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Superovulatory response and embryo development in ewes treated with two doses of bovine somatotropin
Animal Reproduction Science 151 (2014) 105–111
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Superovulatory response and embryo development in ewes treated with two doses of bovine somatotropin J.M. Carrera-Chávez a,1 , J. Hernández-Cerón b , M.A. López-Carlos a , R.R. Lozano-Domínguez a , F. Molinar c,d , F.G. Echavarría-Cháirez a , a ˜ R. Banuelos-Valenzuela , C.F. Aréchiga-Flores a,∗ a Unidad Académica de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Zacatecas, Carretera Panamericana Zacatecas-Fresnillo Km 31.5, 98500 El Cordovel Enrique Estrada, Zacatecas, Mexico b Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Av. Universidad 3000, 04510 México, DF, Mexico c United States Department of Agriculture, 11940 Don Haskins Dr. Suite E-3, El Paso, TX 79936, USA d Departamento de Ciencias Veterinarias, Universidad Autónoma de Ciudad Juárez, Henry Dunant s/n Zona Pronaf, 32315 Ciudad Juárez, Chihuahua, Mexico
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Article history: Received 2 August 2013 Received in revised form 7 October 2014 Accepted 9 October 2014 Available online 22 October 2014 Keywords: Embryo transfer Ewe Somatotropin Insulin IGF-1
a b s t r a c t This study evaluated whether the administration of 50 and 100 mg bovine somatotropin (bST) at the start of synchronization and at the time of natural mating in ewes improves the ovulation rate, embryonic development and pregnancy rate of transferred embryos. Fortyeight donors were assigned to three treatments: the bST-100 treatment (n = 15) received 100 mg bST at the start of synchronization and at natural mating, the bST-50 treatment (n = 15) received 50 mg bST on the same schedule as the previous group, and the control (n = 18) did not receive any bST. Two embryos were transferred to each recipient (n = 121): 35 received embryos from bST-100; 50 received embryos from bST-50, and 36 received embryos from the control. The superovulatory rate, percentage of recovered structures, cleavage rate, percentage of transferable embryos, embryo quality and development and pregnancy rate were analyzed using the GENMOD procedure of SAS. The number of corpora lutea and the cell number were analyzed using the GLM procedure of SAS. The insulin and IGF-1 concentrations were analyzed with ANOVA for repeated measures. The bST application did not affect the superovulatory rate, number of corpora lutea and recovered structures (P > 0.05). The numbers of transferable embryos and embryos reaching the blastocyst were higher (P ≤ 0.01) in the bST-50 (96.4 ± 3.6% and 69.0 ± 7.8%) than the bST-100 (93.0 ± 4.5% and 27.2 ± 38.9%) and control (87.7 ± 5.4% and 50.4 ± 6.4%) groups. The insulin and IGF-1 concentrations were higher (P < 0.05) in the bST-treated groups, but the insulin concentration was higher (P < 0.05) in the bST-100 group than in the bST-50 group. The pregnancy rate was similar (P = 0.21) in ewes receiving embryos from the two treatments [bST-50, (70.0%); bST-100, (62.5%), and control, (56.6%)]. The administration of 50 mg bST at the start of synchronization and at natural mating in superovulated ewes was concluded to enhance the proportion and development of transferable embryos. However, bST did not affect the pregnancy rate of transferred embryos. © 2014 Published by Elsevier B.V.
∗ Corresponding author. Tel.: +52 47 89 85 12 55; fax: +52 47 89 85 02 02. E-mail addresses: [email protected] (J.M. Carrera-Chávez), [email protected] (C.F. Aréchiga-Flores). 1 Present address of First author: Departamento de Ciencias Veterinarias, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo Envolvente del Pronaf y Estocolmo s/n, Zona Pronaf 35315 Ciudad Juárez, Chihuahua, México. Tel.: +52 656 639 8800. http://dx.doi.org/10.1016/j.anireprosci.2014.10.009 0378-4320/© 2014 Published by Elsevier B.V.
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1. Introduction Multiple ovulation and embryo transfer (MOET) is a tool to maximize the progeny of high genetic merit ewes. However, the great variability in the superovulatory rate affects the efficiency of quality embryo production (Oliveira, 2011). The exogenous administration of bovine somatotropin (bST) increases the circulating concentration of insulin and insulin-like growth factor 1 (IGF-1) in sheep (Gong et al., 1996; Joyce et al., 1998; Montero-Pardo et al., 2011). Research suggests that insulin and IGF-1 are responsible for the effects of bST on reproduction (Joyce et al., 1998; Carrillo et al., 2007; Camacho et al., 2008). These effects include an increase in the number of recruited follicles (Gong et al., 1996; Ramón et al., 1998) and an enhancement in the number and development of embryos produced (Montero-Pardo et al., 2011; Mejía et al., 2012). Insulin and IGF-1 receptors have been detected in sheep follicles (Scaramuzzi et al., 2010). The insulin on follicular cells enhances the glucose and amino acid metabolism, stimulates cell proliferation and growth, inhibits follicular steroid secretion (Gallet et al., 2011), and modulates the ˜ function of the gonadotrophin receptor (Munoz-Gutierrez et al., 2002; Somchit et al., 2007). Insulin-like growth factor-1 synergizes with FSH in granulosa cells (Beg and Ginther, 2006) to improve hormonal activities, such as the secretion of follistatin, activin-A, and inhibin-A; the proliferation and differentiation of granulosa cells; the production of estradiol, and the regulation of aromatase activity (Silva et al., 2009). Overall, IGF-1 protects and promotes the maturation of the oocyte (Neira et al., 2010). Research on the effects of bST in ruminants indicates that the hormone improves embryonic development and therefore increases the reproductive efficiency (Ribeiro et al., 2014). bST itself and the resulting IGF-1 concentration enhance the nuclear maturation rate and metabolism of pyruvate, have antiapoptotic effects during the in vitro development of bovine embryos (Stefanello et al., 2006), and increase the number of cells of the blastocyst (Moreira et al., 2002b; Sirisathien et al., 2003). Moreover, these hormones modulate the synthesis of PGF2 ␣ synthesis (Badinga et al., 2002). In studies of sheep, the commercially available bovine dose of bST (500 mg) is commonly divided in four equal portions of 125 mg each. Several studies have administered this dose before or at the time of mating. The results of these studies indicated an improvement in the litter size (Carrillo et al., 2007) and embryonic development in sheep (Montero-Pardo et al., 2011; Mejía et al., 2012). Moreover, another study utilizing the same dose in anestrous goats reported an increase in the pregnancy rate (Martínez et al., 2011). However, several studies reported contradictory effects on the superovulatory response and pregnancy rate after the application of bST (Eckery et al., 1994; Hasler et al., 2003; Bilby et al., 2004; Camacho et al., 2008; Rivera et al., 2010). Other authors have suggested that this variability may be due to several factors, such as the bST dosage, physiological condition, and the resulting serum
concentration of IGF-1 (Block et al., 2005; Velazquez et al., 2009; Ribeiro et al., 2014). For example, the bST treatment that favors conception rate in lactating cows (Bilby et al., 2006) decreases the fertility rate in non-lactating cows (Bilby et al., 2004). The deleterious effect of bST on the pregnancy rate in non-lactating cows may be related to the hyperstimulation of blood insulin and IGF-1 secretion (Bilby et al., 2004). In sheep, the different responses found in several studies may be attributed to the bST dosage. The objective of this study was to evaluate whether the administration of 50 and 100 mg bovine somatotropin to superovulated ewes at the start of synchronization and at the time of natural mating improve the ovulation rate, embryonic development, and pregnancy rate of transferred embryos in sheep. 2. Materials and methods 2.1. Animals and treatments This study was performed during the breeding season (autumn and winter) in Zacatecas, Mexico. The donors were 48 cyclic, adult Dorper ewes (3–5 years old), and the recipients were 121 cyclic, adult hair sheep ewes (Pelibuey, Blackbelly, Katahdin, Dorper and their crosses) (3–5 years old). Both the donors and recipients were housed in total confinement and fed oats and alfalfa hay, ground yellow corn, soybean meal, and minerals. All ewes had at least one parturition prior to the experiment and a body condition score between 3 and 4 on a scale of 5 (Russel et al., 1969). The ewes were synchronized with controlled release intravaginal devices (CIDR, DEC Manufacturing, Hamilton, New Zealand), which remained in place for 12 days. Simultaneously to the application of CIDR (day 0), all ewes received an intramuscular injection of 0.05 mg/kg body weight of selenium and 0.05 mg/kg body weight of vitamin E (Mu-Se, MSD Animal Health, Mexico). After the removal of the intravaginal device, all recipients received an intramuscular injection of 250 IU of equine chorionic gonadotropin (Pregnecol, Bioniche Life Sciences Inc., Australia). On day 0, the donors were randomly assigned to three treatments: (1) the bST-100 treatment (n = 15) received a subcutaneous injection of 100 mg bST (Boostin-S, MSD Animal Health, Mexico) at the start of synchronization and a second injection at natural mating; (2) the bST-50 treatment (n = 15) received a subcutaneous injection of 50 mg bST (Boostin-S, MSD Animal Health, Mexico) according to the same program as previous group; and (3) the control (n = 18) received saline solution instead of bST. Superovulation started 10 days after the CIDR placement. Superovulation was induced with a total of 164 mg of pFSH (Folltropin-V, Bioniche, Ontario, Canada) administered as eight decreasing doses every 12 h. Two days after CIDR removal, the ewes were served by rams of proven fertility every 1 h starting at 09:00 h until each donor received five services. Three or more CL per ewe was considered a superovulatory response (Folch et al., 2001). The embryos were collected using a semi-laparoscopic technique (Bari et al., 2000). The same experienced technician conducted all collections. The embryos were classified according
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Table 1 Superovulatory rate, embryo development, and pregnancy rate of embryos obtained from superovulated ewes treated with 0, 50 and 100 mg of somatotropin (bST) at the start of estrous synchronization and at time of mating. Variable
Superovulatory rate Corpora lutea (n) Recovered structuresc (%) Cleavage rate (%) Transferable embryosd (%) Morula Expanded blastocysts Hatched blastocysts Cells per blastocyst (n) Pregnancy rate
bST dose 0 mg
50 mg
100 mg
72.22% (13/18)a 11.23 ± 1.51a 10.46 ± 1.34 (90.7 ± 4.3)a 10.08 ± 1.37 (95.7 ± 2.5)a 8.69 ± 1.29 (87.7 ± 5.4)b 52.21% (59/113)b 45.13% (51/113)b 2.65% (3/113)b 102.68 ± 3.99a 55.56% (21/36)a
93.33% (14/15)a 14.43 ± 1.46a 12.57 ± 1.27 (86.1 ± 4.7)a 11.86 ± 1.40 (97.9 ± 5.1)a 11.71 ± 1.47 (96.4 ± 3.6)a 29.26% (48/164)a 58.53% (96/164)a 12.19% (20/164)a 107.44 ± 4.09a 70.00 (35/50)%a
80.00% (12/15)a 12.50 ± 1.57a 11.17 ± 2.12 (86.4 ± 4.7)a 8.50 ± 1.98 (79.0 ± 8.9)b 7.83 ± 2.00 (93.0 ± 4.5)b 61.70% (58/94)b 37.23% (35/94)b 1.06% (1/94)b 99.08 ± 4.09a 62.50 (22/35)%a
Different letters (a, b) within columns indicate significant contrasts (P < 0.05). c Total oocytes or embryos recovered. d Quality 1 and 2 embryos.
to their stage of development and morphology (quality grade 1: excellent or good; quality grade 2: fair; quality grade 3: poor; and quality grade 4: dead or degenerating) (Stringfellow and Seidel, 2000). The same technician isolated and classified the ova and embryos. The collection and embryo transfer procedures were performed 6 days after mating. The embryos were transferred using a semi-laparoscopic technique (Bari et al., 2000). The technique introduces two fresh embryos to the uterine horn ipsilateral to the ovary with CL presence. Only transferable embryos (quality grades 1 or 2) were used. Embryos were transferred to 121 recipients: 35 received embryos from the bST-100 treatment, 50 received embryos from the bST-50 treatment, and 36 received embryos from the control. Pregnancy was diagnosed on day 40 after embryo transfer with a real-time ultrasound equipped with a 5.0 MHz convex transducer (Logic 100 PRO VET, GE Medical, Bangalore, India). 2.2. Blood samples and hormonal determinations Five ewes were randomly selected per treatment, and the blood samples were collected in tubes with a clot activator (BD Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The samples were taken from the jugular vein every 48 h after the CIDR insertion (day 0) and until the day of embryo collection (day 20). All samples were collected before the morning meal at 0800 h. The blood samples were centrifuged at 1500 × g for 10 min, and the serum was separated and kept frozen at −20 ◦ C until laboratory analysis. The serum insulin concentration was quantified with a commercial antibody radioimmunoassay (Insulin-CT, Cis-Bio International, Gif sur Yvette, France) with an intra-assay variation coefficient of 3.5%. The serum IGF-1 concentration was determined with a commercial double antibody radioimmunoassay (IGF-1-RIACT, Cis-Bio International, Gif sur Yvette, France) with intra-assay variation coefficients of 0.65% and 14.8% for the low and high control, respectively. 2.3. Cell count A total of 102 embryos were counted (n = 34 embryos per treatment). The cells were counted by permeabilizing
and staining the expanded blastocysts with a 0.2% solution of Triton X-100 (Sigma–Aldrich X100) in maintenance medium (EmCare, ICPBio Limited, Auckland, New Zealand) containing 30 g/ml propodium iodide (Sigma–Aldrich 81845) for 20 s. Immediately after staining, the embryos were washed twice in maintenance medium and fixed in ice-cold methanol (Sigma–Aldrich 322415) containing 10 g/ml bisbenzimide (Hoechst 33342; 141398 Life Technologies) for 10 min. The embryos were transferred into a 50:50 solution of methanol:glycerol and were mounted on small droplets of glycerol (JT Baker 2136-02) (modified from Fouladi-Nashta et al., 2007). The mounted embryos were covered with a coverslip and pressed lightly to extend the embryo in order to count the number of cells. The cells were counted with a Leica epiflourescent microscope (DM IL Leica Microsystems, Wetzlar, Germany). 2.4. Statistical analysis The superovulatory rate, percentage of recovered structures (oocytes or embryos), cleavage rate, percentage of transferable embryos, percentage of embryos ranked by their quality, percentage of embryos per degree of development, and pregnancy rate of transferred embryos were analyzed with the GENMOD procedure of SAS (SAS/STAT version 9.3, SAS Institute Inc., Cary, NC). The number of CL and blastocyst cell number were analyzed with the GLM procedure of SAS. The concentrations of insulin and IGF1 among treatments were analyzed with an ANOVA for repeated measures. The areas under the curve (AUC) of insulin and IGF-1 were calculated with the trapezoidal rule method. The AUC data for both hormones were analyzed with an ANOVA. Differences among treatments were evaluated with a t-test. 3. Results The superovulatory rate (P = 0.14), number of CL (P = 0.13) and percentage of recovered structures (P = 0.07) were not affected by neither of the treatments with bST (50 or 100 mg). However, cleavage rate was lower in the bST-100 treatment (P < 0.0001) when compared to the control and bST-50 treatments. The percentage of transferable
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Fig. 1. Insulin concentration (mean ± standard error) in ewes control, treated with 50 mg of bST or 100 mg of bST at the start of estrous synchronization and at the time of mating. Insulin concentration differ between treatments (literalsa,b,c ) on days marked with *(P = 0.02). Day 0 corresponds to the start of estrous synchronization.
embryos per donor (P = 0.01) and proportion of embryos reaching the blastocyst stage (expanded and hatched) (P < 0.001) were greater in the bST-50 treatment than in the control and bST-100 treatment. The number of cells per blastocyst (P = 0.15) and pregnancy rate of recipients were not affected by either of the bST treatments (P = 0.21) (Table 1). The insulin concentration in ewes treated with bST increased 48 h after the first injection of the hormone. The bST-100 treatment yielded the highest insulin concentration (P = 0.02) followed by the bST-50 treatment (P = 0.02). The insulin concentration returned to the baseline level six days after the first injection of both treatments. However, the insulin concentration increased 24 h after the second injection of the hormone in the bST-100 treatment (P = 0.02). Similarly, the concentration returned to the baseline level 96 h after the second injection (Fig. 1). The IGF-1 concentration in ewes treated with bST increased 48 h after the first injection of bST (P < 0.0001). The IGF-1 concentration in both treatments returned to the baseline level eight days after the initial injection. However, both treatments experienced an increase in the IGF-1 concentration after the second injection, but this increment was less pronounced than that due to the first injection (Fig. 2).
Fig. 2. IGF-l concentration (mean ± standard error) in ewes control, treated with 50 mg of bST or 100 mg of bST at the start of estrous synchronization and at the time of mating. The concentration of IGF-1 differ between treatments (literalsa,b ) on days marked with *(P = 0.0l). Day 0 corresponds to the start of estrous synchronization.
The AUC values for insulin (P = 0.02) and IGF-1 (P = 0.03) were higher in the first injection than in the second injection across both treatments. The AUC values for insulin of the bST-50 treatment were 120.9 ± 65.9 and 25.2 ± 24.2 UI*day/mL for the first and second injection, respectively. Likewise, the AUC values for IGF-1 were 4164.0 ± 531.0 and 2533.7 ± 816.5 ng*day/mL for the first and second injection, respectively. For the bST-100 treatment, the AUC values for insulin were 200.5 ± 117.1 and 46.3 ± 36.0 UI*day/mL for the first and second injection, respectively. Likewise the AUC values for IGF-1 were 4141.6 ± 1065.6 and 2499.3 ± 1030.9 ng*day/mL for the first and second injection, respectively. 4. Discussion The administration of bST increased the peripheral concentration of insulin and IGF-1 in agreement with the results of other studies (Gong et al., 1996; Joyce et al., 1998; Carrillo et al., 2007; Camacho et al., 2008; Montero-Pardo et al., 2011). Interestingly, the serum IGF-1 concentration measured in this study was similar for both bST doses (50 mg or 100 mg). In contrast, the serum insulin concentration was higher for the bST-100 treatment. Overall, the insulin and IGF-1 concentrations were higher for the first injection than the second bST injection. Although the reason for the higher concentration of insulin and IGF-1 in response to the first injection of bST is unknown, some authors have found a similar pattern of insulin concentration in cycling non-lactating cows (Bilby et al., 2004). Spencer et al. (1994) found a similar pattern of IGF-1 concentration in lambs; however, bST was applied daily. These findings suggest that the first bST injection may trigger the production of antibodies against this hormone; therefore, these antibodies may rapidly remove the bST applied in the second injection. The statistical analysis of the present study indicated that neither (50 and 100 mg) bST treatment improved the superovulatory rate; in contrast, Navarrete-Sierra et al. (2008) reported an improvement in response to 125 mg bST at the end of the superovulation protocol. Although the difference among treatments was not statistically significant, the numerical differences indicate that the superovulatory rate of the bST-50 treatment was higher (21% and 13% more) than that of the control and the bST-100 treatment. Likewise, the number of CL by donor or percentage of recovered structures (oocytes or embryos) did not differ between groups. In this regard, previous studies report a favorable effect of bST on the number of follicles or CL generated (Gong et al., 1996; Folch et al., 2001; NavarreteSierra et al., 2008; Mejía et al., 2012). However, other studies report that the administration of bST does not increase the quantity of CL (Driancourt and Disenhaus, 1997; Joyce et al., 1998; Hasler et al., 2003) or the number of structures recovered in ewes (Mejía et al., 2012). In this study, the application of 100 mg bST in donors reduced the percentage of cleaved embryos, which is an adverse effect that could be related to an increase in the insulin concentration after the application of 100 mg bST. Reports from in vitro studies indicate that the addition of 5 g/ml of insulin to the follicle culture medium reduces
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the percentage of cleaved embryos (Fouladi-Nashta and Campbell, 2006). The application of 50 mg bST significantly increased the percentage of transferable embryos. This increase related the percentages found in the control and the bST-100 treatments. Navarrete-Sierra et al. (2008) reported a percentage increase of transferable embryos in response to a dose of 100 mg bST; however, Montero-Pardo et al. (2011) and Mejía et al. (2012) did not find a percentage increase of transferable embryos in response to a dose of 125 mg of bST. Similarly, embryos obtained from the bST-50 treatment showed improved embryonic development because a greater proportion of embryos were in the advanced stages of development (expanded or hatched blastocyst) compared to the other two treatments. This difference is even more remarkable when considering the most advanced stage of development evaluated, i.e., hatched blastocyst, because the percentage of hatched blastocysts was almost five times greater in the group treated with 50 mg bST than in the control and almost 11 times greater than in the group treated with 100 mg of bST (Table 1). Mejía et al. (2012) found that the administration of 125 mg of bST per donor at the time of mating increases the percentage of embryos in the advanced stages of development. These results are similar to the ones reported by Montero-Pardo et al. (2011), who applied the same dose (125 mg) five days before the end of a treatment with progestin. However, the number of cells per blastocysts in the present study was similar among both treatments and the control. Montero-Pardo et al. (2011) reported that the application of 125 mg of bST five days prior to sponge removal in superovulated ewes increased the number of cells in the embryos, although these authors report a lower number of cells than those found in the present study. Conversely, Block et al. (2008) reported that the addition of 100 ng/ml of IGF-1 to the in vitro culture medium did not affect the total cell number in the production of bovine embryos, suggesting that the effects of IGF-1 on embryo survival in vivo are more likely the result of differences in gene expression rather than changes in cell number. Reports from several studies in bovines (Moreira et al., 2002a; Lee et al., 2007) indicate that the administration of bST to donors increases the pregnancy of the embryos obtained. However, the administration of bST did not affect the pregnancy rate increases. These results are consistent with those reported in sheep by Folch et al. (2001) and in cattle by Neves et al. (2005). The variability among experiments may be related to the dose of bST used and the resultant level of IGF-1 because the concentration of IGF-1 must be within a physiological range (approximately 200 ng/ml) (Bilby et al., 2006; Velazquez et al., 2009). A threshold concentration of IGF-1 could increase the fertility and pregnancy rates (Bilby et al., 2006), but exceeding this threshold concentration of IGF-1 may have a negative impact (Bilby et al., 2004). Recently, Ribeiro et al. (2014) reported that a single treatment with a low dose of bST (325 mg) at AI was not sufficient to alter conceptus development and fertility. However, two
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sequential treatments of 325 mg of bST at IA and 14 days later increased the fertility of lactating dairy cows and reduced pregnancy loss, which reinforcing the importance of GH and IGF-1 during early embryo development. In this study, the transferable embryo production and embryo development responded better in ewes treated with 50 mg bST than in ewes treated with 100 mg bST. Embryos exposed to a high concentration of insulin and IGF-1 have been suggested to undergo apoptosis. Consequently, apoptosis affects embryo implantation and therefore the embryo is reabsorbed (Chi et al., 2000; Betancourt-Alonso et al., 2006). Chi et al. (2000) reported that the addition of high doses of insulin to the culture medium of mouse blastocysts increased apoptosis via DNA fragmentation. Apoptosis is dose-dependent, as the intermediate dose (350 nM) and the high dose (700 nM) caused apoptosis rates of 50 and 70%, respectively. Similarly, Mihalik et al. (2000) reported that the addition of insulin to the culture medium of bovine embryos did not affect embryo development. In a study of cattle of moderate body condition (3.4 in a scale of 6), Adamiak et al. (2005) reported that high insulin concentrations produced fewer follicles and lower blastocyst yields following in vitro fertilization. Fouladi-Nashta and Campbell (2006) reported that the addition of 5 g/ml of insulin to the bovine antral follicle culture medium reduces the proportion of cleaved embryos, although the proportion of cleaved embryos that developed into blastocysts and the quality of embryos (assessed by total cell number) did not differ among groups. These authors suggest that the reduction in the rate of division is due to early cytoplasmic changes, suggesting that the oocytes was hypermature or aged, which lowered the fertilization rate. Nevertheless, the IGF-1 serum concentration in this study was similar between groups treated with 50 or 100 mg of bST, while the insulin serum concentration was higher in the group treated with 100 mg. Because insulin and IGF-1 can cross-react with the corresponding receptors (Augustin et al., 2003), this result could represent an antagonistic effect due to over-stimulation by 100 mg of bST, which could increase IGF-1R expression (Velazquez et al., 2011) and glucose uptake (insulin-dependent) by the embryos (Velazquez et al., 2012) to adversely affect them. In in vitro systems, the consumption and degradation of IGF-1 occurs without peptide renewal. In contrast, in a physiologically occurring high IGF-1 environment (e.g., bST administration), the embryos are continuously exposed to abnormally high levels of IGF-1, which may exacerbate apoptosis and hypertrophic ICM (Velazquez et al., 2011). In conclusion, the administration of 50 mg of bST at the start of estrous synchronization and at the time of mating in superovulated ewes enhanced the proportion and development of transferable embryos. The bST treatment did not affect the pregnancy rate of the transferred embryos in the recipient. Additional research is needed to determine the critical administration timing and dosage of bST to enhance the superovulatory response in superovulated ewes and assess the embryo quality. This research should include resistance to cryopreservation.
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Conflict of interest None declared.
Acknowledgments This study was partly supported by the Fundación Produce Zacatecas A.C. (Zacatecas, México) (Project number: 32-2010-0013). The authors thank MSD Salud Animal México for the donation of somatotropin (Boostin-S) and Dr. Clara Mejía Murcia from Laboratorio de Endocrinología del Departamento de Reproducción de la Facultad de Medicina Veterinaria y Zootecnia de la Universidad Nacional Autónoma de México for her assistance in the determination of hormone levels.
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Superovulatory response and embryo development in ewes treated with two doses of bovine somatotropin J.M. Carrera-Chávez a,1 , J. Hernández-Cerón b , M.A. López-Carlos a , R.R. Lozano-Domínguez a , F. Molinar c,d , F.G. Echavarría-Cháirez a , a ˜ R. Banuelos-Valenzuela , C.F. Aréchiga-Flores a,∗ a Unidad Académica de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Zacatecas, Carretera Panamericana Zacatecas-Fresnillo Km 31.5, 98500 El Cordovel Enrique Estrada, Zacatecas, Mexico b Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Av. Universidad 3000, 04510 México, DF, Mexico c United States Department of Agriculture, 11940 Don Haskins Dr. Suite E-3, El Paso, TX 79936, USA d Departamento de Ciencias Veterinarias, Universidad Autónoma de Ciudad Juárez, Henry Dunant s/n Zona Pronaf, 32315 Ciudad Juárez, Chihuahua, Mexico
a r t i c l e
i n f o
Article history: Received 2 August 2013 Received in revised form 7 October 2014 Accepted 9 October 2014 Available online 22 October 2014 Keywords: Embryo transfer Ewe Somatotropin Insulin IGF-1
a b s t r a c t This study evaluated whether the administration of 50 and 100 mg bovine somatotropin (bST) at the start of synchronization and at the time of natural mating in ewes improves the ovulation rate, embryonic development and pregnancy rate of transferred embryos. Fortyeight donors were assigned to three treatments: the bST-100 treatment (n = 15) received 100 mg bST at the start of synchronization and at natural mating, the bST-50 treatment (n = 15) received 50 mg bST on the same schedule as the previous group, and the control (n = 18) did not receive any bST. Two embryos were transferred to each recipient (n = 121): 35 received embryos from bST-100; 50 received embryos from bST-50, and 36 received embryos from the control. The superovulatory rate, percentage of recovered structures, cleavage rate, percentage of transferable embryos, embryo quality and development and pregnancy rate were analyzed using the GENMOD procedure of SAS. The number of corpora lutea and the cell number were analyzed using the GLM procedure of SAS. The insulin and IGF-1 concentrations were analyzed with ANOVA for repeated measures. The bST application did not affect the superovulatory rate, number of corpora lutea and recovered structures (P > 0.05). The numbers of transferable embryos and embryos reaching the blastocyst were higher (P ≤ 0.01) in the bST-50 (96.4 ± 3.6% and 69.0 ± 7.8%) than the bST-100 (93.0 ± 4.5% and 27.2 ± 38.9%) and control (87.7 ± 5.4% and 50.4 ± 6.4%) groups. The insulin and IGF-1 concentrations were higher (P < 0.05) in the bST-treated groups, but the insulin concentration was higher (P < 0.05) in the bST-100 group than in the bST-50 group. The pregnancy rate was similar (P = 0.21) in ewes receiving embryos from the two treatments [bST-50, (70.0%); bST-100, (62.5%), and control, (56.6%)]. The administration of 50 mg bST at the start of synchronization and at natural mating in superovulated ewes was concluded to enhance the proportion and development of transferable embryos. However, bST did not affect the pregnancy rate of transferred embryos. © 2014 Published by Elsevier B.V.
∗ Corresponding author. Tel.: +52 47 89 85 12 55; fax: +52 47 89 85 02 02. E-mail addresses: [email protected] (J.M. Carrera-Chávez), [email protected] (C.F. Aréchiga-Flores). 1 Present address of First author: Departamento de Ciencias Veterinarias, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo Envolvente del Pronaf y Estocolmo s/n, Zona Pronaf 35315 Ciudad Juárez, Chihuahua, México. Tel.: +52 656 639 8800. http://dx.doi.org/10.1016/j.anireprosci.2014.10.009 0378-4320/© 2014 Published by Elsevier B.V.
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1. Introduction Multiple ovulation and embryo transfer (MOET) is a tool to maximize the progeny of high genetic merit ewes. However, the great variability in the superovulatory rate affects the efficiency of quality embryo production (Oliveira, 2011). The exogenous administration of bovine somatotropin (bST) increases the circulating concentration of insulin and insulin-like growth factor 1 (IGF-1) in sheep (Gong et al., 1996; Joyce et al., 1998; Montero-Pardo et al., 2011). Research suggests that insulin and IGF-1 are responsible for the effects of bST on reproduction (Joyce et al., 1998; Carrillo et al., 2007; Camacho et al., 2008). These effects include an increase in the number of recruited follicles (Gong et al., 1996; Ramón et al., 1998) and an enhancement in the number and development of embryos produced (Montero-Pardo et al., 2011; Mejía et al., 2012). Insulin and IGF-1 receptors have been detected in sheep follicles (Scaramuzzi et al., 2010). The insulin on follicular cells enhances the glucose and amino acid metabolism, stimulates cell proliferation and growth, inhibits follicular steroid secretion (Gallet et al., 2011), and modulates the ˜ function of the gonadotrophin receptor (Munoz-Gutierrez et al., 2002; Somchit et al., 2007). Insulin-like growth factor-1 synergizes with FSH in granulosa cells (Beg and Ginther, 2006) to improve hormonal activities, such as the secretion of follistatin, activin-A, and inhibin-A; the proliferation and differentiation of granulosa cells; the production of estradiol, and the regulation of aromatase activity (Silva et al., 2009). Overall, IGF-1 protects and promotes the maturation of the oocyte (Neira et al., 2010). Research on the effects of bST in ruminants indicates that the hormone improves embryonic development and therefore increases the reproductive efficiency (Ribeiro et al., 2014). bST itself and the resulting IGF-1 concentration enhance the nuclear maturation rate and metabolism of pyruvate, have antiapoptotic effects during the in vitro development of bovine embryos (Stefanello et al., 2006), and increase the number of cells of the blastocyst (Moreira et al., 2002b; Sirisathien et al., 2003). Moreover, these hormones modulate the synthesis of PGF2 ␣ synthesis (Badinga et al., 2002). In studies of sheep, the commercially available bovine dose of bST (500 mg) is commonly divided in four equal portions of 125 mg each. Several studies have administered this dose before or at the time of mating. The results of these studies indicated an improvement in the litter size (Carrillo et al., 2007) and embryonic development in sheep (Montero-Pardo et al., 2011; Mejía et al., 2012). Moreover, another study utilizing the same dose in anestrous goats reported an increase in the pregnancy rate (Martínez et al., 2011). However, several studies reported contradictory effects on the superovulatory response and pregnancy rate after the application of bST (Eckery et al., 1994; Hasler et al., 2003; Bilby et al., 2004; Camacho et al., 2008; Rivera et al., 2010). Other authors have suggested that this variability may be due to several factors, such as the bST dosage, physiological condition, and the resulting serum
concentration of IGF-1 (Block et al., 2005; Velazquez et al., 2009; Ribeiro et al., 2014). For example, the bST treatment that favors conception rate in lactating cows (Bilby et al., 2006) decreases the fertility rate in non-lactating cows (Bilby et al., 2004). The deleterious effect of bST on the pregnancy rate in non-lactating cows may be related to the hyperstimulation of blood insulin and IGF-1 secretion (Bilby et al., 2004). In sheep, the different responses found in several studies may be attributed to the bST dosage. The objective of this study was to evaluate whether the administration of 50 and 100 mg bovine somatotropin to superovulated ewes at the start of synchronization and at the time of natural mating improve the ovulation rate, embryonic development, and pregnancy rate of transferred embryos in sheep. 2. Materials and methods 2.1. Animals and treatments This study was performed during the breeding season (autumn and winter) in Zacatecas, Mexico. The donors were 48 cyclic, adult Dorper ewes (3–5 years old), and the recipients were 121 cyclic, adult hair sheep ewes (Pelibuey, Blackbelly, Katahdin, Dorper and their crosses) (3–5 years old). Both the donors and recipients were housed in total confinement and fed oats and alfalfa hay, ground yellow corn, soybean meal, and minerals. All ewes had at least one parturition prior to the experiment and a body condition score between 3 and 4 on a scale of 5 (Russel et al., 1969). The ewes were synchronized with controlled release intravaginal devices (CIDR, DEC Manufacturing, Hamilton, New Zealand), which remained in place for 12 days. Simultaneously to the application of CIDR (day 0), all ewes received an intramuscular injection of 0.05 mg/kg body weight of selenium and 0.05 mg/kg body weight of vitamin E (Mu-Se, MSD Animal Health, Mexico). After the removal of the intravaginal device, all recipients received an intramuscular injection of 250 IU of equine chorionic gonadotropin (Pregnecol, Bioniche Life Sciences Inc., Australia). On day 0, the donors were randomly assigned to three treatments: (1) the bST-100 treatment (n = 15) received a subcutaneous injection of 100 mg bST (Boostin-S, MSD Animal Health, Mexico) at the start of synchronization and a second injection at natural mating; (2) the bST-50 treatment (n = 15) received a subcutaneous injection of 50 mg bST (Boostin-S, MSD Animal Health, Mexico) according to the same program as previous group; and (3) the control (n = 18) received saline solution instead of bST. Superovulation started 10 days after the CIDR placement. Superovulation was induced with a total of 164 mg of pFSH (Folltropin-V, Bioniche, Ontario, Canada) administered as eight decreasing doses every 12 h. Two days after CIDR removal, the ewes were served by rams of proven fertility every 1 h starting at 09:00 h until each donor received five services. Three or more CL per ewe was considered a superovulatory response (Folch et al., 2001). The embryos were collected using a semi-laparoscopic technique (Bari et al., 2000). The same experienced technician conducted all collections. The embryos were classified according
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Table 1 Superovulatory rate, embryo development, and pregnancy rate of embryos obtained from superovulated ewes treated with 0, 50 and 100 mg of somatotropin (bST) at the start of estrous synchronization and at time of mating. Variable
Superovulatory rate Corpora lutea (n) Recovered structuresc (%) Cleavage rate (%) Transferable embryosd (%) Morula Expanded blastocysts Hatched blastocysts Cells per blastocyst (n) Pregnancy rate
bST dose 0 mg
50 mg
100 mg
72.22% (13/18)a 11.23 ± 1.51a 10.46 ± 1.34 (90.7 ± 4.3)a 10.08 ± 1.37 (95.7 ± 2.5)a 8.69 ± 1.29 (87.7 ± 5.4)b 52.21% (59/113)b 45.13% (51/113)b 2.65% (3/113)b 102.68 ± 3.99a 55.56% (21/36)a
93.33% (14/15)a 14.43 ± 1.46a 12.57 ± 1.27 (86.1 ± 4.7)a 11.86 ± 1.40 (97.9 ± 5.1)a 11.71 ± 1.47 (96.4 ± 3.6)a 29.26% (48/164)a 58.53% (96/164)a 12.19% (20/164)a 107.44 ± 4.09a 70.00 (35/50)%a
80.00% (12/15)a 12.50 ± 1.57a 11.17 ± 2.12 (86.4 ± 4.7)a 8.50 ± 1.98 (79.0 ± 8.9)b 7.83 ± 2.00 (93.0 ± 4.5)b 61.70% (58/94)b 37.23% (35/94)b 1.06% (1/94)b 99.08 ± 4.09a 62.50 (22/35)%a
Different letters (a, b) within columns indicate significant contrasts (P < 0.05). c Total oocytes or embryos recovered. d Quality 1 and 2 embryos.
to their stage of development and morphology (quality grade 1: excellent or good; quality grade 2: fair; quality grade 3: poor; and quality grade 4: dead or degenerating) (Stringfellow and Seidel, 2000). The same technician isolated and classified the ova and embryos. The collection and embryo transfer procedures were performed 6 days after mating. The embryos were transferred using a semi-laparoscopic technique (Bari et al., 2000). The technique introduces two fresh embryos to the uterine horn ipsilateral to the ovary with CL presence. Only transferable embryos (quality grades 1 or 2) were used. Embryos were transferred to 121 recipients: 35 received embryos from the bST-100 treatment, 50 received embryos from the bST-50 treatment, and 36 received embryos from the control. Pregnancy was diagnosed on day 40 after embryo transfer with a real-time ultrasound equipped with a 5.0 MHz convex transducer (Logic 100 PRO VET, GE Medical, Bangalore, India). 2.2. Blood samples and hormonal determinations Five ewes were randomly selected per treatment, and the blood samples were collected in tubes with a clot activator (BD Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The samples were taken from the jugular vein every 48 h after the CIDR insertion (day 0) and until the day of embryo collection (day 20). All samples were collected before the morning meal at 0800 h. The blood samples were centrifuged at 1500 × g for 10 min, and the serum was separated and kept frozen at −20 ◦ C until laboratory analysis. The serum insulin concentration was quantified with a commercial antibody radioimmunoassay (Insulin-CT, Cis-Bio International, Gif sur Yvette, France) with an intra-assay variation coefficient of 3.5%. The serum IGF-1 concentration was determined with a commercial double antibody radioimmunoassay (IGF-1-RIACT, Cis-Bio International, Gif sur Yvette, France) with intra-assay variation coefficients of 0.65% and 14.8% for the low and high control, respectively. 2.3. Cell count A total of 102 embryos were counted (n = 34 embryos per treatment). The cells were counted by permeabilizing
and staining the expanded blastocysts with a 0.2% solution of Triton X-100 (Sigma–Aldrich X100) in maintenance medium (EmCare, ICPBio Limited, Auckland, New Zealand) containing 30 g/ml propodium iodide (Sigma–Aldrich 81845) for 20 s. Immediately after staining, the embryos were washed twice in maintenance medium and fixed in ice-cold methanol (Sigma–Aldrich 322415) containing 10 g/ml bisbenzimide (Hoechst 33342; 141398 Life Technologies) for 10 min. The embryos were transferred into a 50:50 solution of methanol:glycerol and were mounted on small droplets of glycerol (JT Baker 2136-02) (modified from Fouladi-Nashta et al., 2007). The mounted embryos were covered with a coverslip and pressed lightly to extend the embryo in order to count the number of cells. The cells were counted with a Leica epiflourescent microscope (DM IL Leica Microsystems, Wetzlar, Germany). 2.4. Statistical analysis The superovulatory rate, percentage of recovered structures (oocytes or embryos), cleavage rate, percentage of transferable embryos, percentage of embryos ranked by their quality, percentage of embryos per degree of development, and pregnancy rate of transferred embryos were analyzed with the GENMOD procedure of SAS (SAS/STAT version 9.3, SAS Institute Inc., Cary, NC). The number of CL and blastocyst cell number were analyzed with the GLM procedure of SAS. The concentrations of insulin and IGF1 among treatments were analyzed with an ANOVA for repeated measures. The areas under the curve (AUC) of insulin and IGF-1 were calculated with the trapezoidal rule method. The AUC data for both hormones were analyzed with an ANOVA. Differences among treatments were evaluated with a t-test. 3. Results The superovulatory rate (P = 0.14), number of CL (P = 0.13) and percentage of recovered structures (P = 0.07) were not affected by neither of the treatments with bST (50 or 100 mg). However, cleavage rate was lower in the bST-100 treatment (P < 0.0001) when compared to the control and bST-50 treatments. The percentage of transferable
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Fig. 1. Insulin concentration (mean ± standard error) in ewes control, treated with 50 mg of bST or 100 mg of bST at the start of estrous synchronization and at the time of mating. Insulin concentration differ between treatments (literalsa,b,c ) on days marked with *(P = 0.02). Day 0 corresponds to the start of estrous synchronization.
embryos per donor (P = 0.01) and proportion of embryos reaching the blastocyst stage (expanded and hatched) (P < 0.001) were greater in the bST-50 treatment than in the control and bST-100 treatment. The number of cells per blastocyst (P = 0.15) and pregnancy rate of recipients were not affected by either of the bST treatments (P = 0.21) (Table 1). The insulin concentration in ewes treated with bST increased 48 h after the first injection of the hormone. The bST-100 treatment yielded the highest insulin concentration (P = 0.02) followed by the bST-50 treatment (P = 0.02). The insulin concentration returned to the baseline level six days after the first injection of both treatments. However, the insulin concentration increased 24 h after the second injection of the hormone in the bST-100 treatment (P = 0.02). Similarly, the concentration returned to the baseline level 96 h after the second injection (Fig. 1). The IGF-1 concentration in ewes treated with bST increased 48 h after the first injection of bST (P < 0.0001). The IGF-1 concentration in both treatments returned to the baseline level eight days after the initial injection. However, both treatments experienced an increase in the IGF-1 concentration after the second injection, but this increment was less pronounced than that due to the first injection (Fig. 2).
Fig. 2. IGF-l concentration (mean ± standard error) in ewes control, treated with 50 mg of bST or 100 mg of bST at the start of estrous synchronization and at the time of mating. The concentration of IGF-1 differ between treatments (literalsa,b ) on days marked with *(P = 0.0l). Day 0 corresponds to the start of estrous synchronization.
The AUC values for insulin (P = 0.02) and IGF-1 (P = 0.03) were higher in the first injection than in the second injection across both treatments. The AUC values for insulin of the bST-50 treatment were 120.9 ± 65.9 and 25.2 ± 24.2 UI*day/mL for the first and second injection, respectively. Likewise, the AUC values for IGF-1 were 4164.0 ± 531.0 and 2533.7 ± 816.5 ng*day/mL for the first and second injection, respectively. For the bST-100 treatment, the AUC values for insulin were 200.5 ± 117.1 and 46.3 ± 36.0 UI*day/mL for the first and second injection, respectively. Likewise the AUC values for IGF-1 were 4141.6 ± 1065.6 and 2499.3 ± 1030.9 ng*day/mL for the first and second injection, respectively. 4. Discussion The administration of bST increased the peripheral concentration of insulin and IGF-1 in agreement with the results of other studies (Gong et al., 1996; Joyce et al., 1998; Carrillo et al., 2007; Camacho et al., 2008; Montero-Pardo et al., 2011). Interestingly, the serum IGF-1 concentration measured in this study was similar for both bST doses (50 mg or 100 mg). In contrast, the serum insulin concentration was higher for the bST-100 treatment. Overall, the insulin and IGF-1 concentrations were higher for the first injection than the second bST injection. Although the reason for the higher concentration of insulin and IGF-1 in response to the first injection of bST is unknown, some authors have found a similar pattern of insulin concentration in cycling non-lactating cows (Bilby et al., 2004). Spencer et al. (1994) found a similar pattern of IGF-1 concentration in lambs; however, bST was applied daily. These findings suggest that the first bST injection may trigger the production of antibodies against this hormone; therefore, these antibodies may rapidly remove the bST applied in the second injection. The statistical analysis of the present study indicated that neither (50 and 100 mg) bST treatment improved the superovulatory rate; in contrast, Navarrete-Sierra et al. (2008) reported an improvement in response to 125 mg bST at the end of the superovulation protocol. Although the difference among treatments was not statistically significant, the numerical differences indicate that the superovulatory rate of the bST-50 treatment was higher (21% and 13% more) than that of the control and the bST-100 treatment. Likewise, the number of CL by donor or percentage of recovered structures (oocytes or embryos) did not differ between groups. In this regard, previous studies report a favorable effect of bST on the number of follicles or CL generated (Gong et al., 1996; Folch et al., 2001; NavarreteSierra et al., 2008; Mejía et al., 2012). However, other studies report that the administration of bST does not increase the quantity of CL (Driancourt and Disenhaus, 1997; Joyce et al., 1998; Hasler et al., 2003) or the number of structures recovered in ewes (Mejía et al., 2012). In this study, the application of 100 mg bST in donors reduced the percentage of cleaved embryos, which is an adverse effect that could be related to an increase in the insulin concentration after the application of 100 mg bST. Reports from in vitro studies indicate that the addition of 5 g/ml of insulin to the follicle culture medium reduces
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the percentage of cleaved embryos (Fouladi-Nashta and Campbell, 2006). The application of 50 mg bST significantly increased the percentage of transferable embryos. This increase related the percentages found in the control and the bST-100 treatments. Navarrete-Sierra et al. (2008) reported a percentage increase of transferable embryos in response to a dose of 100 mg bST; however, Montero-Pardo et al. (2011) and Mejía et al. (2012) did not find a percentage increase of transferable embryos in response to a dose of 125 mg of bST. Similarly, embryos obtained from the bST-50 treatment showed improved embryonic development because a greater proportion of embryos were in the advanced stages of development (expanded or hatched blastocyst) compared to the other two treatments. This difference is even more remarkable when considering the most advanced stage of development evaluated, i.e., hatched blastocyst, because the percentage of hatched blastocysts was almost five times greater in the group treated with 50 mg bST than in the control and almost 11 times greater than in the group treated with 100 mg of bST (Table 1). Mejía et al. (2012) found that the administration of 125 mg of bST per donor at the time of mating increases the percentage of embryos in the advanced stages of development. These results are similar to the ones reported by Montero-Pardo et al. (2011), who applied the same dose (125 mg) five days before the end of a treatment with progestin. However, the number of cells per blastocysts in the present study was similar among both treatments and the control. Montero-Pardo et al. (2011) reported that the application of 125 mg of bST five days prior to sponge removal in superovulated ewes increased the number of cells in the embryos, although these authors report a lower number of cells than those found in the present study. Conversely, Block et al. (2008) reported that the addition of 100 ng/ml of IGF-1 to the in vitro culture medium did not affect the total cell number in the production of bovine embryos, suggesting that the effects of IGF-1 on embryo survival in vivo are more likely the result of differences in gene expression rather than changes in cell number. Reports from several studies in bovines (Moreira et al., 2002a; Lee et al., 2007) indicate that the administration of bST to donors increases the pregnancy of the embryos obtained. However, the administration of bST did not affect the pregnancy rate increases. These results are consistent with those reported in sheep by Folch et al. (2001) and in cattle by Neves et al. (2005). The variability among experiments may be related to the dose of bST used and the resultant level of IGF-1 because the concentration of IGF-1 must be within a physiological range (approximately 200 ng/ml) (Bilby et al., 2006; Velazquez et al., 2009). A threshold concentration of IGF-1 could increase the fertility and pregnancy rates (Bilby et al., 2006), but exceeding this threshold concentration of IGF-1 may have a negative impact (Bilby et al., 2004). Recently, Ribeiro et al. (2014) reported that a single treatment with a low dose of bST (325 mg) at AI was not sufficient to alter conceptus development and fertility. However, two
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sequential treatments of 325 mg of bST at IA and 14 days later increased the fertility of lactating dairy cows and reduced pregnancy loss, which reinforcing the importance of GH and IGF-1 during early embryo development. In this study, the transferable embryo production and embryo development responded better in ewes treated with 50 mg bST than in ewes treated with 100 mg bST. Embryos exposed to a high concentration of insulin and IGF-1 have been suggested to undergo apoptosis. Consequently, apoptosis affects embryo implantation and therefore the embryo is reabsorbed (Chi et al., 2000; Betancourt-Alonso et al., 2006). Chi et al. (2000) reported that the addition of high doses of insulin to the culture medium of mouse blastocysts increased apoptosis via DNA fragmentation. Apoptosis is dose-dependent, as the intermediate dose (350 nM) and the high dose (700 nM) caused apoptosis rates of 50 and 70%, respectively. Similarly, Mihalik et al. (2000) reported that the addition of insulin to the culture medium of bovine embryos did not affect embryo development. In a study of cattle of moderate body condition (3.4 in a scale of 6), Adamiak et al. (2005) reported that high insulin concentrations produced fewer follicles and lower blastocyst yields following in vitro fertilization. Fouladi-Nashta and Campbell (2006) reported that the addition of 5 g/ml of insulin to the bovine antral follicle culture medium reduces the proportion of cleaved embryos, although the proportion of cleaved embryos that developed into blastocysts and the quality of embryos (assessed by total cell number) did not differ among groups. These authors suggest that the reduction in the rate of division is due to early cytoplasmic changes, suggesting that the oocytes was hypermature or aged, which lowered the fertilization rate. Nevertheless, the IGF-1 serum concentration in this study was similar between groups treated with 50 or 100 mg of bST, while the insulin serum concentration was higher in the group treated with 100 mg. Because insulin and IGF-1 can cross-react with the corresponding receptors (Augustin et al., 2003), this result could represent an antagonistic effect due to over-stimulation by 100 mg of bST, which could increase IGF-1R expression (Velazquez et al., 2011) and glucose uptake (insulin-dependent) by the embryos (Velazquez et al., 2012) to adversely affect them. In in vitro systems, the consumption and degradation of IGF-1 occurs without peptide renewal. In contrast, in a physiologically occurring high IGF-1 environment (e.g., bST administration), the embryos are continuously exposed to abnormally high levels of IGF-1, which may exacerbate apoptosis and hypertrophic ICM (Velazquez et al., 2011). In conclusion, the administration of 50 mg of bST at the start of estrous synchronization and at the time of mating in superovulated ewes enhanced the proportion and development of transferable embryos. The bST treatment did not affect the pregnancy rate of the transferred embryos in the recipient. Additional research is needed to determine the critical administration timing and dosage of bST to enhance the superovulatory response in superovulated ewes and assess the embryo quality. This research should include resistance to cryopreservation.
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Conflict of interest None declared.
Acknowledgments This study was partly supported by the Fundación Produce Zacatecas A.C. (Zacatecas, México) (Project number: 32-2010-0013). The authors thank MSD Salud Animal México for the donation of somatotropin (Boostin-S) and Dr. Clara Mejía Murcia from Laboratorio de Endocrinología del Departamento de Reproducción de la Facultad de Medicina Veterinaria y Zootecnia de la Universidad Nacional Autónoma de México for her assistance in the determination of hormone levels.
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