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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* FBN Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Germany;
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Research Institute for Animal Breeding and Nutrition, 2053 Herceghalom, Hungary; and
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L&R Research Associates, 30677 Watkinsville, GA
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: gonadotropin-releasing hormone agonist • luteinizing hormone • ovulation • pig
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Schally et al., 1971) enabled synthesis of several LHRH (GnRH) agonists such as buserelin, goserelin, leuprorelin, nafarelin, and triptorelin, which have had widespread use in human medicine but limited application in farm animals. Native GnRH and GnRH agonists have been shown to induce ovulation in gilts and sows (Webel and Rippel, 1975; Holtz et al., 1977; Brüssow et al., 1996); however, they are not widely used in pig production. Worldwide, few farms use pharmacological methods to synchronize estrus and ovulation, although their benefits were demonstrated in research conducted on German pig farms by Hühn et al. (1996). However, it is possible that the costs associated with the use of reproductive drugs in pig production may be greater than the economic benefits derived. One way to lower such costs is the use of generic products. The generic GnRH agonist Fertilan [goserelin acetate, (D-Ser (But)6, Azgly10]-LH; Bremer-Pharma, Bremerhaven, Germany) was manufactured and marketed for induction of ovulation in farm animals.
Because there is no information on the effective doses of Fertilan needed for inducing LH release in pigs, the objectives of the current study were to 1) examine the effect of various doses of Fertilan on secretion of LH in barrows, to select doses to investigate in gilts (Exp. 1); 2) determine doses of Fertilan that would induce a preovulatory-like rise of LH in gilts (Exp. 2); and 3) determine the time of ovulation after Fertilan treatment (Exp. 3). Responses to Fertilan treatment were compared with that of Gonavet (depherelin, D-Phe6-LHRH; Veyx-Pharma Schwarzenborn, Germany; control group), a commercial preparation with known efficacy (Brüssow et al., 1990b, 1994, 1996) and used currently in German commercial pig production.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All procedures involving animal handling and treatment were approved by the Committee for Animal Use and Care of the Agricultural Ministerial Department of Mecklenburg-Vorpommern, Germany.
Exp. 1.
German Landrace barrows (n = 10), 160 d of age, and with BW of 81 ± 2 kg, were fitted surgically with an indwelling jugular venous catheter (Rodriguez and Kunavongkrit, 1983), and allocated to 5 treatment groups: 10 (n = 2), 20 (n = 2), and 40 µg (n = 2) of Fertilan, 50 µg of Gonavet (n = 2), or saline (controls, n = 2). Each GnRH agonist dose was diluted in 10 mL of saline and injected i.v. via the jugular catheter. Doses of Fertilan were based on those of the nonapeptide, buserelin (Receptal, Intervet, Unterschleissheim, Germany).
Three days after jugular venous catheterization (d 1), 5-mL blood samples were collected into heparinized tubes immediately before (0800) and at 30, 60, 90, 120, 150, 180, 240, 300, 360, and 480 min after i.v. administration of the GnRH agonists or saline for determination of plasma concentrations of LH. Blood collection and GnRH agonist injection was repeated on d 4, 7, 10, and 13, and each barrow received the same treatment as on d 1. During the intervening 2 d between GnRH injections, blood samples were collected at 0800, 1200, and 1600. After every blood collection, the catheter was filled with 3 mL of 3% Na-citrate. Before blood collection, the citrate within the catheter, together with the first 3 mL of blood, was discarded. Blood samples were centrifuged immediately at 1,880 x g for 15 min. Plasma aliquots were pipetted into Eppendorf tubes (1.5 mL, T330-7N, Simport, Beloeil, Canada) and stored at –20°C until analyses.
Exp. 2. Estrus was synchronized in 12 German Landrace gilts averaging 225 ± 2 d of age and 134 ± 5 kg of BW. All had displayed 1 or more estrous cycles. Synchronization was performed by feeding Regumate for 15 d (16 mg of altrenogest/gilt·d–1; Serumwerk Bernburg, Bernburg, Germany) and an i.m. injection of 1,000 IU of eCG (Pregmagon, Impfstoffwerk Dessau-Tornau, Germany) 24 h after the last feeding of Regumate. Ovulation was stimulated 80 h after eCG with 10 (n = 4) or 20 µg of Fertilan (n = 4) or 50 µg of Gonavet (controls, n = 4). Gilts were observed for estrous behavior daily in the presence of a teaser boar from d 2 to 6 and by using the standing reflex in response to back pressure exerted by a human handler.
Four days before the last Regumate feeding, gilts were surgically fitted with jugular venous catheters to collect blood for LH, estradiol, and progesterone analysis. Blood samples (10 mL) were collected twice daily (0800 and 1600) from the last day of Regumate feeding until GnRH agonist administration. Each dose of GnRH agonist was diluted in 10 mL of saline and injected i.m. 80 h after eCG (at 1600 on d 3 posteCG). After GnRH agonist injection, blood samples were taken every 30 to 60 min until 2000, and then every 2 h until 0600 of d 4 posteCG. Thereafter, blood samples were drawn at 0800 and 1600 on d 3 to 5 and at 0800 on d 6 to 13 posteCG.
On d 6 posteCG, all gilts were examined endoscopically for ovulation (Brüssow et al., 1990a; Rátky et al., 1998). Briefly, gilts were anaesthetized by i.v. administration of ketamin/xylazin (20 mg of Ursotamin/kg of BW and 1.8 mg of Xylazin/kg of BW; Serumwerk Bernburg, Germany) and placed in a supine position. After producing a pneumoperitoneum and automatically insufflating about 5 L of CO2 (Endo Tech, Mü nchen, Germany), one 10-mm trocar for the endoscope and two 5-mm trocars for the grasping forceps (Storz, Tuttlingen, Germany) were inserted into the abdomen. Ovaries were observed using a 0° endoscope (ETB, Berlin, Germany) equipped with a video camera (Endo Tech). Each ovary was grasped gently, and the infundibulum was reflected from the ovary with atraumatic forceps (Ergo-LAP, Bowa-electronic, Gomaringen, Germany). Ovarian features (i.e., follicles, corpora haemorrhagica, corpora lutea) were identified (Schnurrbusch et al., 1981) and counted.
Exp. 3.
This part of the study was conducted to determine the onset and termination of ovulation in gilts after Fertilan treatment. Estrus was synchronized in 12 German Landrace gilts (225 d of age and 128 ± 9 kg of BW), as described in Exp. 2, and the gilts were observed for estrous behavior. Treatments to induce ovulation were given 80 h after eCG, with an i.m. injection of 20 µg of Fertilan (n = 8) or 50 µg of Gonavet (n = 4; control group). Time of ovulation was determined endoscopically at 34, 38, and 42 h postGnRH agonist administration as described above. Ovulatory status was characterized as nonovulated (only preovulatory follicles observed), ovulation in process (minimum of 1 follicle ovulated/ovary), and ovulated (
85% of ovulated follicles; Brüssow and Bergfeld, 1984).
Assay Methodology
Plasma concentrations of LH were determined by a nonisotopic electrochemiluminescence immunoassay (ECLIA) as described by Schneider et al. (2004). The ECLIA was performed in 12 x 75-mm polypropylene tubes (Minisorb PE, Nunc, Roskilde, Denmark). An N-hydroxy-succinimide ester of ruthenium(II)-tris-bipyridine chelate (Ruester) was used to label the monoclonal antibovine LH antibody 517 B7 (b-LH; Matteri et al., 1987), as recommended by the manufacturer (BioVeris, Gaithersburg, MD). The standard porcine LH (p-LH; iodination grade) and polyclonal rabbit-anti-p-LH were provided by Biotrend (Cologne, Germany).
The Ru-labeled monoclonal antibody (25 µL) and polyclonal antibody (50 µL) were allowed to bind to the hormone (standard of sample, 20 µL). The standard curve was generated from tubes with the greatest concentration (12.8 ng/mL) and serially diluted through 0.025 ng/mL. After overnight incubation, a second antibody, sheep anti-rabbit IgG, coupled to magnetic beads (50 µL; Dynal, Oslo, Norway), was added. Finally, chemiluminescence was measured in the Origen 1.5 analyzer (BioVeris). The assay required approximately 1 h per carousel sample changer, which held 50 samples. Sensitivity was 0.03 ng/mL. The intra- and interassay CV were 6.1 and 8.7%, respectively.
Peripheral steroid concentrations were measured by RIA for progesterone and estradiol, as previously described (Schneider et al., 2002). The [1,2,6,7-3H] progesterone (tracer) was purchased from Amersham Pharmacia Biotech (Freiburg, Germany). The first antibody was raised in rabbits, and the standard hormone was obtained from Jenapharm (Jena, Germany). The range of the standard curve was 6.25 to 800 pg/mL. The progesterone RIA was performed without extraction in 50 µL of sample. Assays were incubated at 37°C for 30 min followed by 4°C for 2 h. Separation of bound/free hormone fractions was by the dextran-coated charcoal method. Radioactivity was counted in a liquid scintillation counter and calculated with an integrated RIA program (Rackbeta 1219, Wallac, Finland). The sensitivity of the assay was 10 pg/mL. Intra- and interassay CV were 7.6 and 9.8%, respectively.
Estradiol-17ß was measured in 1-mL duplicates, each extracted with 5 mL of diethyl ether. The tracer was [2,4,6,7-3H] estradiol-17ß (Amersham Pharmacia Biotech), and a rabbit-antiserum (FBN, Dummerstorf, Germany) was used. The standard curve ranged from 3.75 to 480 pg/mL, and the assay sensitivity was 3 pg/mL. Incubation, bound/free separation, and counting of radioactivity were accomplished as described for progesterone. Intra- and interassay CV were 6.2 and 9.1%, respectively.
Statistical Analysis
Secretion characteristics of LH [maximal plasma LH concentration of the induced LH surge (LHMAX), the duration of the induced LH surge, the LH area over baseline (LHAOB), and the interval from GnRH agonist administration to LHMAX] were calculated by the PUL-SAR program (Merriam and Wachter, 1982). The following G-values were used: G1, 3.8; G2, 3.2; G3, 2.7; G4, 2.4; and G5, 1.8. Hereby, a 10% criterion of variation was applied, and the assay parameters (sensitivity and frequency of sampling) were used to calculate the respective secretion parameters.
Data from Exp. 1 were evaluated by ANOVA using the MIXED procedure (SAS Inst. Inc., Cary, NC). The repeated measures ANOVA model for the response variable plasma LH concentration between time 0 to 480 min after injection on d 1, 4, 7, 10, and 13, contained the fixed effect of the treatment groups (10, 20, or 40 µg of Fertilan; 50 µg of Gonavet; and saline control) and the repeated factors of day and time and their corresponding interactions. Repeated measures on the animals were taken into account by the repeated statement of the MIXED procedure and the subject = animal option to specify the residual covariance matrix in the model, and estimated by the minimum variance quadratic unbiased estimation of the covariance parameters (MIVQUE0) method. In addition, least squares means and their SE were calculated for each effect in the model. The repeated measures ANOVA for the response variable plasma LH concentration in Exp. 1 was conducted with the GLM procedure of SAS using 2 models. One model with the fixed effect of the treatment groups (10, 20, or 40 µg of Fertilan; 50 µg of Gonavet; and saline control) and the repeated factor day by each time and one model with the fixed effect of the treatment groups and the repeated factor time by each day.
The other response variables (LHMAX, duration of induced LH surge, LHAOB) of Exp. 1 were evaluated by ANOVA using the GLM procedure of SAS/STAT software. The repeated measures ANOVA model contained the fixed effect of the treatment groups (10, 20, and 40 µg of Fertilan; 50 µg of Gonavet; and saline control) and the repeated factor of day (d 1, 4, 7, 10, and 13). Additionally, least squares means and their SE were calculated for all response variables in each treatment group at each day.
Data from Exp. 2 were evaluated by ANOVA using the GLM procedure of SAS. The repeated measures ANOVA model of Exp. 2 contained the fixed effect of the treatment groups (10 and 20 µg of Fertilan and 50 µg of Gonavet) and the response variables plasma LH, estradiol-17ß, and progesterone concentration measured on d –1 to 14 after eCG (repeated factor). Additionally, least squares means and their SE for all response variables in each treatment group at each day were calculated.
The frequency data of Exp. 3 were analyzed using the GENMOD procedure of SAS, with the treatment groups (20 µg of Fertilan and 50 µg of Gonavet) and the repeated factor of time (34, 38, and 42 h).
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Injection of GnRH agonist induced increases in plasma LH concentrations (P < 0.05) in all barrows at all treatment d (d 1, 4, 7, 10, and 13) compared with saline controls (Figure 1). The day of treatment influenced (P < 0.05) GnRH agonist-induced LH release, but no day x treatment or day x time x treatment interactions were found. However, although LH release was decreasing after repeated injections of GnRH agonist, dose-dependent increases in plasma concentrations of LH continued to be observed (Table 1). Administration of 20 µg of Fertilan and 50 µg of Gonavet triggered greater maximal plasma LH concentrations and larger LH surges (LHAOB) compared with the remaining treatments (P < 0.05). Duration of the induced LH surge was different among treatment groups (P < 0.05).
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Based on the LH surge characteristics observed in barrows at doses of 20 and 10 µg, Fertilan at these doses was chosen for treatment of gilts in Exp. 2. Injection of GnRH agonists induced an LH surge in 3 of 4 gilts treated with 10 and 20 µg of Fertilan, and in 4 of 4 gilts treated with Gonavet (Figure 2). No GnRH-induced LH release was observed in gilt No. 4 (10 µg of Fertilan) and No. 7 (20 µg of Fertilan). One animal in the 20 µg of Fertilan group (gilt No. 8) exhibited a second LH surge of greater magnitude than the induced surge. Mean LHMAX concentrations of the induced LH surge did not differ between doses of 10 and 20 µg of Fertilan and 50 µg of Gonavet and were 5.0 ± 0.9, 4.6 ± 1.8, and 6.6 ± 1.1 ng/mL, respectively. No differences were observed in the duration of the induced LH surge (19.3 ± 2.0, 17.0 ± 4.0, and 20.0 ± 2.0 h), the LHAOB (42.7 ± 1.9, 37.8 ± 10.4, and 55.2 ± 6.0 ng/mL), and the interval from GnRH injection to LHMAX (4.0 ± 2.0, 6.7 ± 1.3, and 5.8 ± 1.6 h).
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). Lower concentrations of estradiol (P < 0.05) were measured on d 1 to 4 after eCG in gilts of the Gonavet group compared with gilts in the Fertilan groups. After GnRH agonist injection, plasma estradiol concentrations decreased to basal concentrations in each treatment group. Plasma progesterone concentrations increased after ovulation from basal concentrations on d 5 after eCG to mean maximal concentrations of 7 to 17 ng/mL on d 13 to 14 posteCG. Mean plasma progesterone concentrations were greater (P < 0.05) in gilts treated with 20 µg Fertilan compared with gilts in the remaining treatment groups. These gilts tended to have a greater number of ovulations (21.3 ± 3.6) compared with gilts receiving 10 µg of Fertilan (17.3 ± 4.1) or 50 µg of Gonavet (17.5 ± 3.2). However, all gilts had ovulated on d 6 posteCG as revealed by endoscopy.
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Exp. 3
Ovulations occurred in gilts of both groups by 34 to 42 h after GnRH agonist injection. The percentage of gilts that ovulated increased with time after both GnRH agonist treatments. However, the proportion of gilts that ovulated was less (P < 0.05) in the Fertilan group than in the Gonavet group at 38 h but not at 42 h after injection (Figure 4). The mean number of preovulatory follicles was not different between the treatment groups (18.6 ± 2.0 vs. 23.0 ± 1.8 in Fertilan and Gonavet-treated gilts, respectively). All gilts showed signs of estrus (vulva reddening and swelling), but the standing reflex could not be recorded due to the anesthetic and endoscopic intervention.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; von Kaufmann and Holtz, 1982; Bergfeld et al., 1984a) and sows (Bergfeld et al., 1984b; Szabo et al., 1992; Baer and Bilkei, 2004). Although several potent GnRH analogue preparations are available, to our knowledge only the GnRH agonist D-Phe6-LHRH (Gonavet, Veyx-Pharma Schwarzenborn, and Oestracton, IDT, Dessau-Tornau, Germany) has been permitted in the pig and probably only in Germany. Receptal (buserelin; D-Ser [BUt]6-EA10-LHRH), which is the most widely used agonist in veterinary medicine (Peters, 2005), has been tested in swine (von Kaufmann and Holtz, 1982; Varadin et al., 1983; Möller-Holtkamp et al., 1995) but has not found practical application in the swine industry. Goserelin has been successfully used in human gynecology (West and Baird, 1987; Geber et al., 2002; Vlaisavljevic et al., 2003), breast cancer treatment (Mitchell, 2004; Rody et al., 2005), and in very limited studies in boars (Almond et al., 1992), gilts (Peltoniemi et al., 1995), and mares (Fitzgerald et al., 1993a,b; Mumford et al., 1994) and exclusively via a long-term delivery formulation.
Barrows are an appropriate model to test GnRH preparations for induction of LH release (Möller-Holtkamp et al., 1995; Brüssow and Schneider, 2001; Kauffold et al., 2005). Each dose of Fertilan and Gonavet consistently stimulated LH release (significant time x treatment interaction) with maximum peak concentrations ranging from 1.1 to 5.8 ng/mL after each injection. Mean duration of the induced LH release was dose-dependent and lasted between 78 and 180 min. Similar LH release characteristics were reported by Möller-Holtkamp et al. (1995) using 2 to 20 µg of Receptal or 10 to 50 µg of Gonavet. However, in our study, LH surge variables (i.e., LHMAX and LHAOB) decreased with repeated treatment. Similar observations were made in a previous study (K.-P. Brüssow, unpublished observations) where shorter injection intervals (every day or every second day) progressively decreased the amount of LH released. This likely occurred due to downregulation of GnRH receptors on pituitary gonadotrophs. Although GnRH agonist-induced surge characteristics in barrows differ from those in gilts and sows during the preovulatory period (less maximal release of LH and shorter duration of surge), results from the experiments with barrows were able to provide an indication of GnRH agonist doses that would be effective in females. Based on results of Exp. 1, 10 and 20 µg of Fertilan were chosen for investigation in gilts.
In Exp. 2, both Fertilan doses induced a preovulatory-like LH surge in 6 of 8 gilts, whereas Gonavet induced a LH surge in all treated gilts. Gilt No. 4 did not exhibit any LH release, but ovulation and increasing progesterone concentrations after treatment appeared to be similar to the other gilts. We have no plausible explanation for this observation. Gilt No. 7, which failed to have an induced LH release, showed an endogenous LH surge 1 d later followed by an appropriate increase in postovulatory progesterone concentrations. On the other hand, all LH surge variables (LHMAX, LH surge duration, LHAOB, and interval from GnRH agonist injection to maximum serum LH concentration) of the remaining gilts were not different among treatment groups. Therefore, we concluded that 10 and 20 µg of Fertilan were able to stimulate adequate surges of LH from the pituitary gland to induce ovulation. Maximal plasma concentrations of LH released in response to 10 and 20 µg of Fertilan or 50 µg of Gonavet were in the range reported for natural (van de Wiel et al., 1981; Soede et al., 1994; Waberski et al., 1997) and estrus-synchronized sows (George et al., 1989; Brüssow et al., 1994; Egerszegi et al., 2003). The 9 to 24 h duration of GnRH agonist-induced LH surge was in the range as reported for estrus-synchronized gilts and sows (4 to 18 h; George et al., 1989; Ogasa et al., 1991; Egerszegi et al., 2003) but seems to be shorter than in sows exhibiting spontaneous estrus (22 to 59 h; Blair et al., 1994; Waberski et al., 1997; van Rens et al., 2000). We do not have an explanation for the lower estradiol concentrations detected in the Gonavet group, but perhaps it was due to the limited number of gilts used to examine this variable.
In Exp. 2, endoscopic examination of the ovaries revealed that all gilts had ovulated by d 6 after eCG. However, for fixed-time insemination to be practical, knowledge of the precise time period of ovulation is important. Therefore, in Exp. 3, the onset and termination of ovulation was followed by repeated endoscopic observation. One advantage of this method, compared with ultrasound imaging, is that the quality of the ovarian features (structure, shape, color, and vascularization of follicles and corpora lutea) can be evaluated (Brüssow et al., 1990a; Rátky et al., 1998). Gilts treated with 20 µg of Fertilan commenced ovulation between 38 and 42 h postGnRH agonist injection (6/8 gilts), and ovulation terminated in 50% of these gilts by 42 h. In the Gonavet-treated group, 75% of gilts had ovulated by 42 h postGnRH. However, one must consider that the number of animals treated was limited. Nevertheless, the results indicate that after Fertilan treatment ovulation occurred by about 42 h after administration. This is in accordance with the literature in which time of ovulation following GnRH in estrus-synchronized gilts was between 35 and 42 h (Brüssow et al., 1990b, 1994). A similar temporal relationship was reported with gilts in spontaneous estrus, in which the time between onset of the LH surge (this time point is equivalent to that of GnRH administration in estrus-synchronized gilts), and ovulation was reported to be 37 to 49 h by Soede et al. (1994), 40 to 49 h by Martinat-Botte et al. (1995), and 38 to 45 h by Waberski et al. (1997). Based on the preliminary results, we would suggest a fixed-time double insemination at 24 to 26 h and 38 to 40 h after GnRH agonist administration in gilts, synchronized with Regumate and eCG. This would fit into the time schedule as used in German pig production (Brüssow et al. 1996; Hühn et al., 1996). However, this should be confirmed by additional large experiments, which include AI and subsequent parturition of sows on commercial pig farms.
The results of these experiments indicate that 1) barrows are an appropriate model for determining GnRH doses, which might be effective in inducing a preovulatory-like LH surge in females; 2) the generic GnRH agonist Fertilan (goserelin) at doses of 10 to 20 µg will stimulate a preovulatory-like LH surge with subsequent ovulation in gilts similarly to 50 µg of Gonavet; and 3) Fertilan-induced ovulation within a period of 34 to 42 h could be used for fixed-time insemination.
1 Corresponding author: bruessow{at}fbn-dummerstorf.de
Received for publication May 2, 2006. Accepted for publication August 16, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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—) µg of Fertilan or 50 µg of Gonavet (—
—). Arrows indicate injection of GnRH agonists.