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a INRA, Unité Gonadotropines, URA CNRS 1291, Station PRMD, 37380 Nouzilly, France
b Sanofi Santé Nutrition Animale, La Ballastière, BP 126, 33501 Libourne Cedex, France
c INRA, Laboratoire de Génétique Biochimique et de Cytogénétique, Département de Génétique Animale, 78352 Jouy en Josas, France
d INRA, Station PII, 37380 Nouzilly, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Equine CG, a member of the family of heterodimeric glycoprotein hormones, is synthesized and secreted by trophoblastic cells between Days 40 and 130 of gestation in the mare [2]. It is composed of two dissimilar, noncovalently associated 
and ß subunits [3], both of which are needed for full biological activity. The molecular mass of eCG is estimated at about 45 kDa, 15 kDa for the subunit and 30 kDa for the ß subunit [4]. A unique feature of this hormone is that eCG displays both LH- and FSH-like activities when used in species other than the horse [4–6]. Moreover, with a carbohydrate content of 41% [7], eCG is the most heavily glycosylated of all mammalian glycoprotein hormones, with terminal sialic acid residues accounting for its prolonged plasma half-life (up to 60 h in ewes [8] and 6 days in horses [9]). The dual activity of eCG, its long half-life, and its availability in large quantities have led to its widespread use as a convenient exogenous hormone in treatments for the stimulation of ovarian follicular growth [10] in various species, including the goat.
Because of its molecular size, its high level of glycosylation, and its heterologous origin, eCG is potentially immunogenic. Immunomapping studies of eCG with monoclonal antibodies have localized a large antigenic site, which includes the site of interaction of eCG with the LH and FSH receptors [11, 12].
In practice, repeated use of eCG treatment for induction of ovulation is generally followed by decreasing fertility in goats. Baril et al. [13] reported that the percentage of goats exhibiting delayed estrus increased proportionally with the number of eCG treatments. Moreover, goats that came into estrus only 30 h or more after the end of the progestogen treatment exhibited a significantly lower kidding rate after AI at a fixed time [13]. Such negative effects were even more dramatic after a second eCG treatment in the same breeding season [14]. Decreased fertility rates after repeated eCG treatments were assumed to be associated with the presence of circulating anti-eCG antibodies in the plasma of treated goats, on the basis of 125I-eCG binding measured just before and 25 days after eCG injection [14, 15]. A significant decrease in fertility was observed when 125I-eCG binding before treatment was higher than 10% under the conditions used [15]. However, it has not been confirmed that a humoral immune response did occur after eCG treatment in goats. Nevertheless, ovarian refractoriness to subsequent eCG stimulation has been associated with the presence of eCG-binding immunoglobulins in other species such as the rhesus monkey [16], cat [17, 18], cow [19], and sheep [20]. Only Swanson et al. [18] reported kinetics of the humoral reaction, which demonstrated variable responses in cats with repeated eCG-hCG treatments.
In the present report, we demonstrate the presence of anti-eCG antibodies in plasma of treated goats and show the kinetics of their appearance with a quantitative ELISA. The variability of anti-eCG humoral immune responses in goats corresponded with their major histocompatibility complex (MHC)-DRB genotypes and their subsequent fertility.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies of preimmune and immune plasmas from nontreated (n = 3) or eCG-treated goats (n = 3) were affinity-purified on HiTrap G protein columns (Pharmacia, Uppsala, Sweden) exhibiting a high binding capacity for goat immunoglobulins G (IgG) [21]. Briefly, 1.5 ml of plasma previously dialyzed against 20 mM sodium-phosphate pH 7 was loaded onto a HiTrap G protein column, and the wash-through (containing the IgA and IgM fractions) was collected. Bound IgG was eluted using 0.1 M glycine buffer pH 2.7. The eluted fractions were collected and immediately neutralized with 1 M Tris pH 9. The fractions were then pooled, dialyzed overnight at 4°C against PBS pH 7.4, concentrated to 8 mg/ml using a Minicon B15 ultrafiltration cartridge (Amicon Inc., Beverly, MA), aliquoted in 50% glycerol, and stored at -20°C.
Production of an Anti-eCG Polyclonal Antibody Standard for ELISA Calibration
Two castrated Ile-de-France rams received injections at multiple intradermal (i.d.) sites of 1 mg eCG (6500 IU/mg, purified in our lab) prepared in 1 ml complete Freund's adjuvant. Four monthly booster i.d. injections were administrated using 1 mg eCG (6500 IU/mg) followed by two other i.d. injections of 1 mg highly purified eCG (10 000 IU/mg) every 2 mo in incomplete Freund's adjuvant. Two weeks after each of the three last injections, sera were collected, pooled, and stored at -20°C. The polyclonal anti-eCG antibody was affinity-purified on a HiTrap G protein column as described above, and this antibody was concentrated to 8 mg/ml as determined by the method of Bradford [22]. The polyclonal anti-eCG antibody was then used for ELISA calibration.
Measurement of Anti-eCG Antibody Concentrations by a Quantitative ELISA
Plasma anti-eCG antibodies were quantified by ELISA. Microtiter plates (CML, Angers, France) were coated with 250 ng of a commercial preparation of eCG (Syncro part, batch 13054A1; Sanofi Santé Nutrition Animale [SSNA], Libourne, France) diluted in 100 µl PBS pH 7.4 and were incubated overnight at room temperature. After five washings with PBS containing 0.1% Tween 20 (PBS Tw), the plates were incubated for 45 min at 37°C with 1% BSA (Organon, Boxtel, Holland) prepared in PBS Tw to block unsaturated binding sites. After the plates were emptied, plasma samples and standards (dilutions of anti-eCG polyclonal antibody) were distributed (100 µl/well) in duplicate and incubated for 90 min at 37°C. Plasma samples were initially diluted from 1:50 to 1:200 in PBS Tw containing 5% normal rabbit serum (PBS Tw 5% R). The use of plasma from the same species as the enzyme-labeled antibody prevents goat/rabbit IgG interspecies cross-reaction. Standard anti-eCG polyclonal IgG was serially diluted from 1000 to 7.5 ng/ml in PBS Tw 5% R supplemented with 0.5–2% of negative goat plasma free of anti-eCG antibodies. This supplementation was conducted according to the dilution range of plasma samples (from 1:50 to 1:200) to mimic any "plasma effect" in the assay. After washing five times, a peroxidase-conjugated rabbit anti-goat IgG polyclonal antibody (Jackson Lab., West Grove, PA) was added (final dilution 1:5000, 100 µl/well) and incubated for 1 h at 37°C. The peroxidase activity was detected with 100 µl/well of a substrate mixture containing 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid [ABTS]; Sigma, St. Louis, MO) at 0.04 M in 0.05 M citrate phosphate buffer pH 4.0 and 0.01% H2O2. After incubation for 30 min at room temperature, the absorbance at 405 nm was measured with an automated microtiter plate reader Multiskan MK II (Labsystems, Helsinki, Finland).
The eCG-specific IgG antibody concentration present in plasma was calculated from the linear part of the standard curve. Correction was made for plasma dilution, and antibody concentration was expressed in µg/ml of plasma. For each microtiter plate, the assay included antigen, plasma, and enzyme conjugate controls. A pooled plasma from eCG-treated goats was used on each plate as a positive control. Intra- (n = 10) and interassay (n = 20) coefficients of variation were 7.1% and 11.5%, respectively. The cross-reactivity of the anti-goat IgG conjugated antibody for sheep IgG was verified by preincubation of this conjugate in sheep serum prepared in PBS Tw (1:1) before incubating it with eCG-bound goat antibodies. In this case, no signal was obtained. Therefore, conjugated antibodies cross-reacted with both goat and sheep IgG. To compare this ELISA and the previous 125I-eCG binding method [14], levels of anti-eCG antibody were determined for 96 goat plasma samples at a dilution of 1:50. The coefficient of correlation was 0.557 (p < 0.05).
Determination of Goat Anti-eCG Ig Isotypes by ELISA
The four anti-eCG antibody isotypes (IgG1, IgG2, IgM, and IgA) were assayed in plasma by the use of an ELISA as described above, with slight modifications. Ig isotypes were determined on eCG-coated plates. Plasma samples (serial 1:3 dilutions) were distributed (100 µl/well) in duplicate and incubated for 90 min at 37°C. Then, 100 µl of appropriate dilutions of mouse monoclonal antibodies to sheep IgG1, IgG2, IgA [23], and sheep IgM [24] were added (ascitic fluids kindly provided by Dr. K.J. Beh, CSIRO, Division of Animal Health, McMaster Laboratory, Glebe, Australia). After a 90-min incubation at 37°C, monoclonal antibody binding was detected with peroxidase-conjugated goat anti-mouse IgG (H and L chains; Dako, Glostrup, Denmark) diluted 1:5000 in PBS Tw and incubated for 90 min at 37°C. Between each incubation step, plates were washed five times with PBS Tw. The chromogen-substrate mixture was ABTS and H2O2. Plates were read using the previously described system. Results were expressed as antibody titers. Antibody titers were equivalent to the base 3 logarithm (log3) of the sample dilution for which the point of inflection of the corresponding ELISA logistic curve was reached [25].
Anti-eCG Humoral Immune Response
Experiment 1 Forty-four Alpine goats (1.5–7.5 yr of age; 15 without previous eCG treatment and 29 with one to three previous eCG treatments) from an experimental farm of the Institut National de la Recherche Agronomique (INRA) at Rouillé, France, received intramuscular (i.m.) injection of a single dose of 500 IU eCG (Syncro part, batch 13054A1; SSNA), out of the breeding season, to induce estrus. Jugular vein blood samples were collected into 5-ml heparinized tubes just before the injection and then twice weekly over 8 wk. Plasma samples were stored at -20°C until measurement of anti-eCG antibody concentration by ELISA.
Experiment 2 Twelve other Alpine goats (1.5–2.5 yr of age; without previous eCG treatment) from an experimental farm of INRA (Nouzilly) were treated once per year out of the breeding season with a single i.m. injection of 500 IU eCG. This treatment was repeated over four consecutive years. For each experiment, blood samples were collected just before the eCG injection and then twice weekly over 8 wk. Plasma samples were stored at -20°C until determination of anti-eCG antibody concentration.
Microsatellite Analysis of the MHC Polymorphism
Animals Seventy-eight unrelated Alpine goats from nine herds were selected on the basis of either their low (< 6 µg/ml, n = 40) or high (> 30 µg/ml, n = 38) humoral immune responses to eCG. The selection was done according to the anti-eCG antibody concentration at the maximum of the humoral immune response at Day 10.
DNA extraction Genomic DNA from the white blood cells was prepared as described by Jeanpierre [26]. Venous blood was collected into vacutainer tubes containing EDTA. In a 50-ml centrifuge tube, 15 ml of blood and 35 ml of NE buffer (10 mM NaCl, 10 mM EDTA, pH 7.5) were mixed and incubated overnight at 4°C. After centrifugation (2000 x g for 20 min at 4°C), the supernatant was discarded and the white cells were washed in 45 ml of NE buffer. After centrifugation, the pellet was homogenized in 14 ml of 6 M guanidine hydrochloride and 1 ml of 7.5 M ammonium acetate. About 1 ml of 20% N-lauryl sarkosine (Sigma) and 150 µl of proteinase K (Sigma; 10 mg/ml) were added, and the mixture was heated at 60°C overnight. Then 30 ml of ice cold ethanol was added to precipitate DNA, which was then washed by 70% ice-cold ethanol. Finally, purified DNA was resuspended in TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 7.5).
Polymerase chain reaction (PCR) amplification OLADRB and BM1258 microsatellites, which are located 13 cM (centimorgans) apart on caprine chromosome 23, inside and outside the MHC region, respectively, were amplified using the previously described primers [27]. The PCR amplification was carried out in a Biometra thermocycler: 100 ng of genomic DNA and 0.2 µM of each primer (one 5'-end-labeled with fluorescent dyes: Hex and 6-Fam for OLADRB and BM1258, respectively) were incubated for 2 min at 94°C. Then, a mixture containing 1.6 mM MgCl2, 67 mM Tris HCl pH 8.8, 16 mM (NH4)2SO4, 150 µM of each dNTP, and 1.2 units of Taq DNA polymerase (Eurobio, Les Ulis, France) was added at 60°C to allow the hot-start technique [28]. This was followed by 30 cycles of 94°C for 30 sec, 58°C for 30 sec, and 72°C for 30 sec. Then, 1.5 µl of each PCR product was denatured for 2 min at 94°C after adding of 3.5 µl of internal standard Tamra-500 (Perkin Elmer, Foster City, CA) prepared in formamide dye solution. Finally, 1.5 µl of denatured mixture was separated on a 4% denaturing polyacrylamide gel using an ABI 377 automated sequencer (Perkin Elmer). Sizing of amplified DNA fragments was performed using the Genescan software program (Perkin Elmer).
Relationship between Anti-eCG Antibody Concentration and Fertility in Goats
Animals and treatments
Three hundred and fifteen Alpine goats (1.5–7.5 yr of age) were from the same nine herds as noted in the previous paragraph. The 78 goats subjected to microsatellite analysis belonged to this experimental group. All goats received a hormonal treatment (Syncro part; SSNA) for induction and synchronization of estrus and ovulation as previously described [1]. Briefly, the females were treated for 11 days with progestogen (vaginal sponge impregnated with 45 mg FGA). On the 9th day of this treatment, they received i.m. injections of 50 µg of a PGF2 analogue (cloprostenol; Coopers, Meaux, France) and 500 IU eCG. Forty-eight hours later, vaginal sponges were removed. The occurrence of the onset of estrus was detected with bucks at 4-h intervals, from 12 h to 72 h after the end of the progestogen treatment. Females were cervically inseminated with a total of 108 frozen/thawed spermatozoa at 43 ± 1 h after the progestogen treatment. Blood samples were collected before eCG treatment (at the time of sponge insertion, Day 0) and 10 and 25 days afterwards (Day 10 and Day 25). Plasma samples were stored at -20°C until measurement of anti-eCG antibody concentration by ELISA.
The time of occurrence of the LH surge was determined in three flocks (n = 89). Blood samples were collected every 4 h, from 12 h after sponge removal until 24 h after occurrence of estrus. Plasma samples were stored at -20°C until assay of LH concentration by ELISA (Reprokit; SSNA) [29].
The following parameters were recorded for each goat: herd, age, number of previous eCG treatments, corporeal state, dairy production, number of previous kiddings, number of kids at the preceding kidding, time interval between AI and the preceding kidding, anti-eCG antibody concentration before (Day 0) and after (Day 10, Day 25) treatment, pregnancy diagnosis result, fertility, and prolificacy after AI. For seventy females (two herds), anti-eCG antibody concentrations at Day 10 were missing. In order to compare our fertility results to those of a previous study [15], females were distributed (60%, 20%, and 20%) into three arbitrary classes of anti-eCG antibody concentration.
Statistical Analysis
Average values are presented as means ± SEM. Allele frequencies were determined by direct counting, and frequency distributions were compared using chi-square analysis for the size of classes > 5. For anti-eCG antibody concentration at Day 0, Day 10, and Day 25, the analysis of the distribution and searches for variation factors were carried out by multiple linear regression. The percentages of fertility were compared using chi-square analysis. Differences in anti-eCG antibody concentrations for each parameter analyzed according to several classes were determined by Student's t-test (two groups) or ANOVA (three groups or more). To verify the effect of one main parameter on any other parameter (herd, age, number of previous eCG treatments, dairy production, number of previous kiddings, time interval between AI and the preceding kidding, anti-eCG antibody concentration before [Day 0] and after [Day 10, Day 25] treatment), a multivariate analysis was performed and the fertility analysis was carried out using logistic regression. All the interactions between the different parameters were tested. The conditions under which each test was applied were checked. Differences were considered significant for p values of 0.05 or less.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In order to ensure that the binding of eCG to plasma proteins was due to the presence of anti-eCG antibodies, plasmas collected from goats immediately before and 2 wk after eCG treatment were subjected to protein G affinity chromatography. No anti-eCG antibodies were detected in either unretained or eluted fractions of preimmune plasmas (n = 3; Fig. 1). In contrast, ELISA data for eCG binding indicated that the eluted fractions from immune plasmas (n = 3) contained anti-eCG antibodies (Fig. 1).
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After development of a quantitative ELISA, a typical calibration curve (semi-log plot) displayed absorbance values ranking from 0.2 (blank value) to 1.2 (1000 ng/ml) under optimized experimental conditions (Fig. 2). The linear part of the calibration curve between 7.5 and 500 ng/ml was taken as the working range. The detection limit, as calculated from the mean absorbance of ten replicates of zero standard plus SEM, was 7 ng/ml. To test specificity of the assay, the ELISA was conducted using plates coated with BSA (at 2.5 µg/ml in PBS) instead of eCG. In this case, no antibody binding was obtained with standard anti-eCG IgG, suggesting high specificity for eCG (Fig. 2).
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Kinetics of Humoral Immune Response Induced after One or More eCG Treatments
A first experiment was performed to follow the kinetics of the humoral immune response induced after an eCG injection (Fig. 3A). The 15 goats treated for the first time with eCG exhibited an increase in anti-eCG antibody concentration 10 days (Day 10) after eCG injection. Maximum values were reached between Day 10 and Day 17; then a progressive decrease in anti-eCG levels occurred over 2 mo. Goats previously treated (one or more times) with eCG (n = 29) displayed similar humoral immune response kinetics, except that they exhibited an earlier increase in their antibody concentration at Day 7 and a longer decreasing phase of the antibody concentration. In this study, mainly secondary and also primary responses were composed of anti-eCG IgG1. IgG2 and IgA were very low or absent (data not shown). We were not able to analyze IgM antibodies because of a constant background in this specific ELISA assay. In spite of identical times of increase of antibody concentration in goats with either primary or secondary immune responses, each female of the two groups differed markedly in her anti-eCG concentrations (Fig. 3A). Indeed, maximal anti-eCG antibody concentrations varied from 0.7 to 102 µg/ml in goats treated for the first time and from 3.0 to 219 µg/ml in goats treated several times. The anti-eCG antibodies, already present at the time of eCG injection, were defined as residual antibodies. They were the result of the previous immune response induced by the last eCG injection about 1 yr before.
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Variable Humoral Immune Response among Individual Goats after eCG Treatment
The second experiment evaluated the evolution of the immune response in the same animals eCG-treated once per year over four consecutive years (Fig. 3B). Interestingly, goats eliciting a low humoral immune response upon the first eCG treatment were also poor responders after the following treatments. Conversely, goats with a strong immune response at the first treatment systematically yielded high immune responses upon the following treatments. The mean anti-eCG antibody concentrations at the maximum of the humoral response after each treatment were 2.0, 3.6, 3.9, and 2.2 µg/ml for the low responders and 21.5, 37.7, 144.0, and 92.8 µg/ml for the high responders, respectively.
The origin of the variation in the anti-eCG antibody response was investigated. The MHC, implicated in antigen presentation, was considered as a good candidate. Since low, medium, and high responder goats had been identified, an association between polymorphism of the MHC region and the variability of the anti-eCG immune response was examined. The polymorphism in the DRB region of the MHC was analyzed by PCR amplification of a specific caprine OLADRB microsatellite (Fig. 4A) [27]. Alpine goats were distributed into two groups according to their high (n = 38) and low (n = 40) anti-eCG antibody concentrations at Day 10. The DRB region was found to be highly polymorphic in these two experimental groups. A total of twelve OLADRB alleles were identified, ranging in sizes from 276 to 306 basepairs (bp). Representative allele frequency distributions according to high and low anti-eCG antibody response showed considerable variation among alleles, but three of them were particularly interesting (Fig. 4B). Allele 280 bp was significantly associated with a low-response phenotype (p < 0.05), since 68% of goats with this allele had a low anti-eCG antibody concentration (< 6 µg/ml). By contrast, alleles 295 bp and 288 bp were associated with a high-response phenotype (p < 0.05); 79% and 67% of goats with these two alleles, respectively, were high anti-eCG responders (> 30 µg/ml). BM1258, a microsatellite located outside the MHC region at 13 cM from the OLADRB marker, was used as a control. A total of five BM1258 alleles were identified, ranging in sizes from 101 to 113 bp (Fig. 4C). In contrast to OLADRB, BM1258 allele frequency distributions presented no difference between high and low responders.
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Interference of Anti-eCG Antibody with Fertility in Treated Goats
To analyze the effect of anti-eCG antibodies on subsequent fertility, 315 Alpine goats from nine different herds were examined. In nontreated goats (n = 70), the mean value of antibody concentration at the time of the eCG injection was significantly lower than in goats treated one or several times with eCG (0.6 ± 0.7 µg/ml vs. 2.3 ± 2.7 µg/ml, respectively, p < 0.05).
Among all the parameters tested, only the number of previous eCG treatments had a significant positive effect (p < 0.001) on residual antibody concentration (at Day 0; Table 1). Concentration of anti-eCG antibody measured at Day 25 was significantly related to the residual antibody concentration (p < 0.001). The overall fertility of inseminated goats was 61.9%. Percentage of kidding varied between 31.8% and 77.5% with a significant herd effect (p < 0.001). Fertility significantly decreased with 1) the age of goats (p = 0.01), 2) the number of previous eCG treatments (p = 0.03), and 3) the number of previous kiddings (p = 0.02) (Table 1). Goats inseminated less than 180 days after the previous kidding demonstrated a significant decrease (p < 0.01) in their fertility rate (Table 1). For antibody concentrations measured at Day 0, Day 10, and Day 25, only those at the time of eCG injection (Day 0) had a significant negative effect on subsequent fertility. In fact, when females were distributed in three classes of residual antibody concentrations, a significantly lower fertility rate (42.9%, p = 0.02) was shown in goats with antibody concentration higher than 2.5 µg/ml (Table 2). In contrast, anti-eCG antibody concentrations after treatment (Day 10 and Day 25) were not significant parameters. Prolificacy was not affected whatever the anti-eCG antibody concentration (Table 2). Because parameters probably interacted with each other, a multivariate analysis was performed. Taking into account all parameters, this analysis showed that fertility was significantly (p < 0.01) reduced in goats with a high residual antibody concentration or a short previous kidding-AI interval. To explain these lower fertility results, time of occurrence of estrus and preovulatory LH surge were determined for 89 goats from three flocks. Late (> 36 h after sponge removal) or absent estrus paralleled decline of fertility rate (p < 0.001) and high mean residual antibody concentration (Fig. 5A). For 18% of the goats, no LH surge at all was detected during the observed period of time. A highly significant relationship (p < 0.001) was found between the time of occurrence of the LH surge and fertility (Fig. 5B). In fact, AI was performed at a fixed time, and fertility dramatically dropped under 35% in goats without an LH surge or with a late LH surge (
40 h after the end of the progestogen treatment). Moreover, an absent or a late LH surge was found to be associated with a high mean residual anti-eCG antibody concentration (p < 0.001; Fig. 5B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The reliable and quantitative ELISA described herein was indispensable for the measurement of plasma antibody concentrations and for the comparison of the strength of immune responses in numerous goats treated with eCG. Kinetics showed that a single 500-IU i.m. injection of eCG led to an immediate increase in antibody concentration with typical primary or secondary humoral immune responses. Comparative analysis of the antibody production indicated more rapid secondary immune responses in goats than in cats (1 wk vs. 1–2 wk), especially in cats injected i.m. with a 3-fold eCG dose (relative to weight) [18].
The large variation in anti-eCG antibody concentrations was probably due to a large variability among females in immunoglobulin secretion following eCG injection. Moreover, results obtained after successive eCG treatments 1) indicate that the high or low plasma antibody concentration is an inherent and repeatable characteristic of each individual and 2) demonstrate the occurrence of different populations of goats, classified as low, medium, and high responders.
The MHC, involved in antigen presentation and the regulation of the immune response [30–32], was an obvious potential candidate to explain the variability of the humoral response to eCG. In particular, MHC class II genes encode dimeric glycoproteins involved in antigen presentation to CD4+ T cells, which help B cells to produce appropriate immunoglobulins [33]. Class II genes are among the most polymorphic genes [34, 35], and this extensive polymorphism confers to MHC class II molecules the ability to complex with a large array of different antigen-derived peptides. Although serologic definition of lymphocyte antigens has been described [36], serologic typing errors of up to 25% have been noticed [37]. In goats particularly, serological typing of MHC antigens remains difficult because of the scarcity of immunological reagents. To overcome these difficulties, we decided to develop a microsatellite methodology, which appears to be a more simplified and efficient genetic typing method. For this purpose, we used two microsatellites located in goat chromosome 23 [27]: OLADRB, the only marker in the caprine MHC, and BM1258, the closest marker (at 13 cM) outside the MHC, which was used as control. The interesting feature of this work was the significant correlation between anti-eCG response and some MHC-DRB alleles in unrelated goats. This association is otherwise known as linkage disequilibrium. Although such an association between antibody response and MHC polymorphism has seldom been demonstrated, it has been previously reported for malaria antigen [38] and hepatitis B antigen [39]. At the present time, it is premature to hypothesize that observed linkage disequilibrium between low responders and allele 280 may be the result of a high pressure of selection for high milk production and/or high fertility. For this purpose, it is necessary to type sires used for AI, which are low in numbers, leave many descendants, and could thus transmit alleles corresponding to high (288- and 295-bp) or low (280-bp) phenotypes. Moreover, with the highly polymorphic OLADRB microsatellite, only 69% of goats in our experimental groups had at least one of the three following alleles: 280, 288, and 295 bp. In order to screen the entire goat population, other caprine microsatellites located inside the MHC are under investigation. Additional experiments upon the MHC region, which regulates the immune response, are needed to obtain a thorough understanding of the variability of the anti-eCG antibody response that affects fertility rate after AI at a fixed time. Then, the genetic typing of MHC may be used for the prediction of females with high anti-eCG antibody concentration likely not to be pregnant after AI at a fixed time.
In fact, repeated eCG treatments induced anti-eCG antibodies that clearly have negative effects on reproduction of goats inseminated at a fixed time after treatment. High antibody concentration at Day 0 (> 2.5 µg/ml) was correlated with decreased fertility. This agrees with previous data based on 125I-eCG binding measurement in goats [15]. These anti-eCG antibodies already present at the time of eCG injection were defined as residual antibodies. As shown in this study, humoral immune response started seven days after eCG injection. Consequently, the newly secreted antibodies could not interfere with the bioactivity of the injected eCG. Moreover, antibody concentrations measured between Day 10 and Day 25 showed no significant correlation with fertility. Observations of physiological factors such as time of occurrence of estrus and preovulatory LH peak were a useful strategy for understanding the decreased fertility. Previous results have shown that late estrus was correlated with a high 125I-eCG binding level [15]. However, the LH surge is a much more precise and efficient tool to determine ovulation time in females. In fact, the time interval between the LH surge and ovulation is rather constant [40] in contrast to the time interval between estrus and ovulation, which displays a large variability. Our results showed that females displaying high residual antibody concentrations had a delayed or absent LH surge. These findings suggest that anti-eCG immunoglobulins must interfere with endocrine events and ovarian stimulation. Two hypotheses can be put forward to explain these statements. First, secreted anti-eCG antibodies could exhibit weak affinities and may interfere only partially with the hormone. Accordingly, only the free fraction of eCG would bind to ovarian LH and FSH receptors, leading to diminished stimulation and delayed follicular steroidogenesis. Second, anti-eCG antibodies may interfere with endogenous LH and/or FSH, resulting in modified ovarian stimulation. However, since cross-reactivity of eCG antibodies with endogenous gonadotropins seems to be minimal in goats (unpublished data), monkeys [16], and cats [17], the first hypothesis appears more likely. Moreover, in several species, normal menstrual cyclicity and gestation, which are critically dependent on endogenous pituitary LH and FSH, were not impaired, despite the presence of eCG-neutralizing immunoglobulins [16, 17].
It is also useful to consider the effects of AI at a fixed time after eCG treatment. Between females, a natural variability in the time of ovulation was reported [41]. A delayed preovulatory LH surge in eCG-treated goats adds to this problem. The breeding schedule ensured that sperm were present in the reproductive tract about 20 h before ovulation began. In goats that ovulated belatedly, the time interval between AI and ovulation was no longer optimum, and therefore the waiting time of spermatozoa in the genital tract of females before fertilization was prolonged. Earlier workers reported that low fertility rates were obtained in goats that came into estrus 30 h after sponge removal [13] or in goats inseminated less than 5 h after the LH peak [42]. In contrast, when goats with late estrus were inseminated with a 6-h delay (at 49 h instead at 43 h after the progestogen treatment), fertility was partially restored (55.5% vs. 42.3%; B. Leboeuf, personal communication).
The results of the present study clearly demonstrate that repeated eCG treatments induce a highly variable humoral immune response among individual Alpine goats. Specific IgG antibodies evoked by the 500-IU eCG injection had a negative effect on subsequent fertility when AI was performed at a predetermined time. This phenomenon correlated with delayed estrus occurrence and a late LH surge. It would be interesting to inseminate females according to the occurrence of LH surge, but this is hardly feasible under field conditions. In order to limit the negative immunological interference, one approach would be to replace eCG by recombinant goat gonadotropins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Marie-Christine Maurel, Unité Gonadotropines, INRA, URA CNRS 1291, Station PRMD, 37380 Nouzilly, France. FAX: 33 2 4742–7743; maurel{at}tours.inra.fr
Accepted: November 6, 1998.
Received: August 19, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and ß-subunits. Biochim Biophys Acta 1992; 1159:74–80.[CrossRef][Medline]This article has been cited by other articles:
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  K H Al-Gubory, S Camous, G Germain, P Bolifraud, A Nicole, and I Ceballos-Picot Reconsideration of the proposed luteotropic and luteoprotective actions of ovine placental lactogen in sheep: in vivo and in vitro studies. J. Endocrinol., March 1, 2006; 188(3): 559 - 568. [Abstract] [Full Text] [PDF]
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 M S Medan, S Akagi, H Kaneko, G Watanabe, C G Tsonis, and K Taya Effects of re-immunization of heifers against inhibin on hormonal profiles and ovulation rate Reproduction, October 1, 2004; 128(4): 475 - 482. [Abstract] [Full Text] [PDF]
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 V. Herve, F. Roy, J. Bertin, F. Guillou, and M.-C. Maurel Antiequine Chorionic Gonadotropin (eCG) Antibodies Generated in Goats Treated with eCG for the Induction of Ovulation Modulate the Luteinizing Hormone and Follicle-Stimulating Hormone Bioactivities of eCG Differently Endocrinology, January 1, 2004; 145(1): 294 - 303. [Abstract] [Full Text] [PDF]
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 E. G. Crichton, E. Bedows, A. K. Miller-Lindholm, D. M. Baldwin, D. L. Armstrong, L. H. Graham, J. J. Ford, J. O. Gjorret, P. Hyttel, C. E. Pope, et al. Efficacy of Porcine Gonadotropins for Repeated Stimulation of Ovarian Activity for Oocyte Retrieval and In Vitro Embryo Production and Cryopreservation in Siberian Tigers (Panthera tigris altaica) Biol Reprod, January 1, 2003; 68(1): 105 - 113. [Abstract] [Full Text] [PDF]
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