Comparison of Luteinizing Hormone and Steroid Hormone Secretion During the Peri- and Post-Ovulatory Periods in Mangalica and Landrace Gilts

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1 Journal of Reproduction and Development, Vol. 49, No. 4, 2003 Original Comparison of Luteinizing Hormone and Steroid Hormone Secretion During the Peri- and Post-Ovulatory Periods in Mangalica and Landrace Gilts Istvan EGERSZEGI 1), Falk SCHNEIDER 2), Jozsef RÁTKY 1), Ferenc SOÓS 1), László SOLTI 3), Noboru MANABE 4) and Klaus-Peter BRÜSSOW 2) 1) Research Institute for Animal Breeding and Nutrition, 2053 Herceghalom, Gesztenyés út 1, Hungary, 2) Research Institute for the Biology of Farm Animals, Dummerstorf, Wilhelm- Stahl-Allee 2, Germany, 3) Faculty of Veterinary Science, Szent Istvan University, 1400 Budapest, Hungary, 4) Deparment of Animal Sciences, Kyoto University, Kyoto , Japan Abstract. The objective of this study was to determine plasma concentrations of luteinizing hormone (LH), progesterone (P4) and estradiol-17β (E2) in Mangalica gilts (M), a Hungarian native breed, and compare them with Landrace gilts (L) during the peri- and post-ovulatory periods. The estrous cycle of gilts was synchronised by Regumate feeding, and ovulation was induced with a gonadotropinreleasing hormone (GnRH) agonist. Blood sampling was carried out via indwelling jugular catheters three times a day and in 2-h intervals during a 16-h period after the GnRH application. The concentrations of LH, E2 and P4 were determined by immunoassays. Gilts of both breeds showed a typical gonadotropin and gonadal hormone secretion pattern. Preovulatory E2 peaks were observed on day 2 (M) and day 4 (L) after the last Regumate feeding. Highest E2 concentration was different between M and L breeds (46.5 ± 5.7 vs ± 6.8 pg/ml, P < 0.05). Maximum LH levels measured up to 6 h after GnRH were not different between M and L breeds (11.5 ± 4.1 vs. 6.6 ± 2.3 ng/ml). Both LH amounts during surge (41.1 ± 15.9 vs ± 6.1 ng/ml) and total over LH release (73.4 ± 22.2 vs ± 8.7 ng/ml) did not differ significantly between M and L breeds. P4 concentrations started to rise on day 6 after Regumate feeding and increased significantly from 0.6 ± 0.3 and 0.7 ± 0.4 ng/ml to maximal 14.0 ± 2.4 and 11.3 ± 2.1 ng/ml in M and L breeds, respectively. Mean P4 secretion was higher in M on days (12.9 ± 2.6 vs. 9.3 ± 2.2 ng/ml; P<0.05). At the same time the number of corpora lutea was lower in M compared to L (10.3 ±1.5 vs ± 5.0, P<0.05). In our experiment, there was no evidence that differences in the secretion of analysed hormones during the peri- and postovulatory periods are a possible cause of usually lower fecundity in Mangalica gils. Key words: Mangalica pig, Luteinizing hormone, Progesterone, Estradiol-17β, Ovary (J. Reprod. Dev. 49: , 2003) angalica is an endangered native swine breed of Hungary. Mangalica pigs are characterised by later sexual maturity and lower fecundity compared with European commercial breeds [1, 2]. Accepted for publication: May 16, 2003 Correspondence: K.-P. Brüssow ( bruessow@fbn-dummerstorf.de) Several efforts have been made during recent years to rescue and propagate the Mangalica pigs [3, 4]. However low fecundity still remains a problem. In previous experiments, it was shown that Mangalica gilts had a lower or nearly similar number of preovulatory follicles as Landrace gilts, a typical European commercial breed. Furthermore,

2 292 EGERSZEGI et al. sustained intrafollicular oocyte development was found [5]. Currently, no information is available on hormone secretion during the estrous cycle of Hungarian Mangalica gilts. Consequently, more profound experiments are necessary to determine possible causes of differing fecundity. Therefore, the aim of the present study was to investigate and compare the pattern of secretion and circulating levels of estradiol-17β (E2), progesterone (P4) and luteinizing hormone (LH) during the peri- and post-ovulatory periods in Mangalica and Landrace gilts. Material and Methods Animals, estrus cycle synchronization and blood sampling Altogether six puberal Mangalica (10 12 months of age of kg body weight) and four Landrace gilts (9 months of kg) were included in the trial. Estrus was synchronised in all gilts by 15 day long feeding of Regumate (16 mg altrenogest/day/gilt; Serumwerk Bernburg, Bernburg, Germany). Twenty-four hours after the last Regumate feeding (8:00 h), each animal received a single intramuscular injection of 1,000 IU pregnant mare s serum gonadotropin (PMSG; Folligon, Intervet, Unterschleissheim, Germany). Ovulation was induced 80 h later by an intramuscular administration of 50 µg gonadotropin-releasing hormone (GnRH) agonist (Depherelin ; Veyx Pharma, Schwarzenborn, Germany). Three days before the end of the Regumate feeding, all gilts were surgically fitted with an indwelling jugular vein catheter. Cannulation was performed as described by Rodriguez and Kunavongkrit [6]. Blood samples (each 10 ml) were collected in heparinised tubes three times a day (8:00, 12:00 and 16:00 h) and in 2-h intervals after GnRH injection over a period of 16 h. The blood samples were centrifuged immediately at 3,000 rpm for 15 min. The plasma samples were pipetted into Eppendorf -microtubes and stored at 20 C until use. After every blood collection, the catheter was filled with 3 ml of 3% Na-citrate. Prior to blood collection, the citrate within the catheter together with the first 3 ml of blood were discarded. Endoscopic observation Ovaries were observed by endoscopy [7] on day 14 after last Regumate feeding to count the number of developed corpora lutea. Electrochemiluminescence immunoassay (ECLIA) for LH Plasma LH level was determined by a nonisotopic ECLIA. For LH measurement, a sandwich type immunoassay was performed according to the method for the determination of bovine LH (b-lh) [8]. The ECLIA was carried out in mm polypropylene tubes (Minisorb PE; Nunc, Roskilde, Denmark). An N-hydroxy-succinimide ester of ruthenium(ii)-tris-bipyridine chelate (Ru-ester) was used to label the monoclonal antibody 517-B7 against b-lh [9] as recommended by the manufacturer (IGEN, Gaithersburg, MD, USA). The labelled antibody was stored in the refrigerator where it is stable for more than 1 year [10]. The standard porcine-lh (p-lh; iodination grade) and polyclonal rabbit-anti plh were provided from Biotrend (Cologne, Germany). The Ru-labelled monoclonal antibody (25 µl/tube) and polyclonal antibody (50 µl/tube) were allowed to bind to the hormone (standard/sample, 20 µ l/tube). Determination of the standard curve was started at 12.8 ng/ml and diluted through to ng/ml. After overnight incubation, 50 µl/tube of secondary antibody, sheep anti-rabbit IgG coupled to magnetic beads (Dynal, Oslo, Norway) was added and mixed. Finally, the intensity of chemiluminescence was measured using an Origen 1.5 analyser (IGEN). The ECLIA ran for approximately 1 h per carousel sample changer fitted with 50 samples. The sensitivity of the method was calculated to be 0.03 ng/ml. The intraand inter-assay coefficients of variation (CV) were 6.1 and 8.7%, respectively. Radioimmunoassay (RIA) for E2 and P4 The peripheral plasma E2 and P4 levels were measured by 3 H- RIA as previously described [11]. The [1,2,6,7-3 H]-progesterone (tracer) was purchased from Amersham Pharmacia Biotech (Freiburg, Germany). The antibody was raised in rabbits. The standard for each steroid hormone was supplied from JenaPharm (Jena, Germany). The range of the standard curve was from 12.5 to 800 pg/ml. The P4 analysis was performed without extraction in 50 µl of the sample. The incubation steps were performed at 37 C for 30 min and at 4 C for 2 h. The B/F separation was

3 LH AND STEROID HORMONES IN MANGALICA PIGS 293 performed by the dextran-charcoal method. Counting of radioactivity was made by a Liquid Scintillation Counter with an integrated RIA program (Rack-β 1219, Wallac, Finland). Intra- and inter-assay CVs were 7.6 and 9.8%, respectively. Plasma E2 level was measured in 1-ml duplicates of samples, each extracted by 5 ml of diethyl ether. The tracer was [2,4,6,7-3 H]-estradiol-17β (Amersham Pharmacia Biotech), and a rabbit-anti- E2 serum (FBN, Dummerstorf, Germany) was used. The standard curve was prepared from 7.50 to 480 pg/ml. Incubation, B/F separation and counting were accomplished as described for P4. Intra- and inter-assay CVs were 6.2 and 9.1%, respectively. Statistics Results were analysed by one way analysis of variance and a pair wise multiple comparison procedure (Tukey test). P-values <0.05 were considered to be significant. Results Two Mangalica gilts were removed from the experiment because one began estrus during the Regumate feeding and the other had infantile ovaries. A typical LH and ovarian steroid secretion pattern were found during the peri- and postovulatory periods in Mangalica and Landrace gilts (Fig. 1). The peak concentrations of peripheral hormones, duration of LH surge, intervals from GnRH application and from peak concentration of E2 to LH peak and ovulation rates are shown in Table 1. Preovulatory E2 peaks were observed on day 2 (M) and day 4 (L) after Regumate. The E2 peak maximum was different between M and L (46.5 ± 5.7 vs ± 6.8 pg/ml, P<0.05). LH maximum levels were found up to 6 h after GnRH and were not different between M and L gilts (11.5 ± 4.1 vs. 6.5 ± 2.5 ng/ml). Both LH amounts during surge (41.1 ± 15.9 vs ± 6.1 ng/ml) and total over LH release (73.4 ± 22.2 vs ± 8.7 ng/ml) did not differ between M and L strains. Starting on day 6 after Regumate, P4 concentrations significantly increased from 0.6 ± 0.3 and 0.7 ± 0.4 ng/ml to maximal 14.0 ± 2.4 and 11.3 ± 2.1 ng/ml in M and L, respectively. Mean P4 secretion was higher in M on days (12.9 ± 2.6 vs. 9.3 ± 2.2 ng/ml; P < 0.05). However, the number of corpora lutea/gilt was lower in M than in L (10.3 ± 1.5 vs ± 5.0; P < 0.05). Discussion To date, there has been no data on secretion patterns of reproductive hormones during the estrous cycle in Hungarian native Mangalica pigs. Therefore, quantitative differences with those of other breeds could be the reason for the low reproductive ability of this breed. Thus, we compared the secretion of reproductive hormones, LH, E2 and P4, of Mangalica with the more prolific Landrace breed. All gilts were synchronised and primed with PMSG/GnRH in order to collect peripheral blood samples at the same times. The number of ovulated follicles was checked by endoscopy, and fewer corpora lutea were found in M gilts (10.3 ± 1.5) than in L gilts (17.8 ± 5.0), which is in accordance with earlier observations [3, 5]. The pattern of reproductive hormone secretion during the estrous cycle was comparable in both breeds. Studies on prolific pigs and on selected ones for higher ovulation rates demonstrated no difference in hormonal secretion compared to less prolific pigs [12 15]. In our study, the LH peak occurred up to 6 h after GnRH in all animals, which was in agreement with previous studies on gilts [16, 17] and sows [18, 19]. The peak height of the LH surge ranged from 4.2 to 16.0 ng/ml and tended to be higher in M than in L gilts (P=0.08). These values were within the range observed in spontaneous or synchronised estrous gilts [16, 17, 20 23]. The LH concentration decreased after the peak consistently and remained low during the experimental period. In the present study, E2 peaks were measured in both breeds before LH surge, however, their characteristics were different. The highest E2 values occurred 2 and 4 days after the last Regumate feeding in M and L gilts, respectively, which corresponds to 48 and h before the LH peak. The E2 peak values were distinct but lower in L (26.0 ± 6.8 vs ± 5.7 pg/ ml). Van de Wiel et al. [24] investigated the maximal E2 concentration (between 32.1 and 56.4 pg/ml) and reported it as occurring at 8 15 h before the time of LH maximum, whereas Enne et al. [21] reported the time between the peaks as 24 h. Blair et al. [25] and Soede et al. [26] found that the E2 peak values, and the interval between the E2 and the LH peak concentrations correlated with embryo

4 294 EGERSZEGI et al. Fig. 1. Changes in LH, E2 and P4 concentrations during the estrous cycle relative to the last Regumate feeding in Mangalica and Landrace gilts. Each value represents mean ± SD (circle ± bars; n=8). survival. Higher E2 peak and longer interval between peaks was associated with lower embryo survival. In contrast to these findings, Hunter et al. [13] reported that a two-fold higher E2 concentration was already found 80 h before the LH peak in the Meishan breed, an extremely prolific Chinese native breed, and the onset of the E2 peak occurred earlier than in the Large White breed. The Meishan breed is characterised by a particularly high ovulation rate, and embryo and foetal survival. Apparently, the Hungarian Mangalica has a similar E2 secretion pattern, however, they are characterised by a low ovulation rate and litter size. Further experiments are needed to clarify whether the E2 secretion pattern found in the present study is typical for Mangalica or only due to the limited number of gilts examined. Concentrations of E2 during the diestrus stage were consitently low in both breeds in the present study. Concentrations of P4 in both breeds followed the pattern reported in several studies [20, 21, 24]. Peripheral blood P4 levels progressively increased from the second day after the ovulatory LH peak to reach their highest values after 8 10 days. The

5 LH AND STEROID HORMONES IN MANGALICA PIGS 295 Table 1. Results of hormone secretion and ovulation in Mangalica and Landrace gilts Mangalica Landrace Mean ± SD Range Mean ± SD Range LH surge duration (h) 11.5 ± ± Time from GnRH to LH peak (h) 2.5 ± 1.0 a ± 2.5 b 4 10 Time from peak E2 to peak LH (h) 13.5 ± ± LH amount during surge (ng/ml) 41.1 ± ± Peak LH (ng/ml) 11.5 ± ± Peak E2 (pg/ml) 46.5 ± 5.7 a ± 6.8 b Peak P4 (ng/ml) 14.0 ± ± Number of corpora lutea/gilt 10.3 ± 1.5 a ± 5.0 b Values with different superscripts within a row denote significant differences (P<0.05). observation of an initial rise in the P4 concentration (> 2 ng/ml) 59 h after hcg treatment reported by Ziecik et al. [27] was confirmed by the present results. In addition, higher P4 levels were measured in M than in L, between days after the last Regumate feeding in spite of the lower number of corpora lutea in Mangalica gilts. It is still speculative whether corpora lutea of Mangalica gilts have a higher capability of synthesis and secretion of P4. Moreover, the higher fat content of Mangalica could be responsible for the different clearance of steroid hormones. In conclusion, the results of the present study do not support the hypothesis that differences in the secretion of analysed reproductive hormones during the peri- and post-ovulatory periods are a possible cause of lower fecundity in Hungarian Mangalica pigs. Acknowledgements We would like to thank Dr. Roser JF (Davis, CA, USA) for the monoclonal antibody against b-lh and Drs. Antkewitz U and Rodewald S for their skilled technical help. Egerszegi I received a short term fellowship from the German Academic Exchange Service (DAAD). The work was supported by project grants from Hungarian OTKA T29992 and T038292, and from German BMVEL. We are grateful to Olmos and Toth Ltd. for providing the Mangalica gilts. References 1. Rácz M. Magyarország mangalicasertés tenyésztése. Különlenyomat Az Állattenyésztek Lapja 1932; Szabó P. A mangalica. Kistermelek Lapja 1999; 12: Rátky J, Brüssow K-P. Ovarian activity in gilts including some characteristics of a native breed. Reprod Dom Anim 1998; 33: Rátky J, Brüssow K-P, Solti L, Torner H, Sarlós P. Ovarian response, embryo recovery and results of embryo transfer in a Hungarian native pig breed. Theriogenology 2001; 56: Egerszegi I, Torner H, Rátky J, Brüssow K-P. Follicular development and preovulatory oocyte maturation in Hungarian Mangalica and Landrace gilts. Arch Tierz 2001; 44: Rodriguez H, Kunavongkrit A. Chronical venous catheterization for frequent blood sampling in unrestrained pigs. Acta Vet Scand 1983; 24: Rátky J, Brüssow K-P, Solti L. Endoscopic methods in swine reproductive research: a review. Acta Vet Hung 1998; 46: Schneider F, Bellmann A, Becker F, Bambang Puernomo S, Rehfeldt C, Nürnberg G, Kanitz W. Gonadotropin release in periovulatory heifers after GnRH analogs measured by two types of immuoassays. Exp Clin Endocrinol Diabet 2002; 110: Matteri RL, Roser JF, Baldwin DM, Lipovetsky V, Papkoff H. Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species. Domest Anim Endocrinol 1987; 4: Schneider F, Kanitz E, Deaver DR, Kanitz W, Brüssow KP. Nonisotopic sandwich-type assays in an electrochemiluminescence detection system. Exp Clin Endocrinol Diabet 1999; 107: 1 2.

6 296 EGERSZEGI et al. 11. Schneider F, Kanitz E, Gerrard DE, Kuhn G, Brüssow KP, Nürnberg K, Fiedler I, Nürnberg G, Ender K, Rehfeldt C. Administration of recombinant porcine somatotropin (rpst) changes hormone and metabolic status during early pregnancy. Domest Anim Endocrinol 2002; 23: Hunter MG, Biggs C, Foxcroft GR, McNeilly AS,Tilton JE. Comparisons of endocrinology and behavioural events during the periovulatory period in Meishan and Large-White hybrid gilts. J Reprod Fertil 1993; 97: Hunter MG, Picton HM, Biggs C, Mann GE, McNeilly AS, Foxcroft GR. Periovulatory endocrinology in high ovulating Meishan sows. J Endocrinol 1996; 150: Mariscal DV, Bergfeld EG, Cupp AS, Kojima FN, Fike KE, Sanchez T, Wehrman ME, JohnsonRK, Kittok RJ, Ford JJ, Kinder JE. Concentrations of gonadotropins, oestradiol and progesterone in sows selected on an index of ovulation rate and embryo survival. Anim Reprod Sci 1998; 54: Van Rens BT, Hazeleger W, van der Lende T. Periovulatory hormone profiles and components of litter size in gilts with different estrogen receptor (ESR) genotypes. Theriogenology 2000; 53: Brüssow K-P, Rátky J, Kanitz E. The influence of exogenous GnRH on the time of ovulation in gilts: an endocrine and laparoscopic study. Arch Tierz 1993; 36: Brüssow K-P, Kanitz E, Rátky J. The dynamic of plasma luteinizing hormone surge and its relationships to the time of ovulation in gilts following exogenous GnRH. Arch Tierz 1994; 37: George G, Brüssow K-P, Bergfeld J, Kanitz E, Blödow G. Experimental endocrinological studies in gilts near the time of ovulation using Gn-RH vet Berlin Chemie. Arch Exp Vet Med 1989; 43: Ogasa A, Tsutsui T, Kawakami E, Sone M, Kawarasaki T, Iwamura S. Response of the lactating and postweaning sow to gonadotropin releasing hormone (GnRH). J Vet Med Sci 1991; 53: Parvizi N, Elsaesser F, Smidt D, Ellendorff F. Plasma luteinizing hormone and progesterone in the adult female pig during the oestrous cycles, late pregnancy and lactation, and after ovariectomy and pentobarbitone treatment. J Endocrinol 1976; 69: Enne G, Perotti L, Delrio G, Inaudi P, d Istria M, Pierantoni R, Citarella F, Musaro MA, Monittola C, Genazzani AR. Endocrine profiles of sows during the oestrous cycle. Reproduccion 1981; 5: Redmer DA, Day BN. Ovarian activity and hormonal patterns in gilts fed allyltrenbolone. J Anim Sci 1981; 53: Ziecik A, Krzymowska H, Tilton JE. Porcine LH levels during the estrous cycle, gestation, parturition and early lactation. J Anim Sci 1982; 54: Van de Wiel DF, Erkens J, Koops W, Vos E, Van Landeghem AA. Periestrous and midluteal time courses of circulating LH, FSH, prolactin, oestradiol- 17β and progesterone in the domestic pig. Biol Reprod 1981; 24: Blair RM, Coughlin CM, Minton JE, Davis DL. Peri-oestrous hormone profiles, embryonic survival and variation in embryonic development in gilts and primiparous sows. J Reprod Fertil 1994; 101: Soede NM, Helmond FA, Kemp B. Periovulatory profiles of estradiol, LH and progesterone in relation to estrus and embryo mortality in multiparous sows using transrectal ultrasonography to detect ovulation. J Reprod Fert 1994; 101: Ziecik A, Tilton JE, Espana F, Weigl R. Effect of human chorionic gonadotropin on preovulatory luteinizing hormone surge and ovarian hormone secretion in gilts. J Anim Sci 1987; 64: