Small Ruminant Research 114 (2013) 272279

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Small Ruminant Research

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Blood leptin, insulin and glucose concentrations in hair sheep raised in a tropical climate
A.G.V. Catunda a , I.C.S. Lima a , G.C. Bandeira a , C.R.F. Gadelha a , E.S. Pereira a , C.S.B. Salmito-Vanderley b , A.A. Arajo b , G.A. Martins a , A.C.N. Campos a,
a b

Department of Animal Science, Federal University Ceara, Fortaleza, Ceara, Brazil Faculty of Veterinary, State University Ceara, Fortaleza, Ceara, Brazil

a r t i c l e

i n f o

a b s t r a c t
The aims of this study were to determine the effects of body condition score (BCS), breed and dietary supplementation on the concentrations of leptin, insulin and glucose found in the blood obtained from hair sheep during the breeding season. A further aim was to investigate the possible association of fertility and prolicacy with these blood metabolites, BCS and body weight (BW). All ewes were grazed in paddocks with ad libitum access to mineral salts and water. A total of 96 ewes were divided into two groups according to breed and treatment: Santa Ines (supplemented or unsupplemented) (24 24) and Morada Nova (supplemented or unsupplemented) (24 24). Blood samples, and BW and BCS information were collected during the breeding season. The statistical analyses were performed using the program PROC GLM from the SAS software. The leptin concentrations in hair sheep raised in a tropical climate were low. Little effect of breed, treatment or sample collection was found for blood insulin concentrations (p < 0.05), although the values were higher in the supplemented groups from both breeds. Signicant differences were observed in glucose concentrations between the breeds in the same sample collections, with the higher concentrations being found in the Santa Ines sheep (p < 0.05). The BCS for ewes that were not pregnant showed the highest correlation with leptin, insulin and glucose concentrations (r = 0.53, 0.52 and 0.43, respectively). In the Morada Nova supplemented sheep (prolicacy: 1.45), there were veried correlations between BCS and BW, BCS and insulin concentration, and also between insulin and leptin concentrations. The present study shows that the Morada Nova breed has a higher reproductive efciency than the Santa Ines breed. In conclusion, leptin was present in low concentrations in hair sheep and did not inuence the reproductive processes in these animals. The dietary supplementation positively affected blood leptin, insulin and glucose concentrations in these breeds of hair sheep, but there was no major effect on the reproductive processes. 2013 Elsevier B.V. All rights reserved.

Article history: Received 1 December 2012 Received in revised form 4 July 2013 Accepted 8 July 2013 Available online 13 August 2013 Keywords: Body condition score Hair sheep Insulin Leptin Reproduction

1. Introduction According to data collected by the Brazilian Institute for Geography and Statistics (IBGE) in 2011, there are

Corresponding author at: Department of Animal Science, Av Mister Hull, 2977, Campus of Pici Block 808, Fortaleza, Ceara, CEP: 60356-000, Brazil. Tel.: +85 3366 9023; fax: +85 3366 9023. E-mail address: acncampos11@gmail.com (A.C.N. Campos). 0921-4488/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.smallrumres.2013.07.008

approximately 9,566,968 sheep in northeastern Brazil. They represent 56.9% of the Brazilian sheep population, and the predominant genetic group is the hair sheep (6070%). Of the various breeds of hair sheep, the Morada Nova and Santa Ines breeds are the most widespread in northeastern Brazil because of their adaptation to the climatic conditions in this region. Sheep husbandry has traditionally been considered to be of low commercial value in Brazil, and it is generally practiced by the informal economy (is the income generated by economic agents that operate informally)

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(Paiva et al., 2005). In the Northeast and Central West of Brazil, regions characterized by long dry periods, hair sheep represent an important source of protein for the local populations. More recently in these regions, market diversication has increasingly allowed sheep husbandry to be considered as a viable and lucrative occupation. Despite extensive livestock methods and the semiarid climate, Morada Nova ewes show good reproductive performance and have a better litter size at lambing than other sheep breeds in the Northeast of Brazil (Selaive-Villarroel and Fernandes, 2000). Nevertheless, there have been few studies on hormonal metabolism and the inuence of nutrition in the reproductive processes in hair sheep raised in the Northeast of Brazil. Several studies have related the reproductive performance of ocks to the nutritional status of individuals (Boland and Lonergan, 2003; Kiyma et al., 2004; Kiker et al., 2007), but how this occurs remains unclear. However, it is known that nutrition might inuence ovarian function, as it modulates the secretion of hormones that control the reproductive processes in females (Chilliard et al., 2005). Previous studies have shown that the number of follicles and the ovulation rate are directly inuenced by nutrition. They can be assessed via changes in the concentrations of certain metabolites in the bloodstream, such as insulin, leptin, glucose and growth hormone (GH) (Davies-Morel and Beck, 2003; Ramin and Majdani, 2005; Scaramuzzi et al., 2006). Insulin is a small globular protein of about 5.7 kDa that differs between species. It is synthesized and secreted from the pancreatic -cells of the islet of Langerhans in all species (Gonzlez and Silva, 2006). In ruminants, due to microbial activity in the rumen, little or no dietary carbohydrate is absorbed as hexose sugar in the small intestine (Mcniven, 1984). For this reason, volatile fatty acids (propionate and butyrate) are more potent than glucose for stimulating insulin secretion (Pineda and Dooley, 2003; Brockman, 2005; Gonzlez and Silva, 2006). Another hormone, leptin, a 16 kDa protein product of the obese (ob) gene is secreted by adipocytes. In normal animals, leptin serves as a metabolic signal to the reproductive system, informing it that sufcient fat stores are available to meet the caloric demands of reproduction (Barash et al., 1996). Thus, leptin expression and secretion are correlated with body condition, physiological status (puberty, pregnancy, lactation), and the age of the animal (Krasnow and Steiner, 2006). Other studies have shown that leptin can be affected by changes in food intake (Williams et al., 2005). Leptin seems to act by modulating the function of several target glands (Moschos et al., 2002). Glucose, another metabolite widely discussed in metabolism studies, plays a key role in animal metabolism. It is an essential source of energy for the maintenance of many tissues, such as the nervous system, red blood cells, the placenta, the mammary gland (Sastradipradja, 1998) and the ovaries (Rabiee et al., 1997; Leroy et al., 2004; Krasnow and Steiner, 2006). It also serves as a precursor for the biosynthesis of essential cell components, and is therefore equally important as a metabolite for all mammals. In the ruminant, most glucose must be synthesized by gluconeogenesis (Sastradipradja, 1998).

The aims of this study were to verify the effects of body condition score (BCS), breed and dietary supplementation on the blood leptin, insulin and glucose concentrations in hair sheep, and also to study the possible association of fertility and prolicacy with these blood metabolites and BCS and body weight (BW) recorded in ewes during the breeding season.
2. Materials and methods 2.1. Experimental site The experiment was conducted on the Experimental Farm of the State University of Vale do Acara (Sobral, Ceara, Brazil), which is located in a semi-arid region at 3 36 S, 40 18 W and is 56 m above sea level. The mean annual temperature in the region is 32 C and average rainfall is 800 mm per year. 2.2. Animals, experimental diets and management A total of 96 non-pregnant adult ewes of different ages were used in this study. There were 48 ewes of the Santa Ines breed and 48 ewes of the Morada Nova breed, with average BW and standard deviation of the initial mean of 43.0 5.6 kg and 25.55 3.1 kg, respectively. First the ewes were tagged with an earring for identication. Then they were weighed and were randomly distributed into two groups, unsupplemented and supplemented, for each breed. During the experimental period, all of the ewes were managed under semi-intensive conditions and were grazed in a paddock used for rotational stocking that had been sown with Tifton 85 bermudagrass (Cynodon spp.). All of the animals had free access to water and mineral salt. For statistical analysis, the animals were grouped in a randomized block design (homogenous weights) with four treatments consisting of two supplementation levels (unsupplemented and supplemented) and two breeds (Santa Ines and Morada Nova). The experimental diet fed to the ewes in the supplemented groups was formulated according to the National Research Council (2007) and consisted of ground Tifton 85 hay (70%), 94.67% ground corn, 5.04% soybean meal and 0.29% limestone. The dietary supplementation was provided two weeks before and throughout the breeding season, which lasted for 45 days. The Santa Ines ewes received 250 g of supplementation daily, whereas the Morada Nova ewes received only 150 g because of their smaller size. The supplementation was provided at 9 a.m. each day. Samples of Tifton 85 forage and concentrates were dried in a forcedair oven at 55 C for 72 h before being ground in a knife mill with a screen with a 1 mm screen (Wiley mill, Arthur H. Thomas, Philadelphia, PA, USA). The samples were analyzed for their dry matter (DM) content (AOAC, 1990; method number 930.15) and crude protein content (CP; AOAC, 1990; method number 984.13). To analyze the neutral detergent ber (NDF), the samples were treated with thermostable alpha-amylase without using sodium sulte. The concentrate consisted of 92% DM, 10% CP, 12% NDF, 0.29% calcium and 0.16% phosphorus. The breeding season for this study was considered to be 45 days long (starting at the end of June and ending in early August 2008). Five mature rams, two of the Santa Ines breed and three of the Morada Nova breed, with normal reproductive status were used during the breeding season. The rams were fed with the same dietary supplementation that had been formulated for the ewes. The ewes remained in the paddock during the day and at the end of the day they were taken to the management center. Here they were presented to a teaser ram that had been painted on its sternum with a vegetable dye mixed in a Vaseline base so that the ewes that had been mounted could be identied by the pigment that had been transferred to their rump. After this, the ewes were presented to rams of the same breed for controlled natural mating. The animals were weighed weekly in order to chart weight gain, and the BCS was measured in all ewes according to Russel et al. (1969). The BCS scale ranges from 1 (thin) to 5 (fat). Blood was sampled from 67 randomly selected ewes. During the trial, samples were collected from each animal by jugular venipuncture in non-anticoagulation gel separator vacuum tubes (Vacuntainer ) to obtain serum and to determine the leptin, insulin and glucose concentrations. Immediately after collection, the samples were centrifuged and the serum was stored at 20 C until it was assayed.

274 2.3. Chemical analysis

A.G.V. Catunda et al. / Small Ruminant Research 114 (2013) 272279 Table 1 Means SD of the blood leptin and glucose concentrations for sample collections and treatments during the breeding season. Collection 1 2 3 4 5 6 7 Average Treatment Supplemented Unsupplemented Supplemented Unsupplemented Supplemented Unsupplemented Supplemented Unsupplemented Supplemented Unsupplemented Supplemented Unsupplemented Supplemented Unsupplemented Leptin (ng/ml) 1.27 0.068aA 1.18 0.070aA 1.26 0.064aA 1.07 0.066bAB 1.18 0.064aB 0.98 0.064aB 1.00 0.066aC 1.04 0.065aAB 0.99 0.065aC 0.92 0.073aB 0.97 0.073aC 1.01 0.071aAB 1.27 0.078aA 1.01 0.067bAB 1.10 0.246 Glucose (mg/dl) 53.88 2.68aBC 45.67 2.76bA 63.51 2.60aA 47.31 2.59bA 53.92 2.52aAB 49.54 2.51bA 53.14 2.61aBC 48.79 2.57aA 55.30 2.56aBC 47.69 2.87bA 52.26 2.86aBC 52.68 2.82aA 49.14 3.06aC 51.38 2.65aA 52.39 9.69

Plasma leptin was determined using double-antibody radioimmunoassay (RIA) procedures. Serum immunoreactive leptin was independently determined in 100 l aliquots of serum samples using a multi-species RIA kit (XL-85K; Linco Research, St. Louis, MO, USA) according to the manufacturers recommendations (Delavaud et al., 2000). The sensitivity limit was 1.0 ng/ml (for 100 l sample size) and the interassay coefcients of variation were 5.3%. The plasma insulin concentration was determined using a RIA kit (Porcine Insulin RIA kit PI-12K; Linco Research, St. Louis, MO, USA) according to Greenwood et al. (2002) and Szymanski et al., 2011. The interassay coefcient of variation was 8.7%. Blood glucose concentrations were determined according to Evans et al. (1975), Udum et al. (2008) and Szymanski et al. (2011). The reaction is based on the enzymatic oxidation of glucose in the presence of glucose oxidase, leading to the formation of hydrogen peroxide. The hydrogen peroxide then reacts with phenol and 4-aminophenazone, in the presence of peroxides, to form a red/violet quinone-imine dye, with the intensity of the color produced being proportional to the glucose concentration. The concentration of glucose in each of the samples was determined in duplicate. The inter-assay coefcient of variation was 9.3%. 2.4. Reproductive performance The reproductive efciency of the ewes was evaluated according to fertility (the number of ewes parity/number of ewes submitted to mating) and prolicacy (number of lambs born/number of ewes parity). These results were expressed in percentage values. 2.5. Statistical analyses A completely randomized design in a 2 2 factorial arrangement was used, with two breeds (Santa Ines and Morada Nova) and two treatments (supplemented and unsupplemented), according to the mathematical model: Yijklm = + ci + rj + ik + tl + eijklm ,

Lowercase letters: comparison between treatments in same sample collection. Uppercase letters: comparison between sample collections in same treatment.

where Yijklm , leptin, insulin and glucose concentrations; , general mean (leptin, insulin and glucose concentrations); ci , body condition class; rj , effect of breed (j, Santa Ines and Morada Nova); ik , age, used with the covariate in the model; tl , supplementation effect (l, supplemented or unsupplemented); eijklm , random error. The data for the parameters of the insulin, leptin and glucose concentrations were squared in order to t the analysis of variance (ANOVA) assumptions. The Pearson correlation coefcient was used to evaluate the degree of association of the variables leptin, insulin, glucose, BCS, and BW with fertility class and ewe prolicacy. The BCS score classes were created so that all of the information obtained would be used. The classes were dened as follows in order to increase the accuracy of these parameters: class 1, ewes with BCS < 2; class 2, ewes with BCS < 3; and class 3, ewes with BCS > 3. The statistical analyses were performed using the PROC GLM program of SAS version 9.1 (SAS, 2003).

3. Results Plasma leptin (p < 0.05), insulin and glucose concentrations were inuenced by the dietary supplementation treatment. There was no signicant effect of BCS class on the leptin, insulin and glucose concentrations during the breeding season, and the effect of breed (p < 0.0001) was signicant only for blood glucose concentration. Also, a signicant correlation between breed, treatment and sample collection was found only for blood insulin concentrations (p < 0.0001). Blood leptin concentrations measured in hair sheep raised in a tropical climate ranged from 0.92 to 1.27 ng/ml (mean 1.10 0.24 ng/ml). During the mating period, the initial (second sample collection) and nal (seventh

sample collection) blood leptin concentrations of the ewes were signicantly different (p < 0.05) between treatments, but not between breeds, with higher concentrations being observed for supplemented ewes. A signicant decrease in blood leptin was observed from the third to the seventh sample collections in supplemented ewes (Table 1). The mean blood insulin concentration was 7.91 1.54 U/ml. A signicant difference (p < 0.05) was observed between treatments in the insulin concentrations of Santa Ines ewes in the third sample collection and Morada Nova ewes in the third, fth and seventh sample collections. In both breeds, the insulin concentrations were higher in the supplemented group. At the nal collection, supplemented Santa Ines ewes had higher insulin concentrations than Morada Nova ewes. However, when the Santa Ines ewes were unsupplemented, the insulin concentration was lower than in the Morada Nova ewes (Table 2). The mean blood glucose concentration was 52.4 9.7 mg/dl. The glucose concentration was higher in the supplemented group and varied from collection to collection (Table 1). There was also a signicant difference in blood glucose concentration between breeds in only the fourth and seventh sample collections, with higher concentrations being found in the Santa Ines ewes than in the Morada Nova ewes (Table 3). The supplemented Morada Nova ewes showed more variation in glucose concentrations than the supplemented Santa Ines ewes. There was no signicant difference between breeds for fertility and prolicacy (Table 4). When fertility was considered to be 0% (non-pregnant) (Table 5), signicant associations (p < 0.05) were observed for leptin with insulin, glucose and BCS, and insulin with glucose, BW and BCS. However, in pregnant ewes, the leptin was correlated only with insulin, and insulin correlated only with BW.

A.G.V. Catunda et al. / Small Ruminant Research 114 (2013) 272279 Table 2 Means SE of the blood insulin concentrations (U/ml) for collections, breed and treatments during the breeding season. Collection Breed Treatment Supplemented 1 2 3 4 5 6 7 SI MN SI MN SI MN SI MN SI MN SI MN SI MN Average 5.27 6.74 10.07 7.15 9.94 10.04 12.86 8.69 12.76 9.78 12.69 11.17 15.96 9.92 1.51d 1.56B 1.44bc 1.50AB 1.46c1 1.48AB1 1.48b1 1.51AB 1.41ba1 1.52AB1 1.65ab1 1.61A1 1.88a1* 1.71AB** Unsupplemented 3.11 6.28 5.95 5.49 3.47 5.58 4.50 5.84 3.85 5.55 6.06 5.67 3.66 8.68 1.68 1.68 1.62 1.60 1.702 1.472 1.672 1.48 1.912 1.602 1.632 1.742 1.592** 1.60*

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Table 4 Fertility and prolicacy in according to breed and treatment in hair sheep. Breed Santa Ines (48) Morada Nova (48) Treatment Supplemented (48) Unsuplemented (48) Fertility (%) 85.41 (41/48) 79.16 (38/48) 81.25 (39/48) 83.33 (40/48) Prolicacy 1.37 (56/41) 1.42 (54/38) 1.38 (54/39) 1.40 (56/40)

Fertility: the number of ewes parity/number of ewes submitted to mating and prolicacy: number of lambs born/number of ewes parity.

7.91 1.54

Lowercase letters: comparison between collections in the Santa Ines (SI) ewes. Uppercase letters: comparison between collections in the Morada Nova (MN) ewes. Number: comparison between treatments in the same breed. Asterisk (*): comparison between breeds in the same treatment.

Table 3 Means SE of the blood glucose concentrations for collections and breeds during the breeding season. Collection 1 2 3 4 5 6 7 Average Breed SI MN SI MN SI MN SI MN SI MN SI MN SI MN Glucose (mg/dl) 46.00 53.55 56.30 54.53 50.73 56.73 57.24 44.69 54.19 48.81 54.85 50.09 57.77 42.75 2.823aB 2.942aA 2.744aA 2.965aB 2.755aAB 2.720aA 2.918aA 2.773bC 3.023aA 2.899aBC 2.942aA 3.126aABC 3.234aA 3.083bC

The prolicacy was divided into four categories: 1.35 (Santa Ines breed, supplemented); 1.36 (Santa Ines breed, unsupplemented); 1.45 (Morada Nova breed, supplemented); and 1.46 (Morada Nova breed, unsupplemented) (Table 5). A signicant positive correlation was found between BCS and insulin for supplemented Santa Ines (p < 0.05, r = 0.32) and Morada Nova (p < 0.05, r = 0.49) ewes, but not between unsupplemented ewes of either breed. The correlations between BW and leptin (p < 0.05, r = 0.32) in supplemented Santa Ines ewes, and between BCS and leptin (p < 0.05, r = 0.57) in supplemented Morada Nova ewes were signicant. For supplemented Morada Nova ewes, a signicant positive correlation was also found between leptin and insulin (p < 0.05, r = 0.41). A signicant positive correlation between leptin and glucose (p < 0.05, r = 0.39) was only found in unsupplemented Santa Ines ewes. 4. Discussion There is no information in the literature on the inuence of blood leptin concentration and action, or of the effect of dietary supplementation, on the reproduction of Brazilian hair sheep breeds during the mating period. This is therefore the rst study to determine blood leptin concentrations and conrm that dietary supplementation during the mating period inuences leptin, insulin and glucose concentrations in hair sheep raised in a tropical climatic. The average blood leptin concentration was 1.10 0.24 ng/ml (Table 1). Studies carried out on wool sheep from temperate climates have shown higher leptin concentrations (average minimum of 2.09 ng/dl) (Delavaud et al., 2000; Marie et al., 2001; Sosa et al., 2009) than those obtained for each of the animals (Santa Ines or Morada Nova breeds) in this study. Leptin is secreted mainly by adipocytes (Barash et al., 1996), but it is known that the

52.39 9.693

Lowercase letters: comparison between breeds in the same collection. Uppercase letters: comparison between collections in the same breed.

Table 5 Pearson correlation coefcient between insulin, glucose, body weight and body condition score according to fertility. Fertility (%) Leptin Insulin Glucose Not pregnant Parity Not pregnant Parity Not pregnant Parity Insulin 0.43762* 0.29550* Glucose 0.36945* 0.08683 0.37541* BW 0.07959 0.12626 0.33571* 0.26488* BCS 0.53929* 0.00369 0.52779* 0.000440 0.45969* 0.07321

Body weight (BW), body condition score (BCS). * Signicant at p < 0.05.

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Santa Ines and Morada Nova hair sheep breeds have little body fat and we believe that this explains the low leptin concentrations found in this study. These results therefore suggest that this hormone has less inuence on the reproduction of hair sheep raised in a tropical environment. In normal animals, leptin serves as a metabolic signal to the reproductive system, informing it that sufcient fat stores are available to meet the caloric demands of reproduction (Barash et al., 1996). Energy status is generally considered to be a major nutritional factor that inuences reproductive processes. In the present study, the effect of dietary supplementation on leptin, insulin and glucose concentrations was investigated (Tables 1 and 2). Dietary supplementation was found to increase blood insulin concentrations in three of the sample collections, particularly in the Santa Ines ewes (Table 2). This result was in agreement with the observations made by Parra and Beltran (2008) that a proper nutrient intake in ruminants resulted in increased plasma insulin and an increase in body reserves, mainly fat. In the female reproductive process, an increase in insulin has been linked to follicular growth modulation, steroidogenesis, oocyte maturation and, consequently, embryonic development by its action on ovarian activity (McCann and Hansel, 1986; Kawashima et al., 2007). In monogastric animals, insulin secretion is mediated by the plasma glucose concentration, some amino acids (arginine, lysine and leucine), fatty acids, and ketones (Gonzlez and Silva, 2006). Because ruminants derive metabolizable energy primarily from volatile fatty acid production in the rumen, little glucose is absorbed from their digestive tracts (Sastradipradja, 1998). Thus, propionate (Johnson et al., 1982) and butyrate (Arcus et al., 1976) are insulinogenic in ruminants. Propionate is a well-known precursor of carbohydrate. The amounts of propionate available from rumen fermentation can theoretically supply the glucose requirements not accounted for by protein sources. Butyrate, the third major fatty acid of rumen fermentation, inuences glucogenesis but does not contribute carbon directly to glucose. Butyrate stimulates glucose production by the liver by increasing phosphorylases and gluconeogenesis. The acetyl-CoA derived from -oxidation of butyrate also activates pyruvate carboxylase, a key gluconeogenic enzyme, which further promotes gluconeogenesis (Kaneko, 2008). The results of the current trial are in agreement with those found in the international literature, because the dietary supplementation promoted an increase in volatile fatty acid formation. Tokuda et al. (2002) found a positive correlation between leptin, insulin and glucose concentrations in sheep. They suggested that plasma leptin and insulin concentrations would be affected by the nutritional condition of the animal. Although moderate, there was a positive correlation between insulin and glucose concentrations (0.37), but only in non-pregnant ewes (Table 5). Although an earlier study had suggested that insulin induced hypoglycemia in non-pregnant ewes (Downing and Scaramuzzi, 1997), our experiment showed that dietary supplementation induced a signicant increase in blood glucose concentration in hair sheep. This result indicates that blood glucose

might induce the secretion of insulin in supplemented hair sheep. Downing and Scaramuzzi (1997) also found that sheep can maintain their blood glucose concentrations by utilizing either endogenous or exogenous sources and that glucose is rarely limiting. However, it has been suggested that the mechanism responsible for regulating gonadotropin-releasing hormone (GnRH) pulses may be relatively sensitive to severe undernutrition, such as during late pregnancy or lactation, when there is an extremely high demand for glucose. Another important aspect is that, in lactating dairy cows, insulin is a positive regulator of plasma leptin (Block et al., 2003). The results of this study suggest that insulin also plays an important role in regulating plasma leptin in nonpregnant hair sheep. This assay also showed that insulin concentration was correlated with leptin. Differences in insulin concentrations (Table 2) and in glucose concentrations (Table 3) were also found between breeds. The reason for this difference is not clear; however, similar results were found by Herselman and van Loggerenberg (2012), who veried that plasma insulin concentration was considerably higher in Namaqua Afrikaner sheep than in all other breeds. According to Matsuzaki et al. (1997), the breed differences observed may be due in part to a distinct breed difference in glucose turnover that is attributable to the rate of hepatic glucose output (gluconeogenesis) and peripheral glucose utilization. We believe that these differences partially explain the higher dependence of the Santa Ines breed on supplementation than the Morada Nova breed. In this trial, the major correlations were found in nonpregnant ewes (Table 5). In all parameters, the BCS showed the highest correlations because it is related to the three metabolites studied. Caldeira et al. (2007) reported that a methodology proposed by Russel et al. (1969) attempted to estimate the quantity of body reserves, which includes fat, the largest source of energy, as well as the major reserve of protein in the body, the skeletal muscle. This information is important for appraising the adequacy of feeding programs, particularly in production systems where food availability is not constant. So, according to Caldeira et al. (2007), it was established that the quantity of body reserves has a denite effect on animal reproductive and production efciency. Hence, the BCS has gained widespread acceptance in sheep production, especially as an indicator of the nutritional status of ewes. Moreover, the ability of ruminants to cope metabolically with the nutritional demands of additional production or seasonal changes in pasture yield and quality has made BCS an indispensable tool for ewe management. In addition, nutritional condition is an important parameter because it inuences the follicular growth rate and maximum diameter, and also affects the ability of the follicle to ovulate (Diskin et al., 2003). In this trial, a correlation was also found between blood leptin and glucose concentrations. This differs from the result reported by Kauter et al. (2000), which suggested that glucose is not an important short-term regulator of blood leptin concentration in sheep. These authors also observed that the hormones that affect blood glucose do not inuence leptin concentration. In our trial the animals received dietary supplementation for 59 days (two weeks before and throughout the breeding season), so

A.G.V. Catunda et al. / Small Ruminant Research 114 (2013) 272279 Table 6 Pearson correlation coefcient between insulin, glucose, body weight and body condition score according to prolicacy. Prolicacy (unid.) Body weight 1.35 1.36 1.45 1.46 1.35 1.36 1.45 1.46 1.35 1.36 1.45 1.46 1.35 1.36 1.45 1.46 BCS 0.29307 0.24439 0.57421* 0.3030 Leptin 0.32637* 0.04675 0.11548 0.01082 0.00794 0.04001 0.21884 0.22088 Insulin 0.25934 0.25487 0.15013 0.02786 0.32376* 0.12613 0.49012* 0.06408 0.28430 0.02449 0.40622* 0.14555

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Glucose 0.26851 0.11119 0.10559 0.08317 0.02095 0.06030 0.07196 0.11483 0.30637 0.39490* 0.07990 0.02928 0.13327 0.20221 0.14700 0.02883

BCS

Leptin

Insulin

Prolicacy: 1.35 (Santa Ines breed supplemented), 1.36 (Santa Ines breed unsupplemented), 1.45 (Morada Nova breed supplemented) and 1.46 (Morada Nova breed unsupplemented). Body condition score (BCS). * Signicant at p < 0.05.

this difference explains the results observed in this study, because these hormones seem to be interdependent when dietary supplementation is provided long-term. A correlation was found between insulin and BW and BCS, and between glucose and BCS (Table 5). These correlations suggest that knowing a ewes BCS is relevant during the breeding season because it is a measure of their reproductive potential. Tokuda et al. (2002) demonstrated that the changes in insulin, leptin and glucose were affected by nutritional status and body fat. Some studies reported that a good BCS in ewes during mating and lambing is important for achieving favorable reproductive rates (Machado et al., 1999). Moreover, ewes with a high BCS had a higher mean luteinizing hormone (LH) pulse frequency than ewes with a low BCS and higher mean follicle-stimulating hormone (FSH) concentrations. It is known that, in ruminants, glucose plays a key role in the metabolic regulation that ne-tunes pulsatile GnRH release (Ohkura et al., 2004). The BCS for ewes that were not pregnant showed the highest correlation with leptin, insulin and glucose concentrations (Table 5). These results were different from those found by Liefers et al. (2003), which showed that BW affected leptin concentrations during pregnancy and lactation in cows. These authors also veried that leptin concentrations were very low 30 days before parturition, and suggested that the recovery of leptin concentrations after parturition seemed to depend on the quantity of fat deposition. Other studies found low leptin concentrations before and after parturition (Block et al., 2001; Barb and Kraeling, 2004), and found that the capacity of leptin to act as an indicator of moderate maternal undernutrition may be limited before lambing in sheep (Yuen et al., 2002). Our study suggests that leptin can regulate the endocrine pancreas of non-pregnant and pregnant ewes, but that its effect is more efcient in nonpregnant ewes. Williams et al. (2002) suggested that leptin can modulate both the hypothalamicpituitary axis and the endocrine pancreas under dened nutritional conditions in cattle and sheep. Hausman et al. (2012) suggested that

leptin is a key metabolic signal synthesized and secreted by fat cells that communicates information about body energy reserves, nutritional state, and metabolic shifts to the reproductive axis. The correlations were also evaluated according to different categories of prolicacy (Table 6). The distribution of correlations was heterogeneous between the categories, but was more numerous in the Morada Nova supplemented ewes (prolicacy 1.45). The results of the present study were different from those reported by Allden (1956), which suggested that differences in prolicacy were not related to the nutritional status of ewes at mating. The present study showed that the Morada Nova ewes were reproductively more efcient than the Santa Ines ewes. These results therefore suggest that the Morada Nova ewes were better adapted to the semi-arid region in a tropical climate than the Santa Ines ewes. Previous studies have reported that in northeastern Brazil, especially in semi-arid regions, some breeds, such as Morada Nova, begin to develop features adapted to the climatic conditions that enable them to reproduce in these environments (Costa et al., 2011; Silva et al., 2007). Johnson et al. (2003) reported that energy requirements for maintenance, traditionally dened as those that ensure the maintenance of BW, may vary between individual animals and between breeds because of the inuence of several physiological factors such as sex, BW, age, BCS, physical activity and genetic traits. The reproductive physiology of the hair sheep therefore requires further study because there have been no specic studies on the hormonal and metabolic characteristics of these breeds. Selaive-Villarroel and Fernandes (2000) observed that the coverage rate and prolicacy of Morada Nova ewes were affected by BW. For this reason, BW may be considered to be more important than age in reproductive efciency. We emphasize that the Morada Nova breed has animals with well-established typical traits, but the Santa Ines breed is still undergoing adaptation.

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