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ARS, USDA U.S. Sheep Experiment Station, Dubois, ID 83423
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Libido • Luteinizing Hormone • Naloxone • Rams • Testosterone
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Hulet et al., 1962; Price et al., 1987). The high breeding capacity of rams seems critical to efficient lamb production (Perkins et al., 1992b). Reproductive inefficiency of low sexually performing rams can generally be overcome by using multiple sires but at an additional expense because of extra ram costs. Long term, it may not be financially feasible to use rams with low sexual performance, especially if they are used in single-sire matings or for AI because libido scores are moderately heritable (0.22 ± 0.04; Snowder et al., 2002). The moderate heritability estimate suggests that sexual performance is in part genetically controlled and supports the report that ram sexual performance can be increased in one generation with high-libido rams (Bench et al., 2001).
Ram sexual performance can be monitored during a timed series of serving capacity tests that consist of observing sexual behaviors for each ram with ewes in estrus (Kilgour and Whale, 1980). However, serving capacity tests are time consuming and labor intensive, often making them impractical. Thus, a blood test based on LH and testosterone after naloxone injection was developed to discriminate between sexually active and inactive mature rams (Perkins et al., 2001). Naloxone (an opioid antagonist) acts by blocking the inhibitory effects of endogenous opioids on LHRH release (Schanbacher, 1985; Ebling et al., 1987). This stimulates LH release and results in increased testosterone concentrations. The youngest age at which this test can be effectively used to classify rams is unknown. It would be advantageous to be able to identify sexual behavior of rams before their first breeding season. Therefore, the objective of this study was to determine whether the naloxone test could be used to detect differences in sexual performance in early postpubertal rams in the breeding season and again at 70 wk of age before onset of the breeding season.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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All rams were kept at the U.S. Sheep Experiment Station, Dubois, ID (lat 44° 14' N, long 112° 11' W). Rams were offered long-stem alfalfa hay (RFV 165; relative feed value [RFV] = [% digestible DM x % DM intake]/1.29 = RFV 165) at 2.2% BW (DM basis) daily, housed as a group in an outside paddock, and given free access to water and trace mineral salt (Redmond T.M. [2,000 ppm of Mn, 3,500 ppm of Zn, 600 ppm of Fe, 300 ppm of Cu, 80 ppm of I, and 50 ppm of Co]; Redmond Minerals, Inc., Redmond, UT) from September to April each year. For the remainder of the year, rams are maintained on native sagebrush grasslands and given free access to water and trace mineral salt (Redmond NTM [5 ppm of Mn, 3 ppm of Zn, 300 ppm of Fe, 3 ppm of Cu, and 10 ppm of I]; Redmond Minerals).
Rams of white-faced crossbreds (n = 38), Polypay (n = 16), and Targhee (n = 49) breeding were treated with 0.75 mg of naloxone/kg of BW (Sigma-Aldrich Corp., St. Louis, MO) at 29 ± 0.1 wk and again at 70 ± 0.1 wk of age. This dose of naloxone was selected because doses of 0.75 and 1.50 mg/kg of BW have been equally effective at opioid disinhibition resulting in LH release and testosterone increase during the breeding season (Perkins et al., 2001). The total number of rams used in this study was based on the variability and incidence of sexually inactive rams from our population of 18-mo-old rams and required the use of several breed types. Rams were identified as either sexually active (n = 56 total [22 white-faced crossbreds, 8 Polypay, and 26 Targhee]) or sexually inactive (n = 47 total [16 white-faced crossbreds, 8 Polypay, and 23 Targhee]) using eight 30-min serving capacity tests (Stellflug and Berardinelli, 2002). Briefly, the serving capacity tests entailed exposing each ram to three unrestrained, estrual ewes in a 2.4 x 2.4-m pen and recording sexual behaviors (mounts and ejaculations). Rams were classified as sexually active if they mounted and ejaculated during the serving capacity tests (the number of ejaculations per 30-min period > 0.0) and as sexually inactive if they failed to mount and ejaculate during all serving capacity tests.
Serving capacity tests were initiated within 3 mo of the naloxone challenge at 70 wk of age and were completed within 1 mo. The serving capacity tests were conducted at 70 wk of age because tests are a better predictor of sexual performance, especially for poor performing rams, than results of serving capacity tests with ram lambs that are the most difficult to interpret (Price, 1987). After serving capacity tests, all rams were given at least one preference test (Stellflug and Berardinelli, 2002) that was similar to one (Price et al., 1988) used to determine whether ram sexual orientation was female- or male-oriented. Ten of the sexually inactive rams, as classed by the serving capacity tests, were exclusively male-oriented, and data were removed from the data set (three white-faced crossbred, three Polypay, and four Targhee) because the test cannot be used to discriminate between sexually active female-oriented and male-oriented rams (Perkins et al., 2001). The data were removed for an additional three rams (one sexually inactive Polypay and two Targhee, one sexually active and one sexually inactive) because of missing preference or hormone data at 70 wk of age.
Naloxone Administration and Blood and Semen Collection
Rams were treated with naloxone on November 9 or 10 when they were approximately 29 wk of age. On November 12, semen was collected from all of the 29-wk-old rams with the aid of an electroejaculator (2.5 in with ram probe; Ideal Instruments Inc., Chicago, IL). The rams were treated again with naloxone on August 23 or 24 when they were approximately 70 wk of age. Four blood samples were taken via indwelling jugular catheters (16 gauge, 1.7 x 133 mm Angiocath, Becton Dickinson, Sandy, UT) at 15-min intervals before treatment. Naloxone was administered through the catheter immediately after the fourth blood sample. Blood samples were collected at 15-min intervals for 2 h after the naloxone treatment. Blood was collected with 6-mL syringes, poured into 5-mL glass tubes containing 2 drops of heparin (50 USP units of H3393 porcine heparin/mL physiological saline; Sigma). The plasma was harvested within 1 to 2 h of collection and stored at -20°C.
Hormone Assays
Plasma LH concentration was quantified using a validated RIA procedure (Perkins et al., 1992a) that included anti-oLH AFP-192279 and oLH AFP-8614B for iodination and standards, which were obtained through the National Hormone Pituitary Program, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. The intra- and interassay CV were 8.2 and 17.7%, respectively, with a sensitivity of 0.25 ng/mL.
Plasma concentrations of testosterone were quantified with RIA kits (Diagnostic Products Corp., Los Angeles, CA) using I125 as tracer. Cross-reactivity of the antibody was 3.3% with 5 
-dihydrostestosterone. Intra- and interassay CV were 11.5 and 10.9%, respectively, with a sensitivity of 0.2 ng/mL.
Statistical Analysis
Plasma LH and testosterone concentrations were analyzed using mixed-model procedures of SAS (SAS Inst., Inc., Cary, NC) for repeated measures. The LH and testosterone data were evaluated for nine time periods, including the time immediately before injection of naloxone and the eight time periods after the naloxone treatment at 29 wk and at 70 wk in separate analyses for each hormone and age. The corresponding average values before naloxone were used as a covariate in the analyses. The main plot included breed-type, sexual activity (sexually active and inactive), and breed-type x sexual activity. The subplot included time, breed-type x time, sexual activity x time, breed-type x sexual activity x time. Breed-type, sexual activity, and breed-type x sexual activity were tested by rams nested within breed-type and sexual activity. The subplot was tested with the residual. Degrees of freedom were calculated using the Kenward-Roger procedure (Kenward and Roger, 1997). The LH values were transformed to the natural logarithm, and testosterone values were transformed to the square root to normalize the variances among rams for the fixed effects. The LH and testosterone least squares means and confidence intervals were changed back to original units after analysis. A standard error for the original units was estimated using the 95% confidence intervals; this is an approximation (approximate SEM) and not appropriate for estimating confidence intervals for means that would asymmetrically match the data distribution. If the main effects or their interactions were significant (P < 0.05), Fisher’s protected least significant difference was used as a post-analysis test to detect differences between individual means.
Hormone responses to naloxone between sexually active and inactive rams from 29- and 70-wk-old rams were evaluated separately using Proc Logistic procedures (Stokes et al., 2000; Johnson, 1998) for modeling the probability that rams were sexually active. The logistic regression model was ram class = LH Sample Time (LHST) 1 to LHST12 and testosterone Sample Time (TST) 1 to TST12/selection = forward slentry (significance level for entry of variables into the model) = 0.1, hierarchy = single. Sexual activity of rams was the response variable, and LH and testosterone concentrations were the explanatory variables.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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All of the rams had live sperm with normal morphology and motility within 2 d after the naloxone treatment.
The covariate for LH before naloxone injection accounted for considerable variability (P < 0.001) and improved the precision of the test for fixed effects after naloxone. A breed-type x sexual activity x time interaction for LH was observed after naloxone treatment (P < 0.05; Figure 1). The interaction seemed to be related to the sexually active Polypay rams having the greatest LH concentrations at 30 min and the sexually inactive Polypay rams having the greatest LH concentrations at 15 min after naloxone treatment (P < 0.05). The sexually active Targhee rams only differed in LH over time with values at 15 to 75 min being greater (P < 0.04) than values at 105 and 120 min after naloxone. The sexually inactive Targhee rams responded to naloxone with increased LH at 30 min (P < 0.05). The sexually active crossbred rams had increased LH at 15 min (P < 0.05), and LH remained increased for 105 min after naloxone. The sexually inactive crossbred rams had lower (P < 0.05) LH concentrations at 90 to 105 min compared with concentrations at 15 to 45 min after naloxone.
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). Breed-type x sexual activity x time interaction was not significant, and testosterone did not differ for sexual activity after the naloxone treatment in the 29-wk-old rams.
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The covariate for LH before naloxone accounted for variability (P < 0.001) and improved the precision of the test for fixed effects after naloxone injection. A breed-type x time interaction for LH concentrations was observed (P < 0.01), but the breed-type x sexual activity x time interaction was not significant. The breed type x time interaction seemed to be related to the lower LH response (P < 0.01) in Targhee rams that peaked at 30 min, compared with the LH increase (P < 0.01) in Polypay and crossbred rams after naloxone (Figure 3). Concentrations of LH did not differ by sexual activity after the naloxone treatment in the 70-wk-old rams.
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). The sexual activity x time interaction seemed to be related to testosterone concentrations being similar until 45 min, with testosterone increasing at 30 min (P < 0.02) in both sexually active and inactive rams. However, at 45 min after naloxone treatment, testosterone concentrations started diverging between the sexually active and inactive rams, with testosterone increasing to greater concentrations in the sexually inactive rams. Testosterone peaked at 75 to 90 min and remained increased (P < 0.05) above pretreatment concentrations for 120 min after naloxone treatment in sexually active and inactive rams.
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Logistic regression was used to model the probability of correctly identifying sexually active rams. The explanatory variable meeting the criteria with a P < 0.10 to enter the model was testosterone at Sample Time 8 (TST8) in August at 70 wk of age, and no response variables were entered for 29-wk-old rams. As an example of the output produced by the model, information from the classification table is given for TST8 analyses in August (Table 1). A sensitivity of 100% indicates that all of the sexually active rams would be classified correctly, and a specificity of 100% indicates that all of the sexually inactive rams would be classified correctly. The test in August was more accurate for identifying sexually active rams than it was for sexually inactive rams. The other logistic analyses for August were similar to the example shown in Table 1 with sensitivity being high (70 to 90%) and specificity being low (0 to 30%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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), the results of the present study indicate that the naloxone challenge test cannot be used to discriminate between sexually active and inactive rams at 29 wk of age during the breeding season. Furthermore, the overall accuracy of predicting sexually active and inactive rams was 53% at 70 wk of age before the breeding season in late August. Separately, 69% of sexually active rams were correctly identified compared with 29% of the sexually inactive rams at 70 wk of age. The present results support a recent report that the naloxone challenge cannot be used to discriminate between sexually active and inactive rams before the fall breeding season in July and August (Stellflug, 2002).
In the July and August trial (Stellflug, 2002), seasonality played a role in rams responding to naloxone with increased testosterone in August compared with the reduced but still significant testosterone response in July. Seasonality is known to influence the hypothalamic-pituitary-gonadal axis (Haynes and Schanbacher, 1983; Schanbacher, 1985). During the seasonal cycle, opioid inhibition of LH secretion is thought to change in rams, resulting in increased frequency of LH pulses as rams approach the breeding season (Ebling, 1987). This finding supports the inference that before the fall breeding season, the hypothalamic-pituitary-gonadal axis is not fully developed, and thus, is possibly less responsive to naloxone disinhibition. This rationale may also explain the results in 70-wk-old rams in late August in this study, which was conducted before the results of our previous report were available (Stellflug, 2002). In addition, a similar rationale may relate to early postpubertal rams and lack of full development of the hypothalamic-pituitary-gonadal axis. This may also explain the minimal response to naloxone at 29 wk of age in November, even though at this stage of their development, their LH secretion is insensitive to photoperiod (Herbosa et al., 1995).
The minimal response in 29-wk-old rams may be related to maturity of rams. The present study infers that breed-type of ram at 29 wk of age is an important factor influencing the response to the naloxone challenge. At 29 wk of age, Polypay rams had a greater testosterone response after naloxone than did Targhee and crossbred rams. In addition, at 70 wk of age, Polypay and crossbred rams were comparable with each other in LH response to naloxone treatment and had a greater response than Targhee rams. One explanation for these results is that Polypay are an early-maturing, more prolific breed compared with the slower-maturing, less prolific Targhee that was documented for ewes in these breeds (Stellflug et al., 1993). Similarly, increased LH secretion was reported for Romanov rams (the more prolific breed) compared with Ile de France (Lafortune et al., 1984). The endocrine system becomes more developed as an animal matures. For instance, testosterone gradually increases in rams until 21 mo of age (Haynes and Schanbacher, 1983). Age-related increases in testosterone may account for the greater libido and dominance often observed in older rams (Illius et al., 1976). Even though the hormone responses to naloxone in the 29- and 70-wk-old rams were not compared directly, perhaps the greater increases for LH and testosterone in the 70-wk-old rams compared with the 29-wk-old rams are also consistent with the endocrine system becoming more fully developed as the animal matures.
At 70 wk of age, testosterone concentrations increased within 30 to 45 min after naloxone treatment and remained increased at 120 min in the sexually active and inactive rams in August before the breeding season. However, the testosterone response after naloxone administration at 70 wk of age was greater in the sexually inactive vs. active rams, supporting the most recent study (Stellflug, 2002) but not an earlier study (Fitzgerald and Perkins, 1994; Perkins et al., 2001). During the breeding season, testosterone concentrations were greater for sexually active vs. inactive mature rams at 45 and 60 min after naloxone administration (Perkins et al., 2001). One explanation was given for an increased testosterone concentration in the previous study (Stellflug, 2002) for the sexually inactive rams responding to naloxone with greater testosterone concentrations than sexually active rams. A case was made that increased testosterone in sexually active rams before naloxone treatment could have suppressed the ram’s ability to respond to naloxone because of an increased proportion of opioid receptors being occupied by endogenous opioids. Along this same line of thought, the sexually inactive rams with lower testosterone before the naloxone treatment had a greater testosterone response. However, in the present study, the difference in testosterone before the naloxone challenge between sexually active and inactive rams did not exist, and sexually inactive rams again had greater testosterone after naloxone than did sexually active rams. In addition, naloxone is a pure antagonist, so it is thought to compete very effectively for opioid receptor binding sites at the doses administered. Perhaps a more plausible explanation for an increased testosterone response in sexually inactive rams vs. sexually active rams is that the effects of opioidergic systems to decrease LH pulse frequency are reduced or absent during the sexually inactive season in rams (Lincoln et al., 1987). The disinhibition of naloxone on the opioidergic system is also influenced by season in stallions; however, LH release is induced outside of, but not during, the breeding season (Aurich et al., 1994).
State of pubertal development may also influence the disinhibition of naloxone on the opioidergic system because the breed-type of rams seems to have had an influence in the present study. Breed-type is expected to influence hormone response to naloxone in the young rams because of differences in rate of maturity among the breeds.
In conclusion, the results of the naloxone challenge test were not adequate to discriminate between sexually active and inactive rams at 29 wk of age during the breeding season in November or at 70 wk of age in late August just before the fall breeding season.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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2 Correspondence: HC 62, Box 2010 (phone: 208-374-5306; fax: 208-374-5528; E-mail: jstellflug{at}pw.ars.usda.gov).
Received for publication December 30, 2002. Accepted for publication April 22, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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This article has been cited by other articles:
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  J. N. Stellflug, A. Perkins, and V. A. LaVoie Testosterone and luteinizing hormone responses to naloxone help predict sexual performance in rams J Anim Sci, November 1, 2004; 82(11): 3380 - 3387. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
-], 4 SIA [-
-]), Targhee (n = 25; SA [-
-], 18; SIA [-•-]), and white-faced Crossbred (n = 22; SA [-
-], 13; SIA [-
-]) rams. A breed-type x sexual activity x time interaction was observed (P < 0.05), but LH did not differ by SA and SIA. The estimate of variability is depicted by estimated standard errors of the least squares means because of data transformation.
