Failure of Therapy to Induce Estrus in Gilts Treated with a GnRH Analog to Suppress Ovarian Activity

Antonio Garcia, DVM, PhD (deceased)a Mark J. Estienne, PhDb Allen F. Harper, PhDb James W. Knight, PhDb aDepartment of Large Animal Clinical Sciences Virginia-Maryland Regional College of Veterinary Medicine Blacksburg, VA 24061 bDepartment of Animal and Poultry Science Virginia Polytechnic Institute and State University Blacksburg, VA 24061

KEY WORDS: gilts, GnRH, gonadotropin, mm follicles 4 days after implant insertion ovarian activity was significantly (P < .05) higher in con- trols than in gilts receiving a deslorelin ABSTRACT implant. The percentage of deslorelin- A study was conducted to determine implanted gilts that displayed a normal whether treatment with deslorelin, a estrous cycle was significantly (P < .05) gonadotropin-releasing hormone (GnRH) lower than in individuals given a sham analog, produced anestrus in gilts and to test implant. There was no significant difference the ability of exogenous gonadotropin treat- in the percentage of implanted-gilts that dis- ment with a combination of 400 IU equine played estrus within 10 days after injection chorionic gonadotropin and 200 IU human of either 400 IU equine chorionic gonadotropin chorionic gonadotropin to induce estrus in and 200 IU human chorionic gonadotropin GnRH analog-treated gilts. Twelve days or deionized water. Treatment with a prod- after the third estrus, 11 gilts each received uct containing a combination of 400 IU a full SC implant containing 2.1 mg deslore- equine chorionic gonadotropin and 200 IU lin, a half implant containing 1.05 mg human chorionic gonadotropin failed to deslorelin, or a sham implant. The implants induce estrus in gilts caused to be in anestrus remained in place for the duration of the by implanting the GnRH analog deslorelin. study. Approximately 23 days after place- ment of implants, GnRH analog-treated gilts INTRODUCTION that had not returned to estrus (n = 18) Gonadotropin-releasing hormone (GnRH) is received IM injections of either 400 IU secreted by the hypophysiotropic neurons of equine chorionic gonadotropin and 200 IU the hypothalamus. The decapeptide induces human chorionic gonadotropin or deionized synthesis and secretion of water. The percentage of gilts with 3- to 4- (LH) and follicle-stimulating hormone (FSH)

Intern J Appl Res Vet Med • Vol. 2, No. 3, 2004 195 from gonadotropes located in the anterior pitu- by the Institutional Animal Care Committee. itary gland.1 Since the initial discovery of Prepubertal Yorkshire × Landrace gilts (n = GnRH as the hypothalamic factor controlling 42) approximately 180 days of age were gonadotropin release, numerous GnRH ago- given an IM injection of P.G. 600 containing nists and analogs have been developed and 400 IU equine chorionic gonadotropin (eCG) studied for their pro-fertility potential.2,3 and 200 IU human chorionic gonadotropin Paradoxically, suppressive effects of (hCG). Gilts were checked for estrus once potent GnRH analogs on reproduction have daily beginning at the time of P.G. 600 been reported. For example, chronic treat- administration. Estrus was defined as the ment of cows with a GnRH analog resulted period during which gilts displayed a lordo- in an initial stimulation of gonadotropin sis response in the presence of a mature release, which was soon followed by sup- boar. Within 7 days after the injection (4.3 ± pressed secretion, and ultimately, inhibition 0.1 days), 83% (35/42) of the gilts displayed of ovarian follicular development.4 The estrus. Of these 35, 97.1% subsequently dis- mechanism by which GnRH analogs cause played a second estrus and 94.3% a third reduced gonadotropin secretion and abated estrus at approximately 21-day intervals. ovarian function presumably involves a Gilts that had displayed three periods of desensitization or down regulation of GnRH estrus (i.e., two estrous cycles) were selected receptors on the anterior pituitary gland. to participate in the study. Miller et al5 reported that treatment of On Day 12 after the third estrus, 11 gilts gilts during the luteal phase of the estrous each received in the base of an ear a full SC cycle with a potent GnRH agonist caused implant containing 2.1 mg deslorelin, a half suppression of ovarian activity that was implant containing 1.05 mg deslorelin, or a reversed 28 days later by exogenous a sham implant. The deslorelin implants gonadotropin administration. Reproductive remained in place for the duration of the efficiency in swine breeding herds could be study. Gilts returning to estrus between Days 19 and 22 of the third estrous cycle enhanced by effective methods of synchro- were considered to have had a cycle of nor- nizing estrus in replacement gilts. In theory, mal duration. gilts could be treated with a GnRH analog Approximately 23 days after insertion of to cause a state of anestrus and then syn- implants (Day 35.4 ± 0.5), deslorin-treated chronized estrus could be induced by gilts that had not returned to estrus (n = 18) administration of gonadotropin. The objec- received IM injections of either P.G. 600 (n tives of this study were to determine = 9) or deionized water (n = 9). Of the nine whether a GnRH analog would produce a gilts that had received the full deslorelin state of anestrus in gilts and to test the abili- implant, four were injected with P.G. 600 ty of exogenous gonadotropin treatment to and five received deionized water. Of the induce estrus in GnRH analog-treated gilts. nine gilts that had received the half implant, Commercially available GnRH analog five were injected with P.G. 600 and four (Ovuplant; Fort Dodge Animal Health) and received deionized water. gonadotropin (P.G. 600; Intervet America) Ultrasonography products were employed. Transrectal ultrasonography of the ovaries MATERIALS AND METHODS was performed 4 days after implant insertion using a real-time, B-mode ultrasound Preparation of Subjects machine (Aloka SSD-500; Aloka) equipped The study was conducted at the Virginia with a linear-array 5-MHz transducer Tech-Tidewater Agricultural Research and designed for intrarectal placement. The gilts Extension Center in Suffolk, VA, and the were restrained in standing position inside a study protocol was reviewed and approved portable scale. The ultrasound transducer was

196 Vol. 2, No. 3, 2004 • Intern J Appl Res Vet Med modified by taping a half-moon polyvinyl lower than for sham-implanted individuals chloride pipe along the dorsal part of the (Table 1). There was no significant effect of probe to control manipulation of the transduc- treatment (P > .05) on the interestrus inter- er in the rectum. The modified transducer was val for gilts displaying estrus after implant well lubricated with corn oil before insertion insertion (Table 1). into the rectum and was gently passed along There were no significant (P > .05) dif- the rectal floor to avoid fecal material resist- ferences between groups in the concentra- ance and to establish good contact between tions of progesterone at implant insertion on the transducer and the rectal mucosa. Day 12 or at Day 16 of the estrous cycle. In Blood Samples and Radioimmunoassay deslorin-treated gilts, concentrations of prog- Beginning at implant insertion and continu- esterone in serum remained low until the ing until estrus or injection of either P.G. time of P.G. 600 or deionized water adminis- 600 or deionized water, blood samples were tration at approximately Day 35 (Table 1). collected via jugular venipuncture at 4-day There was no significant (P > .05) difference intervals. Samples were allowed to clot in the percentage of implanted-gilts that dis- overnight at 4˚C. Serum was harvested fol- played estrus within 10 days after injection of lowing centrifugation and stored at –20˚C either P.G. 600 or deionized water (Table 2). until assayed. Serum progesterone concen- trations were determined using a validated DISCUSSION radioimmunoassay method described for Implants containing the potent GnRH ana- swine by Tarraf and Knight.6 The intraassay log deslorelin were successful at producing and interassay coefficients of variation were anestrus in gilts by inhibiting follicular 4.6% and 8.9%, respectively. Assay sensitiv- growth. Four days after insertion of implants ity was 0.02 ng/mL serum. containing either 1.05 or 2.1 mg deslorelin, suppressed follicular development was Statistical Analysis observed ultrasonographically. The lack of The interestrus interval was defined as the estrous cyclicity was confirmed by the period between the onset of two subsequent observation of low progesterone concentra- periods of standing estrus. The percentage tions in serum for an extended period after of gilts displaying estrus after treatment and implant insertion. The mechanism mediating the percentage of gilts with 3- to 4-mm fol- this prolonged anestrus probably involved licles 4 days after treatment (Day 16 of suppression of gonadotropin secretion and estrous cycle) were compared using chi- ultimately an inhibition of follicular devel- square analysis. The interestrus interval and opment. Although serum concentrations of serum progesterone concentrations were were not determined in this analyzed by analysis of variance using the study, previous work from the authors’ labo- GLM procedure of SAS (SAS Institute). ratory demonstrated that treatment of male swine with deslorelin implants decreased RESULTS secretion of LH, testosterone, and .7 Insertion of implants containing the GnRH Treatment with the GnRH analog analog deslorin at Day 12 of the estrous deslorelin caused a desensitization of the cycle resulted in suppression of follicular female bovine pituitary gland to GnRH.8 growth. The percentage of gilts with 3- to 4- Treatment with deslorelin for 3 days result- mm follicles 4 days after implant insertion ed in a prolonged period of anestrus in post- was significantly (P < .05) higher in con- partum dairy cows implanted within 3 days trols than in gilts receiving a full or half of calving.9 As postulated in cattle, the implant (Table 1). The percentage of mechanism by which deslorelin reduces LH implanted gilts that displayed a normal secretion in pigs could involve a decrease in estrous cycle was significantly (P < .05) gonadotrope concentration of GnRH recep-

Intern J Appl Res Vet Med • Vol. 2, No. 3, 2004 197 Table 1. Reproductive Characteristics of Gilts Given a Full (2.1 mg) or Half (1.05 mg) GnRH Analog (deslorelin) or Placebo Implant on Day 12 of the Estrous Cycle Sham Full Implant Half Implant Variable (n = 11) (n = 11) (n = 11)

Percentage of gilts with 3- to 4-mm 100a 18.2b 18.2b follicles 4 days after implant Percentage of gilts displaying 19- to 100a 18.2b 18.2b 22-day estrous cycle Interestrus interval for gilts displaying 20.8 ± 1.0 20.0 ± 0.0 20.0 ± 1.4 19 to 22 d estrous cycle (days)c Progesterone concentrations (ng/mL serum)c Day 12 44.3 ± 9.5 38.5 ± 6.7 40.6 ± 8.2 Day 16 2.1 ± 2.4 12.0 ± 18.7 1.9 ± 1.3 Day 20 — 0.6 ± 0.9 0.2 ± 0.3 Day 24 — 0.4 ± 0.5 0.2 ± 0.3 Day 28 — 0.2 ± 0.2 0.2 ± 0.4 Day 32 — 0.1 ± 0.1 0.1 ± 0.1 Day 36 — 0.03 ± 0.03 0.1 ± 0.1 a,bValues within rows with different superscripts are significantly different (P < .05). cValues are mean ± SD. Table 2. GnRH Analog-Implanted Gilts in Estrus Within 10 Days after Treatment with P.G. 600 or Deionized Water Treatment Half Implant (1.05 mg deslorelin) Full Implant (2.1 mg deslorelin) No. of Gilts Gilts in Estrus (%) No. of Gilts Gilts in Estrus (%) P.G. 600a 50 425 Deionized water 4 0 5 0 aP.G. 600 = 400 IU equine chorionic gonadotropin and 200 IU human chorionic gonadotropin.

tors and GnRH receptor mRNA.10 The effect from the use of implants containing deslore- of deslorelin on LH β-subunit synthesis lin. In accordance with these findings, could be another mechanism mediating gonadotropin therapy failed to induce folli- reduced secretion of LH after continuous cle growth in gilts with low gonadotropin exposure to GnRH in pigs. When deslorelin levels caused by hypophysectomy, implants were inserted on Day 7 postpartum hypophysial stalk-transection, or immuniza- in diary cows, of the follicles pres- tion against GnRH.12–14 ent was observed in 62% of the animals.11 Miller et al5 gave an IM injection of However, a subsequent block in follicular 7.5 mg of the GnRH agonist to growth and lower plasma progesterone and gilts (Decapeptyl, Ferring) between Days 7 estradiol concentrations were also observed and 14 of the estrous cycle, resulting in a for approximately 50 days, suggestive of suppression of LH and FSH secretion and a down regulation of GnRH receptors. block of follicular development. In contrast The use of deslorelin in combination with results of the present study, however, with 400 IU eCG and 200 IU hCG for syn- follicle growth in triptorelin-treated gilts chronizing estrus in gilts would appear to was stimulated by an injection of 1,500 IU have little value. In the study described here, eCG given 28 days later. Gilts were slaugh- treatment with eCG and hCG (P.G. 600) tered to recover ovaries 72 hours after eCG failed to produce estrus in gilts in anestrus treatment, so it is uncertain whether or not

198 Vol. 2, No. 3, 2004 • Intern J Appl Res Vet Med the gilts would have eventually displayed Gonadotropin-releasing hormone: one polypep- behavioral estrus and ovulated. tide regulates secretion of luteinizing and folli- cle-stimulating hormones. Science 1971; The dichotomous results between the cur- 173:1036–1038. 5 rent study and the report of Miller et al are 3. Karten MJ, Rivier JE: Gonadotropin-releasing not easily explained, but it could be due to hormone analog design. Structure-function stud- the different compounds used to cause a state ies toward the development of agonists and antagonists: Rationale and perspective. of anestrus. Perhaps the implants used in the Endocrinol Rev 1986; 7:44–66. report described here as well as the surgical 4. Gong JG, Campbell BK, Bramley TA, Gutierrez manipulations and treatments previously CG, Peters AR, Webb R: Suppression in the employed caused such a severe suppression secretion of follicle-stimulating hormone and of endogenous LH and/or FSH secretion that luteinizing hormone, and ovarian follicle devel- opment in heifers continuously infused with treatment with exogenous gonadotropins gonadotropin-releasing hormone agonist. Biol could not drive follicular development.12–14 In Reprod 1996; 55:68–74. 15 support of this notion, Kraeling et al report- 5. Miller AT, Picton HM, Hunter MG: Suppression ed that eCG stimulated follicular growth and of ovarian activity in the gilt and reversal by ovulation in hypophysial stalk-transected gilts exogenous gonadotrophin administration. Anim Reprod Sci 1999; 54:179–193. receiving pulsatile administration of GnRH but not in hypophysial stalk-transected 6. Tarraf CG, Knight JW: Effect of intrauterine posi- tion on conceptus development, placental and females receiving saline. Alternatively, the endometrial release of progesterone and estrone doses of gonadotropins used in the current in vitro, and concentration of steroid hormones in study (400 IU eCG and 200 IU hCG) may fetal fluids throughout gestation in swine. Domest Anim Endocrinol 1995; 12:179–187. have been an insufficient stimulus. Miller et al5 administered 1,500 IU eCG. 7. Estienne MJ, Harper AF, Knight JW, Barb CR, Rampacek GB: Sexual behavior after treatment The GnRH analog deslorelin produced a with prostaglandin-F2α in boars with suppressed state of anestrus in gilts, presumably due to a concentrations of gonadal steroids. Appl Anim desensitization of pituitary GnRH receptors, Behav Sci 2004. In press. a subsequent suppression of gonadotropin 8. Padula AM, Trigg TE, Macmillan KL: Deslorelin implants prevent early ovulations in early post- secretion, and ultimately, an inhibition of fol- partum Holstein dairy cattle [Abstract]. Proc licular growth. Treatment with 400 IU eCG Aust Soc Reprod Biol 1999; 30:123. and 200 IU hCG failed to cause estrus in 9. Padula AM, McGowan MR, Verrall R, Trigg TE, gilts that were in anestrus by treatment with Macmillan KL: Absorbable deslorelin implants the GnRH analog. delay resumption of ovulatory cycles in postpar- tum dairy cattle [Abstract]. Proc 4th Intl Congr Anim Reprod; 2000. ACKNOWLEDGMENTS 10. Vizcarra JA, Wettemann RP, Braden TD, Turzillo The authors wish to thank Dr. Jon B. AM, Nett TM: Effect of gonadotropin-releasing Rosenberg from Fort Dodge Animal Health hormone (GnRH) pulse frequency on serum and pituitary concentrations of luteinizing hormone for the generous supply of deslorelin and follicle-stimulating hormone, GnRH recep- implants used in this study and the technical tors, and messenger ribonucleic acid for support of the staff at the Tidewater gonadotropin subunits in cows. Endocrinology Agricultural Research and Extension Center. 1997; 138:594–601. 11. Mattos R, Orlandi C, Williams J, Staples CR, Trigg REFERENCES T, Thatcher WW: Effect of an implant containing the GnRH agonist deslorelin on secretion of LH, 1. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, ovarian activity and milk yield of postpartum dairy Knobil E: Hypophysial responses to continuous cows. Theriogenology 2001; 56:371–386. and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 1978; 12. Kraeling RR, Davis BJ, Gerrits RJ: Ovarian 202:631–633. response of the hypophysectomized pig to preg- nant mare’s serum gonadotropin. Biol Reprod 2. Schally A, Arimura A, Kastin A, et al: 1974; 10:116–119.

Intern J Appl Res Vet Med • Vol. 2, No. 3, 2004 199 13. Kraeling RR, Barb CR, Rampacek GB: Ovarian releasing hormone. J Anim Sci 1987; 65:1768–1774. response of the hypophysial stalk-transected pig 15. Kraeling RR, Kesner JS, Estienne MJ, Estienne to pregnant mare serum gonadotropin. Domest CE, Barb CR, Rampacek GB: Follicle growth Anim Endocrinol 1986; 3:177–183. in hypophysial stalk-transected pigs given 14. Esbenshade KL: Ovarian response to pregnant mare pulsatile GnRH and pregnant mare serum serum gonadotropin and porcine pituitary extract in gonadotropin. Domest Anim Endocrinol 1990; gilts actively immunized against gonadotropin 7:395–402.

200 Vol. 2, No. 3, 2004 • Intern J Appl Res Vet Med