Trop Anim Health Prod DOI 10.1007/s11250-015-0833-4
REGULAR ARTICLES
Supplementation with rumen-protected L-arginine-HCl increased fertility in sheep with synchronized estrus Julio Agustín Ruiz de Chávez 1,2 & Adrian Guzmán 1 & Diana Zamora-Gutiérrez 1,2 & Germán David Mendoza 1 & Luz María Melgoza 3 & Sergio Montes 4 & Ana María Rosales-Torres 1
Received: 8 January 2014 / Accepted: 15 April 2015 # Springer Science+Business Media Dordrecht 2015
Abstract The aim of the present study was to evaluate the effects of L-arginine-HCl supplementation on ovulation rate, fertility, prolificacy, and serum VEGF concentrations in ewes with synchronized oestrus. Thirty Suffolk ewes with a mean body weight of 45±3 kg and a mean body condition score (BCS) of 2.4±0.28 were synchronized for estrus presentation with a progestin-containing sponge (20 mg Chronogest® CR) for 9 days plus PGF2-α (Lutalyse; Pfizer, USA) on day 7 after the insertion of the sponge. The ewes were divided into two groups; i.e., a control group (n=15) that was fed on the native pasture (basal diet) and an L-arginine-HCl group (n=15) that received 7.8 g of rumen-protected L-arginine-HCl from day 5 of the sponge insertion until day 25 after mating plus the basal diet. The L-arginine-HCl was administered daily via an esophageal probe between days 5 and 9 of the synchronization protocol and every third day subsequently. Blood samples were drawn from the jugular vein every 6 days throughout the entire experimental period. The results revealed that the L-arginineHCl supplementation increased fertility during the synchronized estrus (P=0.05). However, no effects were observed
* Ana María Rosales-Torres
[email protected] 1
Departamento de Producción Agrícola y Animal, Universidad Autónoma Metropolitana–Xochimilco, Calzada del Hueso 1100, Col. Villa Quietud, Coyoacán, 04960 México, DF, Mexico
2
Maestría en Ciencias Agropecuarias, Universidad Autónoma Metropolitana–Xochimilco, México, Mexico
3
Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana–Xochimilco, Calzada del Hueso 1100, 04960 México, DF, Mexico
4
Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía, Insurgentes Sur 3877, 14269 México, DF, Mexico
on the final BCS (P=0.78), estrus presentation (P=0.33), multiple ovulations (P=0.24), prolificacy (P=0.63), or serum VEGF concentration. In conclusion, L-arginine-HCl supplementation during the period used in this study increased fertility in sheep with synchronized estrus possibly due to improved embryo-fetal survival during early pregnancy. Keywords Estrus synchronization . Ewe . L-arginine-HCl . VEGF
Introduction The reproductive success in ewes and other domestic species depends on oocyte fertilization, embryo-fetal survival and development, and the birth of live offspring (Fierro et al. 2013). In sows, between 50 and 60 % of fertilized oocytes are lost or eliminated during early embryo development (Foxcroft et al. 2006). The rate of embryonic-fetal loss in ewes has been reported to be 30 % and to cause serious economic losses. Dixon et al. (2007) reported that most embryonic losses in ewes occur prior to day 18 of pregnancy, whereas 9.4 % occur after day 18, and between 1 to 5 % occur after day 30. In mammals, during implantation of the fertilized oocyte, the endometrium and fetal membranes exhibit rapid angiogenesis to supply nutrients and oxygen from the mother to the product and this supply is required for the development of the product (Bazer et al. 2010). A reduction in nutrient supply due to a reduction in angiogenesis during this period might influence embryonic-fetal development and cause decreases in the sizes and weights of products or even their reabsorption and loss (Grazul-Bilska et al. 2010). In rats, experimental inhibition of angiogenesis with suramin (an inhibitor of VEGF) causes embryonic loss and reductions in the weights of the fetus and placenta (Carlström et al. 2009).
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VEGF is the most important inducer of angiogenesis in both physiological and pathological conditions (Reynolds et al. 2005; Vonnahme et al. 2005). During placental development in the ewe, there is a linear increase in VEGF messenger RNA (mRNA) expression in the fetal membranes from day 16 to day 30 of pregnancy (Grazul-Bilska et al. 2011), and this increase might be associated with the increase in vascularity that is observed in this period (Grazul-Bilska et al. 2010). VEGF expression is regulated by several factors, among which hypoxia (Ferrara 2004) and the presence of nitric oxide (NO; Kimura and Esumi 2003) are the most important. For example, in tumor cell lines, NO donors and hypoxia stimulate VEGF expression via the activation of HIF-1 (Kimura et al. 2001). L-arginine-HCl plays multiple roles in animal metabolism. It is a substrate for protein synthesis, an intermediate in the urea cycle in the liver, and a precursor of NO (Wu et al. 2010). In sows, embryonic and fetal development have been shown to improve following L-arginine supplementation of the diet (Mateo et al. 2007; Gao et al. 2012), which is associated with an increase in VEGF (Wu et al. 2012). Supplementation with L-arginine during the last third of pregnancy increases the number of piglets that are born alive, their corporal weights, their VEGF serum concentrations, and the mRNA expression of this growth factor in the allantochorion tissue (Wu et al. 2012). The effects of L-arginine-HCl supplementation during the third trimester of pregnancy in polytocous species are clear (Mateo et al. 2007; Gao et al. 2012); however, the effect of such supplementation in early pregnancy is not because of the variable effects that have been described. For example, in sows, L-arginine supplementation (16 g/animal/day) from days 0 to 24 of pregnancy reduces the number of corpora lutea and the number of piglets born alive (Li et al. 2010). In contrast, L-arginine supplementation in rats during the first seven days of pregnancy increases the number of implantation sites and the litter size (Zeng et al. 2008). In ewes, little information about the effects of L-arginine supplementation on reproductive function is available. It has been reported that a positive effect of L-arginine on ovulation rates (Bulbarela-García et al. 2009) and L-arginine supplementation during late gestation have been shown to increase the total number of lambs that are born alive (Lassala et al. 2011); however, no data about the effects of L-arginine on reproductive and endocrine functions during early pregnancy are available. Because L-arginine is a precursor of NO, and NO is an inducer of VEGF synthesis, we propose that the supplementation of ewes with L-arginine will increase serum VEGF levels and thereby improve reproductive responses. Therefore, the objective of this study was to evaluate the effects of the L-arginine-HCl supplementation of ewes on the serum concentrations of VEGF, the percentages of multiple ovulations, the fertility of synchronized estrus, and prolificacy.
Material and methods Animals and management The Ethics and Animal Welfare Committee of the Universidad Autónoma Metropolitana–Xochimilco approved all animal procedures. The study was conducted on a commercial farm in a sub-humid temperate area of Mexico. Thirty Suffolk sheep with an average body weight of 45±3 kg and an average body condition score (BCS) of 2.4±0.28 (on a scale of 1–5) were used in this study. The animals were grazed in native pasture (basal diet) throughout the experimental period. Bypass L-arginine-HCl pellets Our laboratory modified L-arginine HCl pellets (Adyfarm, México) to avoid ruminal degradation. We used inert cores of sugar (Dow Chemical Company, Midland, MI, USA) coated with two different layers. The first layer consisted of Larginine•HCl, talc (Helm, México) and barium sulfate with a non-functional polymer (Opadry® clear YS–1–7006 Colorcon, México). The barium sulfate was obtained from an equimolar reaction between barium chloride and sulfuric acid. Both solutions were mixed, allowed to react until a white precipitate formed, and the phases were then separated by decantation, and the precipitate was washed with water until the pH was neutral. Finally, the precipitate was dried in an oven (Riossa HS, Mexico) at 60 °C. The incorporation of barium sulfate served the purpose of providing density to the pellets to prevent them from being damaged by rumination. The outer layer consisted of a functional polymer (Eudragit® S12.5; Helm, México) with the capacity to dissolve at pHs above 7.2, which thus allowed them to release of Larginine•HCl in the intestine. The ratio between the Larginine-HCl and the polymer was 3:1. The amount of Larginine-HCl in the pellets was evaluated according to the method of Moore and Stein (1954). Briefly, one gram of pellets was pulverized and mixed 200 mL of distilled water and shaken for 15 min. After filtration, the absorbance was recorded at 570 nm. Rumen in situ digestibility Samples of the L-arginine-HCl-containing coated pellets (0.5 g) were placed in the rumen of a steer fed with a mixed diet (70 % roughage and 30 % concentrate) inside Ankom rumen in situ bags (5 cm×10 cm) with pore sizes of 50-μm in duplicate for 12 h to estimate the ruminal degradability of the samples (Vanzant et al. 1998). The amount of L-arginineHCl remaining after the removal of the ruminal bags was compared to the initial amount (0.5 g), and the sample digestibility was calculated taking the initial weight as 100 %.
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Treatments Estrus synchronization was performed in all animals with intra-vaginal sponges with progestin (20 mg Chronogest® CR; MSD Salud Animal México) over 9 days. This procedure was followed by an intramuscular injection PGF2-α (Lutalyse; Pfizer, USA) on day 7 after the insertion of the sponge. The ewes were divided in two groups. The control group (n=15) was fed with a basal diet, and the L-arginineHCl group (n=15) received an additional 7.8 g of rumenprotected L-Arginine-HCl from day 5 after the insertion of the sponge until day 25 after mating (Fig. 1). The Larginine-HCl was administered daily using an esophageal probe gavage between days 5 and 9 of the synchronization protocol and then every third day. Using the equations of the Protein Digestible in the Intestine (PDI) system (INRA 1989), the average amino acid composition of the rumen bacteria (Larginine-HCl, 5.1 %; Clark et al. 1992) and the nondegradable fraction of the basal diet, we calculated that the basal duodenal flow of L-arginine-HCl to be 7.79–8.89 g/day. Therefore, the dose of L-arginine-HCl used for the supplementation of the ewes in these experiments duplicated the normal flow of L-arginine. The BCSs of the animals were evaluated at the beginning and the end of the supplementation period. After sponge withdrawal, the detection of estrus was performed with a male with an apron for 72 h, and natural mating was allowed between hours 6 and 12 following estrus detection (Fig. 1). Ten days after sponge removal, two fertile males were introduced to the females’ facilities for 35 days (breeding period). We recorded the number of corpora lutea (CLs) per female via an Aloka ultrasound (SSD500) machine with a 7.5-MHz linear probe on day 12 after sponge removal (Fig. 1). Fertility in synchronized estrus was calculated as the number of pregnancies during synchronized estrus/the total number of synchronized animals*100. Whereas that fertility of the breeding period was calculated as follows: total pregnancies/total synchronized animals*100. Pregnancies during synchronized estrus or during the breeding period were calculated with the difference between the calving date and the gestation period (in days) for this species (153 days). If this difference coincided with date of service to synchronized estrous, then this animals were considered pregnant at
Fig. 1 The period of L-arginine-HCl supplementation
synchronized estrous, if not, animals were considered pregnant during breeding period. Prolificacy was calculated by dividing the number of lambs delivered by the total number of animals exposed to the synchronization protocol.
VEGF serum quantification The VEGF serum levels were evaluated in seven ewes from the control group and six ewes from the L-arginine-HCl group. These ewes were randomly selected for blood sample collection every 6 days during the entire experimental period. The first sample was collected at the moment of sponge insertion, which was considered to be day 0. Blood from the jugular vein was drawn into vacuum tubes containing clotactivating gel. The samples were placed on ice immediately after collection and then centrifuged at 1000×g for 15 min. The serum was collected in polypropylene tubes and stored at −20 °C until VEGF quantification. The VEGF concentrations were determined by enzyme linked immunosorbent assay (ELISA) following the manufacturer’s instructions (PeproTech no.cat ® Human VEGF 900-K-10). The intraassay coefficient of variation was 2.18 %.
Arginine and citrulline serum quantification The same serum samples were used to measure the levels of VEGF; the concentrations of arginine and citrulline were determined by high-performance liquid chromatography with fluorescence detection after ortho-phthalaldehyde derivatization as described by Pérez-Neri et al. (2007). Briefly, a 250 μl aliquot of serum was mixed with 50 μl concentrated perchloric acid (Suprapur, Merck), vortex-mixed and then 50 μl of saturated potassium carbonate was added to the mixture and vortexed again. Then, 250 μl of 50 % methanol was added and mixed. After 10 min, the mixture was centrifuged at 14,000 rpm for 15 min at 4 °C (Centrifuge Eppendorff 5417R). The resulting supernatant was analyzed into a previously calibrated chromatograph (Agilent 1100, equipped with a fluorescence detector). The autosampler was programed to mix equal volumes of samples and derivatization reagent (Phthalaldehyde 30 mM, Beta-mercaptoethanol 0.1 mM, in 300 mM borate buffer, pH 9.5); reaction products were then separated by using a C-18 reversed-phase column (Adsorbosphere OPA HS, 5 μm particle size, 100 × 4.6 mm i.d., Grace). The chromatographic conditions consisted of 1.5 ml/min flow of a mobile phase containing 1.5 % tetrahydrofuran, 10 % methanol, 0.05 M acetates, pH 5.9. The analytical separation was carried out with a solvent gradient beginning at 10 % and reaching 35 % methanol after 15 min. The quantification limits were 1 μM for both amino acids.
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affected by L-arginine-HCl supplementation across the experimental period (P=0.78).
Statistical analyses The differences between the control group and the L-arginineHCl group in terms of BCS, prolificacy, and the number of ovulations were analyzed with Wilcoxon tests, whereas that numbers of animals in estrus, multiple ovulations, and fertility were analyzed with a chi-square test. VEGF serum concentrations across of the experimental period were calculated as change relative to VEGF serum concentrations previous to L-arginine-HCl supplementation in percentage for each day of sampling, and differences between treatments for each day of sampling were compared with Wilcoxon test.
Results
Response to estrus synchronization and arginine and citrulline serum concentrations The ewes in the L-arginine-HCl group exhibited higher (P= 0.05) fertility in synchronized estrus than did the ewes in the control group (Table 1). The number of animals in estrus, percentage of multiple ovulations, number of ovulations per group, fertility in the breeding period and prolificacy were similar between the L-arginine-HCl group and the control group (Table 1). Similarly the serum concentrations of arginine and citrulline were not affected by L-arginine-HCl treatment (Table 1) Vascular endothelial growth factor
Pellets and digestibility The amount of L-arginine-HCl incorporated per gram of pellets was 294 mg. The functional polymer weight gain was 10 %. The particle size of the final pellets was 1.9 mm with a density of 1.3 g/ml. The in situ digestibility assessment revealed that the Larginine-HCl pellets exhibited a ruminal degradation of 25.7 %, which indicates that the sheep in the L-arginine-HCl group received 5.8 g/d of L-arginine-HCl in addition to that provided by microbial protein synthesis and bypass L-arginine of the basal diet.
Serum levels of VEGF across the experimental period are presented in Table 2. Because the serum VEGF concentrations prior to L-arginine-HCl supplementation were significantly different (P < 0.05) between control and L-arginine-HCl group, the initial VEGF serum concentrations were tested as covariate; however, it was not significant. Thereafter, the VEGF results were analyzed as change in percentage respect to VEGF concentrations prior to L-arginine-HCl supplementation for each treatment group. The results revealed that the serum VEGF concentrations were not affected by L-arginineHCl treatment (P>0.05).
BCS
Discussion At the beginning of experiment, the BCS were similar in both groups (2.30±0.1 and 2.50±0.1 for the control and arginine groups, respectively; P=0.12). The final BCSs of the control (2.46 ± 0.08) and arginine groups (2.50 ± 0.09) were not Table 1 Effects of L-arginineHCl supplementation from day 5 of sponge insertion until day 25 after mating on the responses to the estrous synchronization protocol and serum concentrations of Arginine and Citrulline in sheep
The results revealed that rumen-protected L-arginine-HCl supplementation during the final development of the ovulatory follicle and early pregnancy improved the fertility in ewes
Ewes in estrus (%) Multiple ovulations (%) Number of ovulations Fertility to synchronized estrus (%)a Fertility of breeding (%)b Prolificacyc Arginine serum concentration (μMol/L) Citrulline serum concentration (μMol/L)
Control
L-arginine-HCl
P
93 (14/15) 20 (3/15) 1.2±0.1 53 (8/15) 80 (12/15) 0.8 (12/15) 228.9±4.9 255.3±7.4
100 (15/15) 40 (6/15) 1.5±0.2 87 (13/15) 87 (13/15) 0.9 (13/15) 223.2±4.9 269.6±7.4
0.23 0.22 0.14 0.05 0.62 0.63 0.66 0.43
a
Fertility in synchronized estrus was calculated as the number of pregnancies during synchronized estrus/the total number of synchronized animals*100
b c
Fertility during the breeding period was calculated as total pregnancies/total synchronized animals *100
Prolificacy was calculated by dividing the number of lambs delivered by the total number of animals that were exposed to the synchronization protocol
Trop Anim Health Prod Table 2 Change of serum VEGF concentrations as percentage of day 0 (previous to L-arginine-HCl supplementation) in ewes that were treated with Larginine-HCl and the ewes in the control group from day −5 after the onset of estrus synchronization until day 25 after mating Treatment Days relative to the onset of supplementation
Days relative to mating
Control (change of VEGF respect to day 0 as %)
L-arginine-HCl (change of VEGF respect to day 0 as %)
P value
Day 1 Day 7 Day 13 Day 19 Day 25 Day 31
Day −5 Day 1 Day 7 Day 13 Day 19 Day 25
105.5 129.1 150.7 175.1 134.9 116.2
80.9 100.1 102.1 127.1 114.6 107.1
0.58 0.58 0.25 0.20 0.25 0.78
during synchronized estrus likely because of improved embryo-fetal survival. The pregnancy rate of sheep bred during synchronized estrus is generally lower than that of sheep bred during spontaneous estrus (Lunstra and Christenson 1981; Fierro et al. 2013). In the present study, the fertility in synchronized estrus of the ewes in the control group (57 %) was similar to that which has been reported for ewes that have been synchronized with prostaglandins (63 %), but the animals that were treated with L-arginine-HCl exhibited a higher fertility rate during synchronized estrus (87 %); the latter rate is similar to that which has been reported for ewes bred in spontaneous estrus (88 %) (Fierro et al. 2011). Although many factors can affect fertility in synchronized estrus, embryo-fetal survival might be one of the most important (Fierro et al. 2013). Embryonic mortality is higher in ewes that have been synchronized with progestins (29 %) than in non-synchronized ewes (16 %; Lunstra and Christenson 1981). Based on this evidence and our results, it is possible that the improvement in embryonicfetal survival observed in the present study might have been due to rumen-protected L-arginine-HCl supplementation. This hypothesis is supported by evidence that has shown that the L-arginine supplementation of sows during pregnancy increases placental development and the numbers of piglets that are born alive (Gao et al. 2012). In pregnant rats supplementation with arginine between day 1 and 7 of gestation, the number of implantation sites was increased by 29 % on day 7, compared with the control group (Zeng et al. 2008). Moreover, several lines of evidences in sows support that arginine supplementation can enhance embryonic/fetal survival (Mateo et al. 2007; Gao et al. 2012; Wu et al. 2013). Finally, in sheep, it has shown that intravenous infusion of Larginine during first 15 day after estrus did not affect the ovulation rate but increased number of embryos per ewe (Luther et al. 2008) During early gestation in sheep, there is a high rate of proliferation of the endothelial cells in the placenta that stimulate angiogenesis and promote the flux of nutrients from the
mother to the product (Reynolds et al. 2010). VEGF is the main promoter of angiogenesis (Rosales-Torres and Guzmán-Sánchez 2011), and L-arginine-HCl can stimulate the synthesis of VEGF (Dulak et al. 2000; Wu et al. 2012). For example, in cultures of rat vascular smooth muscle cells, the addition of L-arginine to the media increases VEGF mRNA expression (Dulak et al. 2000). In the sow allantochorion tissues that are collected immediately after birth, supplementation with N-carbamoylglutamate (an Larginine synthesis inducer) increases the mRNA expression of VEGF (Wu et al. 2012). Moreover, arginine is required for the generation of various important molecules. These arginine-derived substances include nitric oxide and polyamines that regulate DNA and protein synthesis, as well as cell proliferation (Wu et al. 2013). Dietary arginine supplementation between day 1 and 7 of the pregnant rats increased nitric oxide in blood as well in the implantation sites of the uterus (Zeng et al. 2008). The nitric oxide is required for increased vascular permeability, normal embryonic development, and uterine quiescence at the sites of blastocyst apposition (Manser et al. 2004). Based on these evidences, we hypothesized that L-arginine-HCl supplementation may improve placenta and uterus function and thus could enhance the embryonic and/or fetal survival. However, further studies should be undertaken to test this hypothesis. The VEGF serum concentrations were not affected by supplementation with L-arginine-HCl in the present experiment. Similarly, in gilts dietary supplementation of the L-arginine synthesis inducer, N-carbamoylglutamate during pregnancy did not modify VEGF blood concentration (Zhang et al. 2014). In contrast, in pregnant sows (Wu et al. 2012) and pregnant mice (Fiorito et al. 2008), the L-arginine supplementation increased the VEGF serum concentrations, whereas that also in pregnant sows arginine or N-carbamoylglutamate supplementation during late gestation reduce VEGF serum concentrations (Liu et al. 2012). In contrast with previous reports (McCoard et al. 2013), in the present experiment L-arginine-HCl treatment did not
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affect the serum concentrations of arginine and citrulline. The main explanation of this differences is that arginine and citrulline were measured in blood samples take 24 or 48 h after Larginine-HCL treatment and due that arginine concentrations returned to the baseline levels at 4 and 5 h after arginine treatment (Wu et al. 2007) was not possible observe changes in the serum concentrations of these amino acids. L-arginine supplementation during the final development of the ovulatory follicle has been shown to increase the ovulation rate in sheep (Bulbarela-García et al. 2009). However, in this study, the percentages of multiple ovulations did not differ between groups. The discrepancy between this and previously reported results might be attributable to the dose used and the protection of the L-arginine employed in the present study. For example, Bulbarela-García et al. (2009) used approximately 13 g of L-arginine per animal, but 5.8 g used in this study. In conclusion, L-arginine-HCl supplementation in ewes increased fertility during synchronized estrus, probably due to the enhanced embryonic-fetal survival. The results of the present study regarding dietary supplementation with L-arginineHCl might be useful for improving the reproductive performance of sheep. Acknowledgments Financial support for this study was provided by Consejo Nacional de Ciencia y Tecnología grant number CB 34410952. JA Ruiz de Chávez and D Zamora-Gutiérrez thank CONACYT for a Master’s student grant. The authors also are grateful for the donation of Opadry® clear YS–1–7006, by Colorcon de Mexico S. de R.L. de C.V., Micronizado Talc and Eudragit® S12.5 by Helm de México S.A. Conflict of interest The authors declare that they have no conflict of interest.
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