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Reproductive performance of ewe lambs in non-breeding season exposed to hCG at day 12 post mating
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Reproductive performance of ewe lambs in non-breeding season exposed to hCG at day 12 post mating R. Catalano a,∗ , M. Teruel a , C. González a , S. Williams b , I. Videla Dorna c , S. Callejas a a b c
Facultad de Ciencias Veterinarias UNCPBA, Tandil, Argentina Facultad de Ciencias Veterinarias UNLP, La Plata, Argentina Laboratorio SYNTEX, Argentina
a r t i c l e
i n f o
Article history: Received 17 September 2014 Received in revised form 19 December 2014 Accepted 31 December 2014 Available online xxx Keywords: Sheep Progesterone Multiple ovulations Pregnancy Fetal growth
a b s t r a c t The objective of this experiment was to determine the effect of hCG treatment at day 12 post mating on ovarian characteristics, reproductive performance and conceptus growth in ewe lambs induced estrus during the non-breeding season. Corriedale ewe lambs 8 months old were pretreated with progestagen sponges for 10 days and 300 IU of eCG. At day 12 post mating, females were treated with either physiological saline (hCG-0; n: 53) or 300 IU of hCG (hCG-300; n: 50). Plasma progesterone concentrations were measured at day 17 post mating by radioimmunoassay. Pregnancy was monitored by transrectal ultrasonography on day 40 post mating. At day 41 post mating, 10 ewe lambs of the hCG-0 group and 9 of the hCG-300 group with single pregnancy (randomly chosen) were euthanized and subjected to a morphometric analysis of ovary, uterine and fetal structures. Plasma progesterone levels in the hCG-300 group were significantly higher than those in the control ewe lambs (P < 0.05). Pregnancy (49.18% and 46.15%), fertility (60.0% and 56.6%) and prolificacy (1.43 and 1.23) did not differ between the hCG treated and non treated ewe lambs. However, a tendency for a high number of ewe lambs with multiple pregnancies was observed in the hCG-300 group (P = 0.08). The percentage of ewe lambs with multiple ovulation was higher in the hCG-300 group compared to the controls (77.8% and 20.0%; P < 0.05), while ovary weight and diameters and corpora lutea area did not differ between groups (P > 0.05). The length of pregnant horn in ewe lambs of the hCG-300 group showed a tendency to be greater compared with that of the hCG-0 group (P = 0.08). Differences were not detected between groups for the uterine weight, number of caruncles and chorionic sac length (P > 0.05). Administration of hCG at day 12 post mating increased cranium-caudal length. However, no difference was observed in biparietal diameter and fetal weight between control and hCG treated ewe lambs. Results indicate that hCG treatment did not improve pregnancy rate, prolificacy or fetal weight, however the increase in the plasma progesterone concentration and the percentage of multiple ovulations observed in ewe lambs during the non-breeding season suggest a positive action of this hormone on reproductive performance. © 2015 Published by Elsevier B.V.
∗ Corresponding author at: Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires. Pinto 399 B7000GHG Tandil, Argentina. Tel.: +54 02494439850. E-mail address: [email protected] (R. Catalano). http://dx.doi.org/10.1016/j.smallrumres.2014.12.014 0921-4488/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Catalano, R., et al., Reproductive performance of ewe lambs in non-breeding season exposed to hCG at day 12 post mating. Small Ruminant Res. (2015), http://dx.doi.org/10.1016/j.smallrumres.2014.12.014
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1. Introduction Insufficient luteal function may be responsible for embryo loss in sheep (Ashworth et al., 1989; Nancarrow, 1994; Wilmut et al., 1986). Inadequate ovarian stimulation by gonadotropins during the periovulatory period (Davies and Beck, 1993) or in the first days post conception (Khan et al., 2003; Nephew et al., 1994) can be related with poor response luteal. The first 3 weeks of pregnancy is the critical period. The embryonic losses which range from 20% to 40% of fertilized eggs in adult ewes reaching 50% in ewe lambs (Quirke et al., 1983) and 63% in ewe lambs mated at synchronized oestrus after a progestagen-eCG treatment (Gordon, 1977) occur in this period. Hormones like progesterone, GnRH analogs and human chorionic gonadotropin (hCG) have been used in sheep and cattle during early pregnancy with the objective to reduce the embryo mortality (Cam et al., 2002; Cam and Kuran, 2004; Khan et al., 2007, 2009; Nephew et al., 1994; Sreenan et al., 1996; Thatcher et al., 2001). Several studies have employed hCG to increase plasma progesterone levels since this hormone is similar to LH in function. Thus, it has been used in ewes to improve lambing rate, litter size and fetal growth (Bobowiec et al., 2012; Cole, 2009; Khan et al., 2003, 2007, 2009; Kittok et al., 1983; Ishida et al., 1999). Adequate progesterone concentrations play an important role in controlling the maternal secretion of nutrients, growth factors and immunosuppressive agents required for successful embryo development and pregnancy recognition (Spencer and Bazer, 2002). Khan et al. (2007, 2009) demonstrated that hCG administrated on day 12 post-mating in cyclic ewes improves plasma progesterone and oestradiol concentrations, weight of corpora lutea, conceptus growth and placental attachment. However, the observed positive effects of hCG administration in adult ewes were less effective in cyclic ewe lambs (Khan et al., 2007, 2009). Anestrous lactating ewes with 20–30 days postpartum (Kittok et al., 1983) and anestrous ewes with low fertility rates (Gomez-Brunet et al., 2007) improved the fertility when they were treated with hCG. This suggests that in those categories of sheep with difficulty to maintain pregnancy, the administration of hCG could improve the reproductive performance. In pre-puberal ewe lambs with estrus induction treatment, the administration of hCG during the luteal phase, increased the luteal area and plasmatic progesterone concentration (Catalano et al., 2012). However, the effect of hCG administered in ewe lambs during the non-breeding season on reproductive performance has not been studied. Therefore, the objective of the present study was to assess the effect of hCG administration at day 12 post mating on reproductive performance in pre-puberal ewe lambs with induction estrus during the non-breeding season. 2. Materials and methods 2.1. Animals and management The experiment was conducted at Field Station of Faculty of Veterinary Sciences UNCPBA, (37◦ S, 60◦ W) Tandil, Argentina during the non-breeding season (spring season).
One hundred and twenty-six Corriedale ewe lambs 8 months old with live weight of 39.2 ± 3.7 kg (mean ± SEM) and body condition score of 3.2 ± 0.5 (mean ± SEM) according to Russel et al. (1969) selected from an overall herd were used. The ewe lambs were continuously exposed to natural photoperiod and handled according to the Animal Welfare Act (Resol. 087/02, Faculty of Veterinary Sciences, UNCPBA) and the Principles of Ethics of the European Commission for experiments involving animals (Directive 2010/63/EU). Transrectal ultrasonography studies were performed before the ovulation induction treatment to confirm absence of luteal structures. Two exams were carried out separated by seven days using a 7.5 MHz transducer (SonoVet 900. Medison, Co, Korea). To induce ovulation, all ewe lambs were pretreated with an intravaginal sponge impregnated with 60 mg of medroxyprogesterone acetate (Progespon® , Laboratorios Syntex S.A., Argentina) for 10 days and 300 IU of equine chorionic gonadotropin (eCG - Novormon® , Laboratorios Syntex S.A., Argentina) were injected intramuscularly upon withdrawal of sponges. Estrus detection and mating were performed in pens with fertile rams (relation ram-to-ewe lambs 1:10). The chest of rams was painted with chalk twice a day during the mating period. Estrus was considered when the ewes stood to be mounted by the rams or when results painted at the rump region. Estrus observation was performed twice daily; 06:00–07:00 h and 18:00–19:00 h during five days from sponge removal. 2.2. Human chorionic gonadotropin (hCG) treatment One hundred and three ewe lambs showing estral characteristics were mated and allocated to two groups on the basis of body weight and condition score using a system of randomized stratification. At day 12 post mating, females were given a single intramuscular injection of either 1 cc of physiological saline (hCG-0; n: 53) or 300 IU of hCG (Ovusyn® , Laboratorio Syntex S.A., Argentina), (hCG-300; n: 50). 2.3. Blood collection and progesterone assay Jugular venous blood was collected for determination of progesterone concentration in all ewe lambs at day 17 post mating. Immediately, plasma was obtained and stored at −20 ◦ C until assayed for progesterone. A radioimmunoassay (RIA) kit (Diagnostic Product Corporation, Los Angeles, CA, USA) was used. The sensitivity of the assay was 0.01 ng/ml, the intraassay coefficient of variation was below 7% for concentrations between 0.1 and 40.0 ng/ml and the inter-assay coefficient of variation was 3.5%. 2.4. Pregnancy diagnosis and related variables Pregnancy was monitored by transrectal ultrasonography using a SonoVet 900 (Medison, Co, Korea), real-time linear-array scanner equipped with 5 MHz transducer on day 40 post mating. Pregnancy rate (number of pregnant ewe lambs/ewe lambs with estrous induction treatment per 100), ewe lambs with multiple pregnancy (females with two fetuses or more/pregnant ewe lambs per 100), prolificacy (number of fetuses/pregnant ewe lambs per 100) and fertility (number of pregnant ewe lambs/ewe lambs with estrus characteristics per 100) were recorded. 2.5. Morphometric analysis of ovarian, uterine and fetal structures At day 41 post mating, 10 ewe lambs of hCG-0 group and 9 ewe lambs of hCG-300 group with single pregnancy (randomly chosen) were euthanized in agreement with the Animal Welfare Act (Resol. 087/02, Faculty of Veterinary Sciences, UNCPBA) and according to the Principles of Ethics of the European Commission for experiments involving animals (Directive 2010/63/EU). Reproductive tracts were collected and a morphometric analysis of ovary, uterine and fetal structures was carried out. The percentage of ewe lambs with multiple ovulations was determined by count of corpora lutea. The area of each corpus luteum was calculated by the formula = × (long diameter/2) × (short diameter/2) according to Hruska (1996). The total luteal area by ewe lamb was determined by the sum of the areas of each corpus luteum. Uteri gravid weight, ovarian diameters and weight (mean of both ovaries by ewe lambs) were registered. Once externalized the conceptus, weight, length and diameter of pregnant uterine horn, caruncles number of pregnant uterine horn, chorionic sac length and amniotic liquid weight were recorded. Fetal body
Please cite this article in press as: Catalano, R., et al., Reproductive performance of ewe lambs in non-breeding season exposed to hCG at day 12 post mating. Small Ruminant Res. (2015), http://dx.doi.org/10.1016/j.smallrumres.2014.12.014
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R. Catalano et al. / Small Ruminant Research xxx (2015) xxx–xxx weight and body measures (cranium-caudal length, crown-rump length and biparietal diameter) were also determined. 2.6. Statistical analysis Ovarian, uterine and fetal variables were shown as mean ± standard error and were analyzed as a completely randomized design (the treatments were characterized by the administration of 0 IU or 300 IU of hCG at day 12 post mating). The percentage of ewe lambs with multiple ovulations, percentage of pregnancy, fertility, prolificacy and the percentage of ewe lambs with multiple pregnancies were analyzed using CATMOD from SAS (1989). Significant differences were considered when P < 0.05.
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Table 2 Ovarian characteristics in ewe lambs administered with hCG at day 12 post mating and euthanized at day 41 of pregnancy. Variable
hCG-0 group
hCG-300 group
Ovarian weight (g ± SEM) Ovarian long diameter (cm ± SEM) Ovarian short diameter (cm ± SEM) Total area of corpus luteum (cm2 ± SEM)
1.03 ± 0.2 1.43 ± 0.1
1.17 ± 0.2 1.50 ± 0.1
1.10 ± 0.1
1.18 ± 0.1
1.25 ± 0.2
1.88 ± 0.3
Table 3 Morphometric analysis of uterine and placental variables in ewe lambs administered with hCG at day 12 post mating and euthanized at day 41 of pregnancy.
3. Results The plasma progesterone concentration evaluated at day 17 post mating (in ewe lambs diagnosed as pregnant at day 40 post mating) was significantly different between groups: 6.30 ± 0.38 ng/ml and 10.89 ± 0.88 ng/ml, for hCG0 and hCG-300 groups, respectively (P < 0.05). The administration of 300 IU of hCG at day 12 post mating did not modify the pregnancy rate, fertility nor the prolificacy compared with the control hCG-0 group (P > 0.05. Table 1). The hCG-300 group showed a trend to a higher percentage of ewe lambs with multiple pregnancies (P = 0.08, Table 1). The percentage of ewe lambs with multiple ovulation was higher in hCG-300 group than in hCG-0 group (77.8% vs. 20.0%; P < 0.05); however no significant differences in the weight and diameters of both ovaries and corpora lutea area between groups were recorded (P > 0.05, Table 2). The length of pregnant horn in ewe lambs of hCG-300 group showed a tendency to be greater compared with that of the hCG-0 group (P = 0.08, Table 3). Differences were not detected between groups for the other parameters evaluated (P > 0.05, Table 3). With respect to the fetal morphometry, the administration of 300 IU of hCG at day 12 post mating did not modify the fetal weight, nor the biparietal diameter (P > 0.05, Table 4). However, the fetuses of the hCG-300 group showed a trend to have greater crown-rump length Table 1 Reproductive response in ewe lambs administered with hCG at day 12 post mating. Variable a
Pregnancy rate Fertilityb Prolificacyc
Percentage of ewe lambs with multiple pregnanciesd
hCG-0 group
hCG-300 group
46.15 (30/65) 56.6 (30/53) 1.23 (37/30) 23.33x (7/30)
49.18 (30/61) 60.0 (30/50) 1.43 (43/30) 43.33y (13/30)
x-y: (P = 0.08). a Number of pregnant ewe lambs/ewe lambs with ovulation induction treatment per 100. b Number of pregnant ewe lambs/ewe lambs with estrus characteristics per 100. c Number of fetuses/pregnant ewe lambs per 100. d Number of ewe lambs with more than one fetuses/pregnant ewe lambs per 100.
Variable Pregnant uterine horn length (cm ± SEM) Pregnant uterine horn diameter (cm ± SEM) Uterine weight (g ± SEM) Pregnant uterine horn weight (g ± SEM) Caruncles in the gravid horn (n ± SEM) Chorionic sac length (cm ± SEM) Amniotic fluid weight (g ± SEM)
hCG-0 group 16.0 ± 0.6x 4.4 ± 0.2
hCG-300 group 17.34 ± 0.4y 4.4 ± 0.1
238.1 ± 14.0 199.6 ± 41.2
241.9 ± 11.5 196.9 ± 9.2
63.3 ± 4.5
65.4 ± 5.4
70.0 ± 3.0
70.4 ± 2.4
32.9 ± 2.6
35.8 ± 1.6
x-y: (P = 0.08). Table 4 Morphometric analysis of fetal variables in ewe lambs treated with hCG at day 12 post mating and euthanized at day 41 of pregnancy. Variable
hCG-0 group
Fetal weight (g ± SEM) Biparietal diameter (cm ± SEM) Crown-rump length (cm ± SEM) Cranium-caudal length (cm ± SEM)
6.5 1.2 4.1 5.0
± ± ± ±
0.2 0.02 0.1x 0.1a
hCG-300 group 6.5 1.2 4.6 5.3
± ± ± ±
0.2 0.04 0.1y 0.1b
a-b: (P = 0.05); x-y:(P = 0.06).
(P = 0.06) and the cranium-caudal length was higher than that in fetuses of the hCG-0 group (P = 0.05, Table 4). 4. Discussion It is well known that an inadequate luteal function is an important cause of reduced reproductive efficiency due to preimplantation losses (Ashworth et al., 1989; Nancarrow, 1994; Wilmut et al., 1986). In order to reduce embryo mortality, supplementation with exogenous progesterone, GnRH analogs and human chorionic gonadotropin (hCG) during early pregnancy have been used in sheep and cattle (Cam et al., 2002; Cam and Kuran, 2004; Khan et al., 2007, 2009; Nephew et al., 1994; Sreenan et al., 1996; Thatcher et al., 2001). In the present study, the ewe lambs treated with 300 IU of hCG at day 12 post mating showed an increase in plasma progesterone concentration and higher percentage of multiple ovulations. Prior findings have reported however, that the administration of hCG during the luteal phase provoke the ovulation of a dominant follicle and the consequent development of an accessory
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corpus luteum in cyclic ewes, but not in cyclic ewe lambs (Khan et al., 2009). This may be attributed to the ewe lambs not having well developed follicles able to be ovulatory in response to hCG treatment (Khan et al., 2009). Probably the higher percentage of multiple ovulations obtained in this work may be due to the accessory corpora lutea in ewe lambs treated with hCG at day 12 post mating. This is in agreement with the report that the administration of hCG at day 12 post mating in goats with estrous synchronization and natural mating increased the number of corpora lutea (Lashari and Tasawar, 2010). The differences observed with the study of the Khan et al. (2009) might be because the race, the reproductive season and the treatment of control of estral cycle used were different. Previous studies indicated that hCG administrated to ewe lambs showed no improvement the reproductive performance by the limited embryonic viability, which limit the response to hormonal treatments and then no improvement the embryonic survival (Beck et al., 1996; Khan et al., 2009). Moreover, it has been noted that the steroids hormone production by the ovary in pregnancy ewe lambs is lower than in pregnancy ewes post hCG treatment (Khan et al., 2007). In the present study, the hCG treatment there was no increase the pregnancy rate or the prolificacy. However, increased progesterone concentration and percentage of ewe lambs with multiple ovulations were observed. These results, in addition to the tendency of an increase in the percentage of ewe lambs with multiple pregnancies, disagree with the previously cited works. These differences could possibly be due to the different reproductive characteristics. In the present study ewe lambs during the non breeding season were used, whereas ewe lambs with estrous synchronized in breeding season were employed by Khan et al. (2007). Human chorionic gonadotropin has been reported to increase the size of conceptus in relation to higher IFN-tau production and plasma progesterone concentrations (Khan et al., 2003; Nephew et al., 1994). This gonadotropin moreover has been shown to increase gravid uterine weight, fetal weight and length and placental weight (Cam and Kuran, 2004). The results in the present study demonstrated that hCG administered to 12 days after service increased the cranium-caudal length, whereas the length in gravid uterine horns and the fetal crown-rump length showed a trend to be higher. The hCG has been postulated to increase the progesterone level and consequently the fetal growth (Garrett et al., 1988; Kleemann et al., 1994), probably by promoting an environment rich in nutrients and growth factors (Barnes, 2000), which could explain the observed results. In conclusion, this study indicates that the administration of hCG at day 12 post mating increased the plasma progesterone concentration, the percentage of multiple ovulations and the cranium-caudal length, however, no significant changes in pregnancy rate, prolificacy or fetal weight were observed. Therefore, further research is necessary to establish the relationship between hCG and maternal/embryonic factors as hormones, growth factors, citoquines that influence growth and differentiation of the conceptus in pre-puberal ewe lambs.
Acknowledgements The authors thank the students for taking part of the project. This research was supported by grants from CIC (Comisión de Investigaciones Científicas de la Provincia de Buenos Aires n◦ 18/2006). References Ashworth, C., Sales, D., Wilmut, I., 1989. Evidence of an association between the survival of embryo and the periovulatory plasma progesterone concentration in the ewes. J. Reprod. Fertil. 87, 23–32. Barnes, F., 2000. The effect of the early uterine environment on the subsequent development of embryo and fetus. Theriogenology 53, 649–658. Beck, N., Davies, M., Davies, B., 1996. A comparison of ovulation rate and late embryonic mortality in ewe lambs and ewes and the role of late embryo loss in ewe lamb subfertility. Anim. Sci. 62, 79–83. ˜ Bobowiec, R., Kosior-Korzecka, U., Patkowski, K., Gruszecki, T., Tusinska, E., 2012. Reproductive performance of PLS and BCP ewes exposed to hCG at the follicular phase of the estrous cycle. Med. Weter. 68, 226–230 [Online] http://www.medycynawet.edu.pl/images/ stories/pdf/pdf2012/042012/201204226230.pdf Cam, M.A., Kuran, M., Yildiz, S., Selcuk, E., 2002. Fetal growth and reproductive performance in ewes administered GnRH agonist on day 12 post-mating. Anim. Reprod. Sci 72, 73–82. Cam, M., Kuran, M., 2004. Effects of a single injection of hCG or GnRH agonist on day 12 post mating on fetal growth and reproductive performance of sheep. Anim. Reprod. Sci. 80, 81–90. Catalano, R., Teruel, M., Gonzalez, C., Williams, S., Videla Dorna, I., Callejas, S., 2012. The effect of hCG treatment on follicular characteristics and luteal function in seasonally anestrous ewes lambs. J. Anim. Vet. Adv. 11, 2091–2096. Cole, L., 2009. New discoveries on the biology and detection of human chorionic gonadotropin. Reprod. Biol. Endocrinol. 7, 8 [Online] http://www.rbej.com/content/7/1/8 Davies, M., Beck, N., 1993. A comparison of plasma prolactin LH and progesterone concentration during estrous and early pregnancy in ewe lambs and ewes. Anim. Prod. 57, 281–286. Garrett, J., Geisert, R., Zavy, M., Morgan, G., 1988. Evidence for maternal regulation of early conceptus growth and development in beef cattle. J. Reprod. Fertil. 84, 437–446. Gomez-Brunet, A., Santiago-Moreno, J., Montoro, V., Garde, J., Pons, P., Gonzalez-Bulnes, A., López-Sebastián, A., 2007. Reproductive performance and progesterone secretion in estrus-induced Manchega ewes treated with hCG at the time of AI. Small Rumin. Res. 71, 117–122. Gordon, I., 1977. Controlled Reproduction in Sheep and Goats. CAB, Slough, pp. 330–346. Hruska, K., 1996. Milk progesterone determination in dairy cows. Reprod. Dom. Anim. 31, 483–485. Ishida, N., Okada, M., Sebata, K., Minato, M., Fukui, Y., 1999. Effect of GnRH and hCG treatments for enhancing corpus luteum function to increase lambing rate of ewes artificially inseminated during the non-breeding season. J. Reprod. Dev. 45, 73–79. Khan, T., Beck, N., Khalid, M., 2007. The effects of GnRH analogue (buserelin) or hCG (Chorulon) on day 12 of pregnancy on ovarian function plasma hormone concentrations conceptus growth and placentation in ewes and ewe lambs. Anim. Reprod. Sci. 102, 247–257. Khan, T., Beck, N., Khalid, M., 2009. The effect of hCG treatment on day 12 post-mating on ovarian function and reproductive performance of ewes and ewe lambs. Anim. Reprod. Sci. 116, 162–168. Khan, T., Hastie, P., Beck, N., Khalid, M., 2003. hCG treatment on day of mating improves embryo viability and fertility in ewe lambs. Anim. Reprod. Sci. 76, 81–89. Kittok, R., Stellflug, J., Lowry, S., 1983. Enhanced progesterone and pregnancy rate after gonadotropin administration in lactating ewes. J. Anim. Sci. 56, 652–655. Kleemann, D., Walker, S., Seamark, R., 1994. Enhanced fetal growth in sheep administered progesterone during the first three days of pregnancy. J. Reprod. Fertil. 102, 411–417. Lashari, M., Tasawar, Z., 2010. The effect of hCG given on day 12 postmating on ovarian function and embryo survival in Beetal goats in southern Punjab Pakistan. Turk. J. Vet. Anim. Sci. 34, 513–517. Nancarrow, C., 1994. Embryonic mortality in the ewe and the doe. In: Zavy, M.T., Geisert, R.D. (Eds.), Embryonic Mortality in Domestic Species. CRC, Press, Boca Ratón, FL, pp. 79–97. Nephew, K., Cárdenas, H., McClure, K., Ott, T., Bazer, F., Pope, W., 1994. Effects of administration of human chorionic gonadotropin or
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Sreenan, J., Diskin, M., Dunne, L., 1996. Embryonic mortality: the major cause of reproductive wastage in cattle. In: Proceedings of the 47th Annual Meeting of the European Association of Animal Production, Lillihammer, August. Páginas. Thatcher, W., Guzeloglu, A., Binelli, M., Hansed, T., Pru, J., 2001. Uterine conceptus interactions and reproductive failure in cattle. Theriogenology 56, 1435–1450. Wilmut, I., Sales, D., Ashworth, C., 1986. Maternal and embryonic factors associated with prenatal loss in mammals. J. Reprod. Fertil. 76, 851–864.
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Reproductive performance of ewe lambs in non-breeding season exposed to hCG at day 12 post mating R. Catalano a,∗ , M. Teruel a , C. González a , S. Williams b , I. Videla Dorna c , S. Callejas a a b c
Facultad de Ciencias Veterinarias UNCPBA, Tandil, Argentina Facultad de Ciencias Veterinarias UNLP, La Plata, Argentina Laboratorio SYNTEX, Argentina
a r t i c l e
i n f o
Article history: Received 17 September 2014 Received in revised form 19 December 2014 Accepted 31 December 2014 Available online xxx Keywords: Sheep Progesterone Multiple ovulations Pregnancy Fetal growth
a b s t r a c t The objective of this experiment was to determine the effect of hCG treatment at day 12 post mating on ovarian characteristics, reproductive performance and conceptus growth in ewe lambs induced estrus during the non-breeding season. Corriedale ewe lambs 8 months old were pretreated with progestagen sponges for 10 days and 300 IU of eCG. At day 12 post mating, females were treated with either physiological saline (hCG-0; n: 53) or 300 IU of hCG (hCG-300; n: 50). Plasma progesterone concentrations were measured at day 17 post mating by radioimmunoassay. Pregnancy was monitored by transrectal ultrasonography on day 40 post mating. At day 41 post mating, 10 ewe lambs of the hCG-0 group and 9 of the hCG-300 group with single pregnancy (randomly chosen) were euthanized and subjected to a morphometric analysis of ovary, uterine and fetal structures. Plasma progesterone levels in the hCG-300 group were significantly higher than those in the control ewe lambs (P < 0.05). Pregnancy (49.18% and 46.15%), fertility (60.0% and 56.6%) and prolificacy (1.43 and 1.23) did not differ between the hCG treated and non treated ewe lambs. However, a tendency for a high number of ewe lambs with multiple pregnancies was observed in the hCG-300 group (P = 0.08). The percentage of ewe lambs with multiple ovulation was higher in the hCG-300 group compared to the controls (77.8% and 20.0%; P < 0.05), while ovary weight and diameters and corpora lutea area did not differ between groups (P > 0.05). The length of pregnant horn in ewe lambs of the hCG-300 group showed a tendency to be greater compared with that of the hCG-0 group (P = 0.08). Differences were not detected between groups for the uterine weight, number of caruncles and chorionic sac length (P > 0.05). Administration of hCG at day 12 post mating increased cranium-caudal length. However, no difference was observed in biparietal diameter and fetal weight between control and hCG treated ewe lambs. Results indicate that hCG treatment did not improve pregnancy rate, prolificacy or fetal weight, however the increase in the plasma progesterone concentration and the percentage of multiple ovulations observed in ewe lambs during the non-breeding season suggest a positive action of this hormone on reproductive performance. © 2015 Published by Elsevier B.V.
∗ Corresponding author at: Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires. Pinto 399 B7000GHG Tandil, Argentina. Tel.: +54 02494439850. E-mail address: [email protected] (R. Catalano). http://dx.doi.org/10.1016/j.smallrumres.2014.12.014 0921-4488/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Catalano, R., et al., Reproductive performance of ewe lambs in non-breeding season exposed to hCG at day 12 post mating. Small Ruminant Res. (2015), http://dx.doi.org/10.1016/j.smallrumres.2014.12.014
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1. Introduction Insufficient luteal function may be responsible for embryo loss in sheep (Ashworth et al., 1989; Nancarrow, 1994; Wilmut et al., 1986). Inadequate ovarian stimulation by gonadotropins during the periovulatory period (Davies and Beck, 1993) or in the first days post conception (Khan et al., 2003; Nephew et al., 1994) can be related with poor response luteal. The first 3 weeks of pregnancy is the critical period. The embryonic losses which range from 20% to 40% of fertilized eggs in adult ewes reaching 50% in ewe lambs (Quirke et al., 1983) and 63% in ewe lambs mated at synchronized oestrus after a progestagen-eCG treatment (Gordon, 1977) occur in this period. Hormones like progesterone, GnRH analogs and human chorionic gonadotropin (hCG) have been used in sheep and cattle during early pregnancy with the objective to reduce the embryo mortality (Cam et al., 2002; Cam and Kuran, 2004; Khan et al., 2007, 2009; Nephew et al., 1994; Sreenan et al., 1996; Thatcher et al., 2001). Several studies have employed hCG to increase plasma progesterone levels since this hormone is similar to LH in function. Thus, it has been used in ewes to improve lambing rate, litter size and fetal growth (Bobowiec et al., 2012; Cole, 2009; Khan et al., 2003, 2007, 2009; Kittok et al., 1983; Ishida et al., 1999). Adequate progesterone concentrations play an important role in controlling the maternal secretion of nutrients, growth factors and immunosuppressive agents required for successful embryo development and pregnancy recognition (Spencer and Bazer, 2002). Khan et al. (2007, 2009) demonstrated that hCG administrated on day 12 post-mating in cyclic ewes improves plasma progesterone and oestradiol concentrations, weight of corpora lutea, conceptus growth and placental attachment. However, the observed positive effects of hCG administration in adult ewes were less effective in cyclic ewe lambs (Khan et al., 2007, 2009). Anestrous lactating ewes with 20–30 days postpartum (Kittok et al., 1983) and anestrous ewes with low fertility rates (Gomez-Brunet et al., 2007) improved the fertility when they were treated with hCG. This suggests that in those categories of sheep with difficulty to maintain pregnancy, the administration of hCG could improve the reproductive performance. In pre-puberal ewe lambs with estrus induction treatment, the administration of hCG during the luteal phase, increased the luteal area and plasmatic progesterone concentration (Catalano et al., 2012). However, the effect of hCG administered in ewe lambs during the non-breeding season on reproductive performance has not been studied. Therefore, the objective of the present study was to assess the effect of hCG administration at day 12 post mating on reproductive performance in pre-puberal ewe lambs with induction estrus during the non-breeding season. 2. Materials and methods 2.1. Animals and management The experiment was conducted at Field Station of Faculty of Veterinary Sciences UNCPBA, (37◦ S, 60◦ W) Tandil, Argentina during the non-breeding season (spring season).
One hundred and twenty-six Corriedale ewe lambs 8 months old with live weight of 39.2 ± 3.7 kg (mean ± SEM) and body condition score of 3.2 ± 0.5 (mean ± SEM) according to Russel et al. (1969) selected from an overall herd were used. The ewe lambs were continuously exposed to natural photoperiod and handled according to the Animal Welfare Act (Resol. 087/02, Faculty of Veterinary Sciences, UNCPBA) and the Principles of Ethics of the European Commission for experiments involving animals (Directive 2010/63/EU). Transrectal ultrasonography studies were performed before the ovulation induction treatment to confirm absence of luteal structures. Two exams were carried out separated by seven days using a 7.5 MHz transducer (SonoVet 900. Medison, Co, Korea). To induce ovulation, all ewe lambs were pretreated with an intravaginal sponge impregnated with 60 mg of medroxyprogesterone acetate (Progespon® , Laboratorios Syntex S.A., Argentina) for 10 days and 300 IU of equine chorionic gonadotropin (eCG - Novormon® , Laboratorios Syntex S.A., Argentina) were injected intramuscularly upon withdrawal of sponges. Estrus detection and mating were performed in pens with fertile rams (relation ram-to-ewe lambs 1:10). The chest of rams was painted with chalk twice a day during the mating period. Estrus was considered when the ewes stood to be mounted by the rams or when results painted at the rump region. Estrus observation was performed twice daily; 06:00–07:00 h and 18:00–19:00 h during five days from sponge removal. 2.2. Human chorionic gonadotropin (hCG) treatment One hundred and three ewe lambs showing estral characteristics were mated and allocated to two groups on the basis of body weight and condition score using a system of randomized stratification. At day 12 post mating, females were given a single intramuscular injection of either 1 cc of physiological saline (hCG-0; n: 53) or 300 IU of hCG (Ovusyn® , Laboratorio Syntex S.A., Argentina), (hCG-300; n: 50). 2.3. Blood collection and progesterone assay Jugular venous blood was collected for determination of progesterone concentration in all ewe lambs at day 17 post mating. Immediately, plasma was obtained and stored at −20 ◦ C until assayed for progesterone. A radioimmunoassay (RIA) kit (Diagnostic Product Corporation, Los Angeles, CA, USA) was used. The sensitivity of the assay was 0.01 ng/ml, the intraassay coefficient of variation was below 7% for concentrations between 0.1 and 40.0 ng/ml and the inter-assay coefficient of variation was 3.5%. 2.4. Pregnancy diagnosis and related variables Pregnancy was monitored by transrectal ultrasonography using a SonoVet 900 (Medison, Co, Korea), real-time linear-array scanner equipped with 5 MHz transducer on day 40 post mating. Pregnancy rate (number of pregnant ewe lambs/ewe lambs with estrous induction treatment per 100), ewe lambs with multiple pregnancy (females with two fetuses or more/pregnant ewe lambs per 100), prolificacy (number of fetuses/pregnant ewe lambs per 100) and fertility (number of pregnant ewe lambs/ewe lambs with estrus characteristics per 100) were recorded. 2.5. Morphometric analysis of ovarian, uterine and fetal structures At day 41 post mating, 10 ewe lambs of hCG-0 group and 9 ewe lambs of hCG-300 group with single pregnancy (randomly chosen) were euthanized in agreement with the Animal Welfare Act (Resol. 087/02, Faculty of Veterinary Sciences, UNCPBA) and according to the Principles of Ethics of the European Commission for experiments involving animals (Directive 2010/63/EU). Reproductive tracts were collected and a morphometric analysis of ovary, uterine and fetal structures was carried out. The percentage of ewe lambs with multiple ovulations was determined by count of corpora lutea. The area of each corpus luteum was calculated by the formula = × (long diameter/2) × (short diameter/2) according to Hruska (1996). The total luteal area by ewe lamb was determined by the sum of the areas of each corpus luteum. Uteri gravid weight, ovarian diameters and weight (mean of both ovaries by ewe lambs) were registered. Once externalized the conceptus, weight, length and diameter of pregnant uterine horn, caruncles number of pregnant uterine horn, chorionic sac length and amniotic liquid weight were recorded. Fetal body
Please cite this article in press as: Catalano, R., et al., Reproductive performance of ewe lambs in non-breeding season exposed to hCG at day 12 post mating. Small Ruminant Res. (2015), http://dx.doi.org/10.1016/j.smallrumres.2014.12.014
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R. Catalano et al. / Small Ruminant Research xxx (2015) xxx–xxx weight and body measures (cranium-caudal length, crown-rump length and biparietal diameter) were also determined. 2.6. Statistical analysis Ovarian, uterine and fetal variables were shown as mean ± standard error and were analyzed as a completely randomized design (the treatments were characterized by the administration of 0 IU or 300 IU of hCG at day 12 post mating). The percentage of ewe lambs with multiple ovulations, percentage of pregnancy, fertility, prolificacy and the percentage of ewe lambs with multiple pregnancies were analyzed using CATMOD from SAS (1989). Significant differences were considered when P < 0.05.
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Table 2 Ovarian characteristics in ewe lambs administered with hCG at day 12 post mating and euthanized at day 41 of pregnancy. Variable
hCG-0 group
hCG-300 group
Ovarian weight (g ± SEM) Ovarian long diameter (cm ± SEM) Ovarian short diameter (cm ± SEM) Total area of corpus luteum (cm2 ± SEM)
1.03 ± 0.2 1.43 ± 0.1
1.17 ± 0.2 1.50 ± 0.1
1.10 ± 0.1
1.18 ± 0.1
1.25 ± 0.2
1.88 ± 0.3
Table 3 Morphometric analysis of uterine and placental variables in ewe lambs administered with hCG at day 12 post mating and euthanized at day 41 of pregnancy.
3. Results The plasma progesterone concentration evaluated at day 17 post mating (in ewe lambs diagnosed as pregnant at day 40 post mating) was significantly different between groups: 6.30 ± 0.38 ng/ml and 10.89 ± 0.88 ng/ml, for hCG0 and hCG-300 groups, respectively (P < 0.05). The administration of 300 IU of hCG at day 12 post mating did not modify the pregnancy rate, fertility nor the prolificacy compared with the control hCG-0 group (P > 0.05. Table 1). The hCG-300 group showed a trend to a higher percentage of ewe lambs with multiple pregnancies (P = 0.08, Table 1). The percentage of ewe lambs with multiple ovulation was higher in hCG-300 group than in hCG-0 group (77.8% vs. 20.0%; P < 0.05); however no significant differences in the weight and diameters of both ovaries and corpora lutea area between groups were recorded (P > 0.05, Table 2). The length of pregnant horn in ewe lambs of hCG-300 group showed a tendency to be greater compared with that of the hCG-0 group (P = 0.08, Table 3). Differences were not detected between groups for the other parameters evaluated (P > 0.05, Table 3). With respect to the fetal morphometry, the administration of 300 IU of hCG at day 12 post mating did not modify the fetal weight, nor the biparietal diameter (P > 0.05, Table 4). However, the fetuses of the hCG-300 group showed a trend to have greater crown-rump length Table 1 Reproductive response in ewe lambs administered with hCG at day 12 post mating. Variable a
Pregnancy rate Fertilityb Prolificacyc
Percentage of ewe lambs with multiple pregnanciesd
hCG-0 group
hCG-300 group
46.15 (30/65) 56.6 (30/53) 1.23 (37/30) 23.33x (7/30)
49.18 (30/61) 60.0 (30/50) 1.43 (43/30) 43.33y (13/30)
x-y: (P = 0.08). a Number of pregnant ewe lambs/ewe lambs with ovulation induction treatment per 100. b Number of pregnant ewe lambs/ewe lambs with estrus characteristics per 100. c Number of fetuses/pregnant ewe lambs per 100. d Number of ewe lambs with more than one fetuses/pregnant ewe lambs per 100.
Variable Pregnant uterine horn length (cm ± SEM) Pregnant uterine horn diameter (cm ± SEM) Uterine weight (g ± SEM) Pregnant uterine horn weight (g ± SEM) Caruncles in the gravid horn (n ± SEM) Chorionic sac length (cm ± SEM) Amniotic fluid weight (g ± SEM)
hCG-0 group 16.0 ± 0.6x 4.4 ± 0.2
hCG-300 group 17.34 ± 0.4y 4.4 ± 0.1
238.1 ± 14.0 199.6 ± 41.2
241.9 ± 11.5 196.9 ± 9.2
63.3 ± 4.5
65.4 ± 5.4
70.0 ± 3.0
70.4 ± 2.4
32.9 ± 2.6
35.8 ± 1.6
x-y: (P = 0.08). Table 4 Morphometric analysis of fetal variables in ewe lambs treated with hCG at day 12 post mating and euthanized at day 41 of pregnancy. Variable
hCG-0 group
Fetal weight (g ± SEM) Biparietal diameter (cm ± SEM) Crown-rump length (cm ± SEM) Cranium-caudal length (cm ± SEM)
6.5 1.2 4.1 5.0
± ± ± ±
0.2 0.02 0.1x 0.1a
hCG-300 group 6.5 1.2 4.6 5.3
± ± ± ±
0.2 0.04 0.1y 0.1b
a-b: (P = 0.05); x-y:(P = 0.06).
(P = 0.06) and the cranium-caudal length was higher than that in fetuses of the hCG-0 group (P = 0.05, Table 4). 4. Discussion It is well known that an inadequate luteal function is an important cause of reduced reproductive efficiency due to preimplantation losses (Ashworth et al., 1989; Nancarrow, 1994; Wilmut et al., 1986). In order to reduce embryo mortality, supplementation with exogenous progesterone, GnRH analogs and human chorionic gonadotropin (hCG) during early pregnancy have been used in sheep and cattle (Cam et al., 2002; Cam and Kuran, 2004; Khan et al., 2007, 2009; Nephew et al., 1994; Sreenan et al., 1996; Thatcher et al., 2001). In the present study, the ewe lambs treated with 300 IU of hCG at day 12 post mating showed an increase in plasma progesterone concentration and higher percentage of multiple ovulations. Prior findings have reported however, that the administration of hCG during the luteal phase provoke the ovulation of a dominant follicle and the consequent development of an accessory
Please cite this article in press as: Catalano, R., et al., Reproductive performance of ewe lambs in non-breeding season exposed to hCG at day 12 post mating. Small Ruminant Res. (2015), http://dx.doi.org/10.1016/j.smallrumres.2014.12.014
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corpus luteum in cyclic ewes, but not in cyclic ewe lambs (Khan et al., 2009). This may be attributed to the ewe lambs not having well developed follicles able to be ovulatory in response to hCG treatment (Khan et al., 2009). Probably the higher percentage of multiple ovulations obtained in this work may be due to the accessory corpora lutea in ewe lambs treated with hCG at day 12 post mating. This is in agreement with the report that the administration of hCG at day 12 post mating in goats with estrous synchronization and natural mating increased the number of corpora lutea (Lashari and Tasawar, 2010). The differences observed with the study of the Khan et al. (2009) might be because the race, the reproductive season and the treatment of control of estral cycle used were different. Previous studies indicated that hCG administrated to ewe lambs showed no improvement the reproductive performance by the limited embryonic viability, which limit the response to hormonal treatments and then no improvement the embryonic survival (Beck et al., 1996; Khan et al., 2009). Moreover, it has been noted that the steroids hormone production by the ovary in pregnancy ewe lambs is lower than in pregnancy ewes post hCG treatment (Khan et al., 2007). In the present study, the hCG treatment there was no increase the pregnancy rate or the prolificacy. However, increased progesterone concentration and percentage of ewe lambs with multiple ovulations were observed. These results, in addition to the tendency of an increase in the percentage of ewe lambs with multiple pregnancies, disagree with the previously cited works. These differences could possibly be due to the different reproductive characteristics. In the present study ewe lambs during the non breeding season were used, whereas ewe lambs with estrous synchronized in breeding season were employed by Khan et al. (2007). Human chorionic gonadotropin has been reported to increase the size of conceptus in relation to higher IFN-tau production and plasma progesterone concentrations (Khan et al., 2003; Nephew et al., 1994). This gonadotropin moreover has been shown to increase gravid uterine weight, fetal weight and length and placental weight (Cam and Kuran, 2004). The results in the present study demonstrated that hCG administered to 12 days after service increased the cranium-caudal length, whereas the length in gravid uterine horns and the fetal crown-rump length showed a trend to be higher. The hCG has been postulated to increase the progesterone level and consequently the fetal growth (Garrett et al., 1988; Kleemann et al., 1994), probably by promoting an environment rich in nutrients and growth factors (Barnes, 2000), which could explain the observed results. In conclusion, this study indicates that the administration of hCG at day 12 post mating increased the plasma progesterone concentration, the percentage of multiple ovulations and the cranium-caudal length, however, no significant changes in pregnancy rate, prolificacy or fetal weight were observed. Therefore, further research is necessary to establish the relationship between hCG and maternal/embryonic factors as hormones, growth factors, citoquines that influence growth and differentiation of the conceptus in pre-puberal ewe lambs.
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