The use of melatonin and progestagen ecg to initiate reproductive activity in prepuberal Awassi ewe lambs

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1 Trop Anim Health Prod (2011) 43: DOI /s SI FAT TAILED SHEEP The use of melatonin and progestagen ecg to initiate reproductive activity in prepuberal Awassi ewe lambs Mohammad N. Sawalha & Rami T. Kridli & Khalil I. Jawasreh & Cesar A. Meza-Herrera Accepted: 12 April 2011 / Published online: 21 April 2011 # Springer Science+Business Media B.V Abstract This experiment was conducted to evaluate the effect of administering hormonal treatments (melatonin and progestagen/equine chronic gonadotropin (ecg)) on advancing puberty in Awassi ewe lambs. Fifty-one 6-monthold ewe lambs of similar body weights (around 28 kg) were randomly assigned into four treatment groups; control (CON; n=14), melatonin (MEL; n=13), melatonin plus progestagen ecg (MELPP; n=11), and progestagen ecg (PP; n=13). Ewe lambs in the PP and MELPP groups were fitted with intravaginal progestagen sponges containing 60 mg medroxyprogesterone acetate for 14 days; 400 IU ecg were administered to each of these ewe lambs on the day of sponge removal. Ewe lambs in the MEL and MELPP groups received subcutaneous melatonin implants (Regulin, 18 mg melatonin) 36 days before sponge insertion. Hormonal treatment had no effect on ewe lamb body weight change. Estrous behavior was greater (p< 0.001) in the PP and MELPP groups than in the CON and MEL ewe lambs. The duration from ram introduction to onset of estrus was shorter (p<0.05) in the CON, PP, and MELPP than in the MEL ewe lambs. The number of ewe lambs showing luteal activity was greater (p<0.001) in the MELPP and PP than in the CON and MEL groups. M. N. Sawalha : R. T. Kridli (*) : K. I. Jawasreh Department of Animal Production, Faculty of Agriculture, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan rkridli@just.edu.jo C. A. Meza-Herrera Universidad Autónoma Chapingo, Unidad Regional Universitaria de Zonas Áridas, Bermejillo, Durango, Mexico Lambing rate from mating during the first cycle following ram introduction differed significantly (p< 0.01) among treatments being greater in the MELPP and PP than in the CON and MEL groups. Based on these results, it can be concluded that progestagen ecg appears to be more effective than melatonin in inducing reproductive activity. However, the majority of ewe lambs in the current study either failed to cycle or stopped cycling following the induced estrus. Such failure in advancing puberty may be related to body weight of ewe lambs at the time of mating. Keywords Puberty. Sheep. Reproduction. Progestagen. Melatonin Introduction The age at which ewe lambs enter the breeding season for the first time in the most traditional sheep production systems in the Middle East region is around 18 months, thus they produce their first lamb at around 2 years of age while ewes are culled at 6 to 7 years of age after having produced five to six lamb crops (Gonzalez-Bulnes et al. 2010). Puberty is the result of a dynamic interaction between genetic factors and environmental cues, all of which lead to the attainment of reproductive capacity (Meza-Herrera et al. 2010a, b). Therefore, significant changes in hormone secretion occur from the prepubertal to the pubertal stage (Meza-Herrera 2008). Advancing puberty age along with early breeding allows ewe lambs to enter the breeding season at around 8 to 9 months of age, thus, obtaining one more lamb crop per female during her productive life.

2 1346 Trop Anim Health Prod (2011) 43: Progesterone is a female hormone produced mainly by the corpus luteum in the ovary and by the placenta. Anderson et al. (1996) reported that short-term progestin exposure will increase secretion of LH following progesterone withdrawal. Melatonin is a universal photoperiodic signal with diverse action. It regulates cyclic activity in ewes by affecting the hypothalamic-hypophyseal axis. These photoperiodic effects on reproduction are mediated via the secretion of melatonin from the pineal gland (Karsch et al. 1984). As melatonin is secreted during night, declining day length is associated with a longer pattern of melatonin secretion, which is stimulatory to reproduction in short-day breeders. As ewe lambs reach the desired body weight, decreasing photoperiod stimulates the release of melatonin which, in turn, leads to hormonal changes that stimulate estrus and ovulation (Kennaway et al. 1985). Melatonin alone may not be the only regulator of puberty induction as several authors indicated that the level of feeding affects the time of reaching puberty in ewe lambs (Epstein 1977; Foster and Olster 1985; Sharafeldin et al. 1969). A high pre-mating feeding level, with ram exposure, was found to induce puberty in Awassi ewe lambs (Kassem et al. 1989). Adequate body weight (35 40 kg) and continuous ram exposure can allow Awassi ewe lambs to lamb at 13 months of age (Kassem et al. 1989). Kridli et al. (2006b) indicated that the melatonin administration in combination with flourogestone acetate sponges and ecg can be successfully used to induce estrus in sheep. Melatonin and progestagen, separately and together, may be used to advance and condense the rates of lambing at the onset of the breeding season (Horoz et al. 2003). Melatonin implants have been shown to improve pregnancy and lambing rates and advance lambing in Sarda ewe lambs (Mura et al. 2010). Additionally, Papachristoforou et al. (2007) reported that melatonin administration to ewe lambs and goat kids during the last month of the spring advances the onset of reproductive activity by 80 days. There are very few reports on the use of exogenous melatonin and progesterone to advance the onset of puberty, hence the breeding season of fat-tailed ewe lambs. Our working hypothesis was that a combination of melatonin along with a progestagen ecg protocol would be more effective in improving reproductive performance in ewe lambs of late-maturity breeds (like Awassi) than either melatonin or induced estrus alone (Laliotis et al. 1997; Stellflugetal.1994). For this reason, this study was designed in an attempt to advance puberty and initiate the breeding season in Awassi ewe lambs through hormonal treatments. Two methods were utilized and compared: melatonin implants and progestagen ecg administration. Materials and methods This experiment was conducted at the National Center for Agricultural Research and Extension to evaluate the effect of administering hormonal treatments (melatonin and progestagen and equine chorionic gonadotropin (ecg)) on advancing puberty in Awassi ewe lambs. Fifty-one 6-month-old ewe lambs of similar body weights (around 28 kg) were randomly assigned into four treatment groups: control (CON; n=14), melatonin (MEL; n=13), progestagen and ecg (PP; n=13), and melatonin plus progestagen and ecg (MELPP; n=11). Ewe lambs were housed in a single barn, with free access to shade, and fed 0.5 kg per head per day of straw and 1 kg per head per day of concentrate mixture (barley, soybean, and wheat bran in addition to mineral and vitamin supplements), divided into morning and evening feedings. Trace mineral blocks and clean drinking water were provided ad libitum. At around 7.5 months of age, ewe lambs in the PP and MELPP groups were fitted with intravaginal progestagen sponges containing 60 mg medroxyprogesterone acetate (MAP; Esponjavet, Laboratories Hipra, Amer (Girona), Spain) for 14 days; 400 IU ecg (Laboratories Hipra, Amer (Girona), Spain) were administered to each of these ewe lambs on the day of sponge removal. Ewe lambs in the MEL and MELPP groups received subcutaneous melatonin implants (Regulin, 18 mg melatonin) 36 days before sponge insertion (around 6.5 months of age). Seven fertile, harnessed Awassi rams were introduced at the time of sponge removal (day 0). Rams were kept in a separate barn around 40 m away before the day they were allowed with the ewe lambs. Ewe lambs lambing up to 155 days following day 0 were considered to have conceived at induced estrus. Body weights were recorded at weaning (70 days of age), at melatonin implant insertion (180 days of age), at ram introduction (230 days of age), and at the end of the study when rams were removed (270 days of age). All experimental procedures were approved by the Institutional Animal Care and Use Committee. Blood samples (5 ml) were collected once weekly from all groups starting on the day of melatonin implant administration (day 50) until the day of ram introduction (day 0). Additional blood samples were collected every 3 days, thereafter, for 2 months to monitor luteal activity. Samples were collected via jugular venipuncture into lithium heparin vaccutainers and centrifuged (3,000 rpm for 15 min) within 1 h of collection. Plasma was harvested using plastic Pasteur pipettes into plastic tubes and stored frozen at 20 C until assayed for progesterone. Concentration of progesterone was determined by commercial radioimmunoassay kits (Immunotech, Marseille, France). Plasma progesterone samples were run in a single assay.

3 Trop Anim Health Prod (2011) 43: Intra-assay coefficient of variation was 5.4%. Sensitivity of the progesterone assay was 0.1 ng/ml. A progesterone concentration of >2 ng/ml was considered to be indicative of luteal activity. The reason for having considered the concentration of 2 ng/ml of progesterone as compared with a concentration of 1 ng/ ml as reported by Chagas e Silva et al. (2003) wasdueto differences in the type of assay between our study and theirs. We used a progesterone kit by Immunotech (France) which appears to over-estimate progesterone values (Oqla 2003). Data were analyzed by analysis of variance for completely randomized design. Differences in body weight and progesterone concentration were analyzed by split-plot analysis of variance for repeated measures. Estrous behavior, pregnancy, and lambing rates were analyzed by Chi-square. The interaction between melatonin and progestagen ecg administration on the various parameters was evaluated and found to be nonsignificant. Unless otherwise stated, data are presented as means±se. All analyses were conducted using the general linear model procedure for SAS (SAS 1994). Results and discussion Body weights As shown in the Fig. 1, no significant differences were observed in body weight of Awassi ewe lambs receiving progesterone ecg, melatonin or their combination when compared with non-treated females from weaning to the end of the experiment. At the beginning of the experiment, mean animal body weight was 28.6±0.8 kg. As expected, Weight (kg) Ram introduction Age (day) CON MEL MELPP PP Fig. 1 Body weight (mean±se) of Awassi ewe lambs, receiving no treatment (CON), melatonin (MEL), melatonin and progestagen plus ecg (MELPP), and progestagen plus ecg (PP) animal body weight increased as the experiment advanced due to body growth. Ewe lambs reached around 31 kg of body weight at the time of ram introduction. Kassem et al. (1989) reported that Awassi ewe lambs may begin to cycle at an early age (7 to 8 months) if they reach kg of body weight with continuous ram exposure starting at 5 months of age. At the time of ram removal (270 days of age) the overall mean body weight reached 36.2±1 kg. Previous studies have shown the mature weight of Awassi ewes to range between 50 and 55 kg (Awawdeh et al. 2009; Titi and Kridli 2008). Induced cycle reproductive responses As shown in Table 1, estrous behavior within 7 days from ram introduction was greater (p<0.001) in the PP and MELPP compared with CON and MEL ewe lambs (1/14 (7%), 2/13 (15%), 8/11 (64%), and 8/13 (62%) in the CON, MEL, MELPP, and PP treatment groups, respectively). Similarly, in an experiment utilizing similar treatments in mature Awassi ewes, Kridli et al. (2006b) reported that the estrous behavior following ram introduction differed among groups. The authors reported greater estrous behavior in PP and MELPP groups than in melatonin-treated or non-treated control ewes. The intervals from ram introduction to estrous behavior differed significantly (p<0.05) among treatments. Estrus was delayed in the MEL group compared with the remaining treatments. The reason that the CON group was similar to the other treatments was that only one ewe lamb expressed estrus within 7 days of ram introduction. These results indicate that treating ewe lambs with progesterone and ecg appears to accelerate the mechanism of follicular growth and development. Zarkawi et al. (1999) reported that estrus was induced within 36 to 48 h post-sponge withdrawal in 82% of Awassi ewes treated with MAP sponges followed by 600 IU ecg. In contrast, Vinoles et al. (2001) reported no significant difference in the interval to estrus occurrence in Polwarth ewes treated with MAP sponges plus ecg compared with ewes treated with MAP sponges alone. In the present study, progesterone priming and ecg were effective in speeding the process of follicular development, thus allowing for estrus to occur earlier. Previous studies have shown that exogenous melatonin (Rajkumar et al. 1989) and progestins (Safranski et al. 1992) promote ovarian activity and follicular development in the anestrous ewe. Additionally, Forcada et al. (2007) have shown that melatonin implants improve the responsiveness of the pituitary gland to GnRH stimulation. According to Chagas e Silva et al. (2003), puberty is considered to have occurred in ewe lambs when progesterone concentration exceeds 1 ng/ml in two successive blood samples. This concentration is indicative of luteal function. Plasma progesterone concentrations were at minimal levels

4 1348 Trop Anim Health Prod (2011) 43: Table 1 Induced estrus reproductive responses and luteal activity in Awassi ewe lambs receiving hormonal treatments Variable Treatment CON (n=14) MEL (n=13) MELPP (n=11) PP (n=13) Estrous behavior (n) 1 b 2 b 8 a 8 a Day of estrus occurrence following ram introduction 3.0 c± d± c± c±0.3 Luteal activity a (n) 0 b 0 b 10 a 10 a Day to first P 4 rise following ram introduction 7.9± ±0.3 Lambing rate (n) 0 d 0 d 3 c 4 c Values within a row with different lowercase letters differ (a and b, p<0.001 and c and d, p<0.05) CON no treatment, MEL melatonin implants only, MELPP, melatonin implants and intravaginal progesterone sponges plus ecg, PP intravaginal progesterone sponges plus ecg only a The number of ewe lambs with progesterone concentration of >2 ng/ml of less than 1 ng/ml from day 48 until day 4 (day 0=ram introduction). Thereafter, progesterone concentration started to increase rapidly in ewe lambs of MELPP and PP groups, and peaked on day 14, then started to decline until day 24 (Fig. 2). Progesterone concentrations increased slightly in ewe lambs of MEL group around day 24 but remained low in the control ewe lambs. None of the CON and MEL ewe lambs showed a significant progesterone rise within 10 days following ram introduction. Luteal activity was greater (p< 0.001) in the MELPP and PP than in the CON and MEL groups (Table 1). Lambing rate from mating during the first cycle following ram introduction differed significantly greater (p<0.01) in MELPP and PP groups (3/11 (27%) and 4/13 (31%), respectively) than in the CON and MEL groups (0/ 14 (0%) and 0/13 (0%), respectively). Only ewe lambs from groups that received progestagen and ecg were lambed. These results contradict the findings of Papachristoforou et al. (2007) who showed that melatonin administration advanced reproductive activity in ewe lambs and doe kids. Such contradiction is probably related to the ram effect, timing of melatonin administration, and the breed of sheep used. The above results also indicate that treating ewe lambs with progestagen and ecg was slightly more effective in inducing fertile estrus earlier during the prepubertal period than treating them with melatonin alone. Although the lambing rates were low (ranging around 30%), somewhat similar lambing rates were reported by Rekik et al. (2002) in ewe lambs treated with progestagen and ecg at 9 months of age. Mura et al. (2010) reported greater pregnancy rates and earlier lambing in melatonin-treated ewe lambs compared 8 7 CON MEL MELPP PP Progesterone concentration (ng/ml) Days from ram introduction Fig. 2 Plasma progesterone concentrations (mean±se) of Awassi ewe lambs receiving no treatment (CON), melatonin (MEL), melatonin and progestagen plus ecg (MELPP), and progestagen plus ecg (PP)

5 Trop Anim Health Prod (2011) 43: with the controls. In the current study, however, melatonin treatment failed to improve reproductive performance in Awassi ewe lambs. Even those animals that were lambed represent a very small percentage of the whole group. The reason behind that may be related to body weight, metabolic status, and age. Certainly, both body weight and metabolic status are a crucial signaling cues activating GnRH neurons and promoting the interaction among the hypothalamic-hypophyseal-gonadal components, resulting in the establishment of reproductive function (Flores-Najera et al. 2010; Guerra-García et al. 2009; Meza-Herrera et al. 2010a, b; Urrutia-Morales et al. 2009). Kridli et al. (2006a) have shown that Awassi ewe lambs reach puberty at around 280 days of age when they reach 36 kg of body weight. Thus, ewe lambs in the current study may not have reached a specific body weight or metabolic status required to activate some specific neural pathways in order to promote to their hypothalamic-hypophyseal axis to be responsive to melatonin stimulation and to maintain the level of cyclicity induced by progestagen ecg. This was evident by the fact that these ewe lambs responded only when exogenous gonadotropin was supplemented. As reported by Nowak and Rodway (1985), melatonin administration does not stimulate estrus if prepubertal ewes have not reached the appropriate body weight and age. Conclusions Based on the current results, it appears that melatonin implants were ineffective in inducing fertile estrus in prepubertal Awassi ewe lambs. Progestagen ecg treatment with or without melatonin resulted in slight improvements in reproductive responses and lambing rate. However, the majority of ewe lambs in the current study either failed to cycle or stopped cycling following the induced estrus. Such failure in advancing puberty may be due to body weight of ewe lambs at the time of ram introduction. Acknowledgments The authors wish to acknowledge the Deanship of Scientific Research at the Jordan University of Science and Technology and The National Center for Agricultural Research and Extension for funding this project. The assistance of Mr. Isam Shurman in data collection is highly appreciated. References Anderson, L.H., McDowell, C.M., and Day, M.L., Progestininduced puberty and secretion of luteinizing hormone in heifers, Biology of Reproductions, 54, Awawdeh, M.S., Obeidat, B.S., and Kridli, R.T., Yellow grease as an alternative energy source for nursing Awassi ewes and their suckling lambs. 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