than younger rams (Wiemer and Ruttle, 1987). The estimated risk of failure of BSE (from any cause) was lowest for the breeds included in the “other” category, followed by the Hampshire and Rambouillet breeds. Breedcategory differences in maturation rate and susceptibility to disease (as well as differences in flock management) might have influenced the disparity among breed categories in BSE failure in our study. In contrast, Ley et al. (1990) failed to detect a difference among 1 yr-old Dorset,
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Hampshire, and Suffolk breeds in failure percentage on a single BSE. We evaluated a subset of rams in this study population by testing for antibodies to B. ovis. Most rams that were seropositive were otherwise normal on BSE—findings that corroborate those of previous studies wherein infection with B. ovis was documented commonly to be subclinical (Cameron, 1976; Webb et al., 1980; Bagley et al., 1984; Kimberling et al., 1986; Kott et al., 1988; Bulgin, 1990a; Dargatz et al., 1990). In light of these findings, serologic testing for B. ovis should continue to be considered as an integral component of the BSE in rams. However, only a small subset of rams in this study was tested for B. ovis infection, and these animals were selected by the owners. Therefore, our serologic data are heavily biased and should not be interpreted as representative of the true population of rams in the Western USA. The proportion of BSEs in the present study that were categorized as passed (71.0%) is similar to that (73.8%) reported by Kimberling and Butler (1986) in a different Colorado study and to that (70.7%) reported by Wiemer and Ruttle (1987) in a New Mexico, USA study. The pass percentage for BSEs in our study is also similar to that reported for rams in Virginia, USA (71.7%) by Ley et al. (1990). The most common reason for failure on BSE in our study was substandard semen parameters, which is similar to the findings of Ley et al. (1990). That those previous studies were performed >20 years ago suggests minimal change in the proportion of rams in the USA that are classified as passed on BSE over that time period, as well as minimal change in the primary reason for failure on BSE.
5. Conclusions Collection of semen with the penis retained in the preputial cavity was associated with higher odds of leukocytes in the semen sample relative to semen collection with the penis extended from the sheath. Because leukospermia can indicate contagious epididymitis caused by B. ovis, semen collection with the penis extended is recommended to prevent spurious contamination of the semen sample with leukocytes from the prepuce. We found that emaciation in rams was associated with an increased risk of BSE failure from substandard semen parameters, but ulcerative posthitis was not. Rams from medium- and large-sized flocks showed the highest odds of BSE failure from inflammatory causes–suggesting potential disparate risk for contagious venereal diseases among flocks of different sizes. As shown in previous studies, rams that are seropositive to B. ovis can be otherwise normal on BSE; therefore, to detect subclinically infected rams, serologic testing for B. ovis should be performed in conjunction with BSE.
Conflict of interest statement The authors of this manuscript have no conflicts of interest to report that are relevant to this work.
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