- Email: [email protected]
The onset of puberty of Pelibuey male hair sheep is not delayed by the short term consumption of Morus alba or Hibiscus rosa-sinensis foliage
Livestock Science 157 (2013) 378–383
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
The onset of puberty of Pelibuey male hair sheep is not delayed by the short term consumption of Morus alba or Hibiscus rosa-sinensis foliage E. Aguilar-Urquizo a,b, J.R. Sanginés-García b, J.A. Delgadillo c, C. Capetillo-Leal a, J.F.J. Torres-Acosta a,n a
FMVZ, Universidad Autónoma de Yucatán, Mérida, Yucatán, Km 15.5, Carretera Mérida-Xmatkuil, Mérida, México División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Conkal, Km 16.3, Antigua Carretera Mérida-Motul, Conkal, Yucatán, México c Centro de Investigación en Reproducción Caprina, Universidad Autónoma Agraria Antonio Narro, Periférico Raúl López Sánchez y Carretera a Santa Fe, C.P. 27054 Torreón, Coahuila, México b
a r t i c l e in f o
abstract
Article history: Received 16 May 2012 Received in revised form 26 April 2013 Accepted 31 May 2013
The present trial evaluated the effect of phytoestrogens consumed from Mulberry (Morus alba) or Hibiscus (Hibiscus rosa-sinensis) foliage, on the onset of puberty of Pelibuey male lambs. The design of the study aimed at identifying possible acute changes on the onset of puberty. Eighteen male lambs were used (10779 days of age, 1673 cm scrotal circumference (SC), 3.470.1 body condition score (BCS) and 1771 kg bodyweight (BW)). At weaning, the lambs were distributed in three groups (n¼6 each): (i) Control group (balanced feed based on grains +star grass), (ii) Mulberry Group (Mulberry foliage+balanced feed) and (iii) Hibiscus Group (Hibiscus foliage+balanced feed). BW, SC and body condition score (BCS) were determined every 14 days. An estrogenized adult female was used to attempt the collection of semen samples from the males two times per week. The onset of puberty was determined as the presence of sperm showing progressive movement in the ejaculate. From then, semen collections continued in the males (once a week) until the first normal ejaculate was obtained. The nutritional value of the foliages, as well as the content of total phenols, total tannins and condensed tannins, was determined. The phytoestrogen content (Equol, 4′7-dimetoxyisoflavone, Genistein, Rutin and Ononin) was determined from the methanolic extract of both plants. Total dry matter consumptions were similar between the Control and Mulberry groups and both were higher than the Hibiscus group (Po0.05). The total consumption of phytoestrogens and tannins was similar in the Mulberry and the Hibiscus group. The short-term consumption of phytoestrogens from Mulberry or Hibiscus foliage failed to delay the onset of puberty. The mean age at the onset of puberty was similar in the three groups (146 days in the control and Mulberry groups and 154 days in the Hibiscus group; P40.05). The mean age at the first normal ejaculate was also similar between groups (167 days for Mulberry and Hibiscus groups and 169 days in the control group; P40.05). The BW (Control: 21.673.1; Mulberry: 20.771.9; Hibiscus: 19.872.8 kg), the BCS and the SC were similar between groups at the onset of puberty. The present trial showed that a short exposure to M. Alba or H. rosa-sinensis foliage through diet did not influence the onset of puberty of Pelibuey male lambs. & 2013 Published by Elsevier B.V.
Keywords: Puberty Male sheep Phytoestrogens Morus alba Hibiscus rosa-sinensis Tropics Tannins
n Corresponding author. Tel.: +52 999 942 3200; fax: +52 999 942 3205. E-mail addresses: [email protected], [email protected] (J.F.J. Torres-Acosta).
1871-1413/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.livsci.2013.05.031
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
1. Introduction The foliage of Morus alba (Mulberry) and Hibiscus rosasinensis (Hibiscus) may become viable substitutes for the imported feed-stuffs used in the diets of growing sheep due to its high foliage productivity under tropical conditions. However, both plants contain phytoestrogens (flavonoids and isoflavonoids) that may affect the development of the reproductive tract of different mammals (Vasudeva and Sharma, 2008; You et al., 2002). Some flavonoids and isoflavonoids may act as antagonists or agonists of the steroidal hormones, because these metabolites may bind to estrogenic receptors (ER α and/or β) (Weihua et al., 2001; Whitten et al., 2002). The devastating effects of phytoestrogens on the reproduction of female ruminants have been recognized. However, the subclinical effects of low concentrations of phytoestrogens are considered now more important than the occasional outbreaks of clinical estrogenism (Adams, 1995). The effect of phytoestrogens consumption on the reproduction of male ruminants has been considered “little” (Adams, 1995), but the magnitude of those effects has been poorly studied for ruminants consuming forages containing phytoestrogens (Retana-Márquez et al., 2012). The effects of phytoestrogens on a given population can only be detected by measuring concentrations of phytoestrogens in the forage, or their effect on exposed animal populations (Adams, 1995). The use of Mulberry or Hibiscus foliages in the diet of weaned male lambs represents an opportunity to study the effect of the consumption of foliages containing low concentrations of phytoestrogens on their reproductive performance. Such effect should be evaluated on (a) the acute phase, by measuring the effect of short term consumption of these forages on the onset of puberty, and (b) the chronic phase by measuring the effect on the reproductive performance/ subclinical signs on adult males. These aspects should be explored before Mulberry and Hibiscus foliages are used extensively in the diets of growing male lambs, especially if those foliages are suggested for the nutrition of young
379
males intended for breeding purposes. We hypothesize that the short-term consumption of phytoestrogens contained in Mulberry and Hibiscus foliages could delay the onset of puberty in Pelibuey male lambs. Therefore, the objective of the study was to determine the effect of phytoestrogen consumption from the foliage of Mulberry or Hibiscus on the onset of puberty of Pelibuey male lambs. 2. Materials and methods This trial was performed in the Instituto Tecnológico de Conkal (ITC), Yucatán, México (21105′ N and 89132′ W) at 7 m above sea level. The predominant climatic conditions are hot, sub-humid with summer rainfall (Aw0(x′)(f)gi) with a mean ambient temperature of 26.5 1C and 900 mm of rainfall (Flores-Guido and Espejel-Carvajal, 1994). 2.1. Experimental animals and groups The experimental protocol was approved by the Academic Committee of the Posgraduate Unit (FMVZ-Universidad Autónoma de Yucatán) and the respective funding body (2547.09-P DGEST). A total of 18 male Pelibuey hair sheep lambs were used (from the same parturition group). Animals were born in June and were all managed under similar conditions in terms of feeding and housing. The animals were randomly assigned to the following three experimental groups (n ¼6): (i) Control Group ¼ Balanced feed, (ii) Mulberry Group¼Mulberry foliage ad-libitum+balanced feed and (iii) Hibiscus Group ¼Hibiscus foliage ad-libitum+balanced feed. The balanced feed was prepared with a mixture of grains that were confirmed to be free of phytoestrogens as described below (Table 1). At the beginning of the study, males of the Control, Mulberry and Hibiscus groups had similar BW (17 72, 18 71 and 17 71 kg, respectively), age (108 79, 106 711 and 106710 days, respectively), body condition score (BCS) (3.4 70.2, 3.570.0 and 3.4 70.2, respectively) and
Table 1 Ingredients used for the experimental diets and the chemical composition including ash, crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF). Experimental groups
Ingredients Sorghum (g kg−1 DM)
Corn (g kg−1 DM)
Meat meal (g kg−1 DM)
Star grass hay (g kg−1 DM)
Mulberry fodder (g kg−1 DM)
Hibiscus fodder (g kg−1 DM)
Control group Mulberry group Hibiscus group
280 140 140
310 140 140
150 0 0
260 120 120
0 600 0
0 0 600
Diet composition
Ash (g kg−1 DM)
Crude protein (g kg−1 DM)
NDF (g kg−1 DM)
ADF (g kg−1 DM)
87.2 40.1 40.1
115.7 89.2 89.2
618.8 490.5 490.5
315.3 186.0 186.0
108.7 100.7
131.8 99.4
375.1 478.7
237.5 281.7
Balanced feed Control group Mulberry group Hibiscus group Foliages Mulberry foliage Hibiscus foliage
380
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
scrotal circumference (SC) (1673, 15 71 and 17 73 cm, respectively). The trial started with a 10-day adaptation period starting on October (beginning of autumn). After that, the average length of time animals were exposed to the diets was 40 days at the onset of puberty, and 60 days at the first normal ejaculate.
glass slide (previously heated at 401) and a cover-slip was placed on top of the sample. (f) The semen was observed under a microscope (40 ) to confirm the presence of spermatozoa with progressive movement.
2.2. Animal management
From the moment when the male sheep were positive to the presence of viable spermatozoa, the individual males were taken every week (Saturday morning only) to perform further semen collections. The same protocol described above was used in further semen collections. These semen samples were obtained to determine the time when the animals were producing their first normal ejaculate. All the semen samples were analyzed to determine the semen volume and the percentage of sperm abnormalities (as described by Baril et al. (1993)). The latter was determined from semen smears stained with eosin–nigrosin (evaluating 200 spermatozoa). The sperm concentration was determined using a computer-assisted semen analysis (CASAs, ISASPROISIER, Spain). The following features were considered in semen samples to be declared normal: at least 0.35 mL of volume, a sperm concentration above 3100 million/mL and at least 65% of normal spermatozoa (Baril et al., 1993).
The Mulberry and Hibiscus group received their balanced feed individually (2% of their body weight (BW) in dry matter (DM)) as well as the respective foliage (2.5% of their BW in DM). The control group received only the balanced feed (4.5% of their BW in DM). The adjustment of the quantity of feed offered was performed every second week (when the BW was determined). Foliage refusal was collected daily. Concentrate feed was refused at a very low level for the duration of the study (nearly 40 g per animal per day). Animals received water and a mineral supplement (Fosforisals, Purina México) ad libitum for the duration of the study (Calcium 4%, Phosphorus 1%, Manganeso 3%, Selenio 3 ppm, Cobalto 5 ppm, Magnesio 1000 ppm, Cobre 2 ppm, Iodo 25 ppm, Zinc1000 ppm, Vit A 60 U.I/kg, Vit E 120 U.I./kg y Vit D 90 U.I./kg). The different groups were offered a diet to achieve a theoretical growth rate of 100 g/day. The composition of the diets is presented in Table 1. 2.3. Animal bodyweight, scrotal circumference, body condition score and average daily gain (ADG) The weight, SC and body condition score (BCS) were measured every 14 days from the beginning of the study. Animals were refrained from feed for 16 h prior to weighing. The SC was determined as described by Fuenmayor et al. (1990) and the BCS as reported by Russel (1984). Animals were also weighed on the day when the puberty was declared. The individual ADG was calculated by subtracting the initial BW to that recorded at the onset of puberty and the difference was divided by the number of days needed to reach puberty. 2.4. Onset of puberty The onset of puberty was declared when spermatozoa with progressive movement were found in the ejaculate of experimental lambs (Valencia et al., 1977). The collection of semen was performed using an artificial vagina and a standing female sheep. For that purpose, the following protocol was performed: (a) an adult female sheep (nonpregnant) was estrogenized. (b) All the experimental lambs were taken individually to the pen of the estrogenized female to establish direct individual contact for 5 min (Tuesdays and Thursdays every week). Males were observed during the 5 min they spent with the female to identify the mating attempts. When a lamb was observed to perform penetration (copula), it was then scheduled for a semen collection on the following morning. (c) The semen collections were performed with an artificial vagina. (d) The respective semen samples were collected in conical glass tubes. (e) A semen sample was placed in a
2.5. First normal ejaculate
2.6. Feed intake and feed analyses The voluntary feed intake (total dry matter intake) was determined three times per week for the duration of the trial. Samples of the different portions of the diet were collected every second day. The samples were used to prepare a bulk-sample per week. The analyses performed were dry matter, ashes, crude protein, neutral detergent fiber and acid detergent fiber (FAO, 2000). The content of total phenols (TP) and total tannins (TT) was determined using the Folin–Ciocalteu method (Makkar, 2003). The quantification of tannins was made using a diode array spectrophotometer (Spectrophotometer UV–vis Genesys 10S, Thermo Scientifics). The quantification of TT and TP was made at 725 nm. The condensed tannin (CT) content was quantified as described by Price et al. (1978). The quantification of CT was made by spectrophotometry at 520 nm. 2.7. Phytoestrogen identification and quantification A qualitative screening technique (Galisteo, 1997) was used to determine the presence/absence of phytoestrogens in every ingredient of the feeds used for the experimental groups. The only ingredients positive to phytoestrogens were the Mulberry and Hibiscus foliages. The phytoestrogens contained in them were identified and quantified. This was performed on five samples obtained from weekly bulk samples of the foliages (managed according to Zhishen et al. (1999)). Phytoestrogen standards evaluated were Equol, 4′7-dimetoxyisoflavone, Genistein, Rutin and Ononin. Firstly, plant methanolic extracts for each of the five weekly bulk samples were prepared (Klump et al., 2001). Extracts were injected in an HPLC system (ClarityChrom-Chromatographic SW. Knauer, Darmstad, Germany) with a C18 reversed-phase column 5 mm (250 4.6 mm) for
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
separation of the metabolites. Mobile phase was a gradient of water/acetic acid (96:4 V/V) (A) and Acetonitrile (B) (0 min 90:10; 1 min 80:20; 7 min 35:65; 15 min 23:77; 23 min 90:10; v/v). Flow rate was set at 1 mL/min. Column was used at a temperature of 25 1C and the readings were performed with a UV detector (wave length of 260 nm). Solutions of standards at various concentrations from 0.5 to 4 mg/mL were injected into the HPLC system and the calibration curves were established for each standard compound with a correlation coefficient of at least 0.998 for each standard.
381
3. Results The total DM daily intake of the Control (786.5 719.8) and Mulberry group (837.4727.2 g DM) were similar and both were higher than the Hibiscus group (701.6727.2 g DM) (P o0.05). This was caused by the low daily foliage intake recorded for the Hibiscus group (348.8 721.9 g DM) compared to the Mulberry group (476.8 721.9 g DM) (P o0.05). The TP content of the two types of foliages (Table 2) was similar (Mulberry: 18.8 72.6 and Hibiscus: 13.1 72.6 g kg−1 DM; P 40.05) and so it was for the TT content (Mulberry: 11.372.0 and. Hibiscus: 8.0 7 2.0 g kg−1 DM; P 40.05) and the CT (Mulberry: 0.3 70.1 and Hibiscus: 0.270.1 g kg−1 DM; P4 0.05). In spite of the difference in foliage intake between the Mulberry and Hibiscus group, the total phytoestrogen consumption was similar in the Mulberry and Hibiscus groups (P40.05). Such similarity in the consumption of phytoestrogens was mainly due to the higher consumption of the flavonoids (rutin and 4′7-dimetoxyisoflavons) in the Hibiscus foliage compared to Mulberry. The isoflavonoid genistein was consumed more by sheep fed Mulberry, and the ononin was consumed more in the animals fed Hibiscus foliage (Po0.05) (Table 3). Age at the onset of puberty in Control Pelibuey lambs and in those fed M. alba or H. rosa-sinensis foliage did not differ (P 40.05; Table 4). Furthermore, the first normal ejaculate was also obtained at the same age in the three experimental groups (P40.05). There was no difference in the BW at the onset of puberty or at the first normal
2.8. Data analyses The mean age and BW at the onset of puberty, as well as the mean feed intake (DM), foliage intake (DM), ADG, BCS and SC were compared between the three groups using the GLM procedure and the post-hoc Tukey test of SAS 9.1 for Windows. The same analyses were performed on the data obtained when the first normal ejaculate was obtained from the males. The GLM procedure was also used to compare the contents of TP, TT and CT of the two foliage types (Mulberry and Hibiscus). The difference in the quantity of the different phytoestrogens consumed and the total quantity of phytoestrogens consumed in the foliage of the two plants were compared with respective non-parametric NPAR1WAY procedures for two samples (SAS 9.1 for Windows) using the Wilcoxon rank sum test.
Table 2 Total phenols (TP), total tannins (TT) and condensed tannins (CT) contained in the foliage of Morus alba and Hibiscus rosa-sinensis. Morus alba foliage
TPn TTn CTT1
Hibiscus rosa-sinensis foliage
Minimum
Maximum
Mean
S.E.
Minimum
Maximum
Mean
S.E.
11.5 6.7 0.2
23.6 13.8 0.5
18.8a 11.3a 0.3a
2.6 2.0 0.1
7.3 3.0 0.1
23.6 16.5 0.3
13.1a 8.1a 0.2a
2.6 2.0 0.1
S.E.¼ Standard error. a Different letters between columns indicate differences (P o0.05). n Expressed as tannic acid equivalent (g kg−1 DM). 1 Expressed as catechin equivalent (g kg−1 DM).
Table 3 Consumption (mg/day) of phytoestrogens, flavonids and isoflavonoids of Pelibuey sheep fed foliage of Morus alba (Mulberry) and Hibiscus rosa-sinensis (Hibiscus). Mulberry group
Phytostrogen Consumption (Total) Flavonoids (Total) Rutin 4′7-Dimetoxyisoflavone Isoflavonoids (Total) Genistein Ononin Equol a,b
Hibiscus group
Min
Max
Median
Min
Max
Median
170.1 39.4 9.4 170.2 109.2 102.7 0.0 0.0
271.2 90.3 6.2 271.3 228.8 181.0 10.3 37.6
209.2a 63.5 41.6a 24.0a 142.1 142.1a 1.5a 3.0a
64.7 34.0 34.0 0.0 29.1 8.3 5.4 1.5
480.1 210.3 210.4 15.0 298.3 85.4 229 31.8
223.3a 134.3 126.8b 2.1b 73.0 43.3b 18.0b 13.5a
Different letters in the same row indicate differences (Po 0.05).
382
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
Table 4 Mean age, body weight, scrotal circumference, body condition score and average daily gain ( 7 standard error) at the onset of puberty and at the moment of the first normal ejaculate in male Pelibuey lambs fed Mulberry and Hibiscus foliage compared to lambs fed a control diet. Variables
Age (days) Body weight (kg) Scrotal circumference (cm) Body condition score (1–5) Average daily gain (g/day) a,b
Onset of puberty
First normal ejaculate
Mulberry
Hibiscus
Control
Mulberry
Hibiscus
Control
146.2 7 8.6a 20.7 7 1.9a 21.6 7 1.3a 3.5 7 0.0a 71.5 7 37.2ab
1547 22.4a 19.87 2.8a 22.57 2.4a 3.57 0.3a 49.07 24.0b
1467 14.4a 21.6 7 3.1a 23.3 7 3.5a 3.5 7 0.0a 102.4 7 36.2a
167.17 10.9a 21.3 7 2.9a 20.87 2.7a 3.5 7 0.0a 60.17 39.0ab
167.0 7 9.8a 19.5 7 2.4a 21.3 7 2.8a 3.5 7 0.0a 34.3 7 16.9b
169.1 78.7a 22.3 71.5a 21.9 72.2a 3.5 70.0a 85.1 728.3a
Different letters in the same row at the respective reproduction stage indicate differences (Po 0.05).
ejaculate in the three groups (Table 4). Likewise, the SC and the BCS were similar throughout the trial in the three experimental groups. Finally, the ADG was similar (P40.05) between the Control and Mulberry groups, and both grew faster than the Hibiscus group during the experimental period (P o0.05; Table 4). 4. Discussion The onset of puberty of Pelibuey lambs was not delayed by the phytoestrogens consumed in the two types of foliage from weaning. Although the Mulberry group consumed more foliage than the Hibiscus group, the total quantity of phytoestrogens consumed was similar in both groups. Individual phytoestrogens consumed with each foliage type were different in composition. The Mulberry group consumed more 4′7dimetoxyisoflavone and Genistein than the Hibiscus group. Meanwhile, the Hibiscus group consumed more Rutin and Ononin than the Mulberry group. However, this difference in composition of phytoestrogens consumed by the Pelibuey lambs did not delay the onset of puberty. Whitten et al. (1992) showed that phytoestrogens cause biological effects in the animals when: (i) they consume material containing high quantities of some specific phytoestrogens (i.e. coumestrol, genisteina), or (ii) they consume low concentrations for a long period of time. Indeed, long periods of consumption of phytoestrogenic materials may decrease sperm concentration in humans (Chavarro et al., 2008). Similarly, high amounts of phytoestrogens from soy foods, have been related to lower testicular weight, germ cell number and Sertoli cell function in Asian men compared with Caucasian and Hispanic individuals (Johnson et al., 1998). Studies performed with rodent models have confirmed that the exposure to phytoestrogen early in life can be associated to decreased testicular weight or size, decreased spermatogenesis and lower FSH levels (Atanassova et al., 2000). However, the effect of exposure of adult males to phytoestrogens varies with different animal species. While some studies performed in male rodents showed negative effects on reproductive activity (Glover and Assinder, 2006), other studies reported a positive effect of phytoestrogen consumption on semen quality (Yousef et al., 2004). Thus, the susceptibility to phytoestrogens on male animals seems to be species-specific (Chavarro et al., 2008). The latter highlights the importance of producing specific evidence for male sheep rather than trying to use data from other animal species. In our study, lack of any negative effect of phytoestrogens on the onset puberty could be explained by at least two non-
exclusive hypotheses. Firstly, the period of consumption of the phytoestrogenic materials might have been too short to delay the onset puberty (40 days from the start of the study) or to first normal ejaculate (60 days from the start of the study). Secondly, all the experimental males of the experiment were in a constant contact with an estrogenized female used in the process of training for semen collection of males using an artificial vagina. Males received a constant stimulus that consisted of visual, olfactory and physical contact twice a week until the moment when they performed their first complete copula with an ejaculate. Thereafter, males were exposed to the same female once a week until the moment when they got their first normal ejaculate. The presence of a female (either receptive or not) can modify the pattern of behavior of male sheep affecting plasmatic testosterone concentrations as well as the frequency and pulsatility of LH especially in males with sexual experience (González et al., 1991a, 1991b). All those aspects may have had stronger influence on the onset of puberty than the consumption of phytoestrogens in the foliage. Changes in sexual behavior have been already described in male rats without sexual experience when exposed to females in estrus (Portillo and Paredes, 2004). Although the animals fed the Hibiscus fodder grew slowest than the group fed Mulberry or the Control group (Po0.05), the onset of puberty was similar in the three groups. This suggested that age was more important than BW or ADG to reach puberty in Pelibuey males. Animals in the Hibiscus group showed a low ingestion of Hibiscus foliage (348.8 g/day) compared to other trials where it was used dry meal of Hibiscus (Mata et al., 2006; Ruiz-Sesma et al., 2006). It is unlikely that the animals suffered any reduction in foliage consumption due to the low TT or CT content of the plants. The mucilage present in the chopped foliage could have caused the low ingestion of Hibiscus fresh foliage (Shimizu et al., 1993). Lack of any acute effect of foliage consumption on puberty should be considered only as a first step and cannot be extrapolated to other stages of the reproductive life of male sheep. 5. Conclusion The present study showed that the short-term consumption of Mulberry or Hibiscus foliages did not affect the onset of puberty of Pelibuey lambs. Also, the first normal ejaculate was obtained at the same age in all the experimental groups. The total consumption of
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
phytoestrogens and tannins was similar in the Mulberry and the Hibiscus group. Thus, the consumption of phytoestrogens from the foliage of Mulberry or Hibiscus for a short time did not affect the reproductive function of Pelibuey males early in their reproductive life. Conflict of interest The authors confirm that they have no conflict of interest that could inappropriately influence, or be perceived to influence the work included in this paper.
Acknowledgments The financial support for the present trial was obtained from the Dirección General de Educación Superior Tecnológica (DGEST) (Project no. 2547.09-P). Aguilar-Urquizo E., received a Doctoral scholarship from CONACYT, Mexico. References Adams, N.R., 1995. Detection of the effects of phytoestrogens on sheep and cattle. J. Anim. Sci. 73, 1509–1515. Atanassova, N., McKinnell, C., Turner, K.J., Walker, M., Fisher, J.S., Morley, M., Millar, M.R., Groome, N.P., Sharpe, R.M., 2000. Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility: evidence for stimulatory effects of low estrogen levels. Endocrinology 141, 3898–3907. Baril, G., Chemineau, P., Cognie, Y., Guérin, Y., Leboeuf, B., Orgeur, P. Vallet, J., 1993. Manuel de formation pour L'insemination artificielle chez les ovins et les caprins. Organisation des Nations Unies pour L'alimentation et L'agriculture, Rome, Italie, 125. Chavarro, J.E., Toth, T.L., Sadio, S.M., Hauser, R., 2008. Soy food and isoflavone intake in relation to semen quality parameters among men from an infertility clinic. Hum. Reprod. 23, 2584–2590. FAO, 2000. Quantification of Tannnins in Tree Foliage. A Laboratory Manual for the FAO/IAEA Coordinated Research Project on “Use of Nuclear and Related Techniques to Develop Simple Tannin Assays for Predicting and Improving the Safety and Efficiency of Feeding Ruminants on Tanniniferous Tree Foliage”. FAO/IAEA Working Document IEA, Vienna. Flores-Guido, J.S., Espejel-Carvajal, I., 1994. Tipos de vegetación de la Península de Yucatán. Etnoflora yucatanense. Universidad Autónoma de Yucatán. Mérida, Yucatán, México. Fuenmayor, F.C., Martínez, A.M., Valle, A., Fuentes, A., Quintana, H., Regueiro, C., 1990. Observaciones sobre el desarrollo testicular y desprendimiento del prepucio en corderos West African, Berbados y Persa Cabeza Negra. Zootec. Trop. 8, 73–84. Galisteo, M.M., 1997. Rosmarinus tomentosus Huber-Morath & Maire: Estudio Farmacognóstico. Tesis Doctoral Inéd. Facultad de Farmacia, Universidad de Granada España. Glover, A., Assinder, S.J., 2006. Acute exposure of adult male rats to dietary phytoestrogens reduces fecundity and alters epididymal steroid hormone receptor expression. J. Endocrinol. 189, 565–573. González, R., Orgeur, P., Poindron, P., Signoret, J.P., 1991a. Female effect in sheep. I. The effects of sexual receptivity of females and sexual experience of rams. Reprod. Nutr. Dev. 31, 97–102.
383
González, R., Levy, F., Orgeur, P., Poindron, P., Signoret, J.P., 1991b. Female effect in sheep. II. Role of volatile substances from the sexually receptive female; implication of the sense of smell. Reprod. Nutr. Dev. 31, 103–109. Johnson, L., Barnard, J.J., Rodriguez, L., Smith, E.C., Swerdloff, R.S., Wand, X.H., Wang, C., 1998. Ethnic difference in testicular structure and spermatogenic potential may predispose testes of Asian men to a heightened sensitivity to steroidal contraceptives. J. Androl. 19, 348–357. Klump, P.S., Allred, C.M., MacDonald, L.J., Ballam, M.J., 2001. Determination of isoflavonen in soy and selected foots containing soy by extraction, saponification, and liquid chromatography: collaborative study. J. AOAC Int. 84, 1865–1883. Makkar, H.P.S., 2003. Quantification of Tannins in Tree and Shrub Foliage. A Laboratory Manual. Food and Agriculture Organization of the United Nations/International Atomic Energy Agency (FAO/IAEA), Vienna, Austria 49–53. Mata, E.M.A., Hernández, S.D., Cobos, P.M.A., Ortega, C.M.E., Mendoza, M.G.D., Arcos-García, J.L., 2006. Comportamiento productivo y fermentación ruminal de corderos suplementados con harina de cocoíte (Gliricidia sepium), morera (Morus alba) y tulipán (Hibiscus rosa-sinensis). Rev. Cient. XVI, 249–256. Portillo, W., Paredes, R.G., 2004. Sexual incentive motivation, olfactory preference, and activation of the vomeronasal protection pathway by sexually relevant cues in non-copulating and naïve male rats. Horm. Behav. 46, 330–340. Price, M.L., Van Scoyoc, S., Butler, L.G., 1978. A critical evaluation of the vanillin reaction assay for tannin in sorghum grain. J. Agric. Food Chem. 26, 1214–1218. Retana-Márquez, S., Hernández, H., Flores, J.A., Muñoz-Gutiérrez, M., Duarte, G., Vielma, J., Fitz-Rodríguez, G., Fernández, I.G., Keller, M., Delgadillo, J.A., 2012. Review. Effects of phytoestrogens on mammalian reproductive physiology. Trop. Subtrop. Agroecosyst. 15, 129–145. Ruiz-Sesma, D.L., Lara-Lara, P.E., Sierra-Vázquez, A.C., Aguilar-Urquizo, E., Magaña-Magaña, M.A., Sanginés-García, J.R., 2006. Evaluación nutritiva y productiva de ovinos alimentados con heno de Hibiscus rosasinensis. Zootec. Trop. 24, 467–482. Russel, A., 1984. Body condition scoring of sheep. Farm Pract. 6, 91–93. Shimizu, N., Tomodo, M., Suzuki, I., Takada, K., 1993. Plant mucilages. XLIII. A representative mucilage with biological activity from the leaves of Hibiscus rosa-sinensis. Biol. Pharm. Bull. 16, 735–739. Valencia, J., Barrón, C., Fernández-Baca, S., 1977. Pubertad en corderos Tabasco x Dorset. Vet. Méx. 8, 127–130. Vasudeva, N., Sharma, S.K., 2008. Post-Coital antifertility activity of Hibiscus rosa-sinensis Linn roots. eCAM 5, 91–94. Weihua, Z., Mäkelä, S., Andersson, L., Salmi, S., Saji, S., Webster, J.I., Jensen, E.V., Nilsson, S., Warner, M., Gustafsson, J., 2001. A role for estrogen receptor B in the regulation of growth of the ventral prostate, 98. 〈www.pnas.org/cgl/dol/10.1073/pnas.111150898〉. Whitten, P.L., Russell, E., Naftolin, F., 1992. Effects of a normal, humanconcentration, phytoestrogen diet on rat uterine growth. Steroids 57, 98–106. Whitten, P.L., Patisaul, H.B., Young, L.J., 2002. Neurobehavioral actions of coumestrol and related isoflavonoids in rodents. Neurotoxicol. Teratol. 24, 47–54. You, L., Sar, M., Bartolucci, E.J., McIntyre, B.S., Sriperumbudur, R., 2002. Modulation of mammary gland development in prepubertal male rats exposed to genistein and methoxychlor. Toxicol. Sci. 66, 216–225. Yousef, M.I., Esmail, A.M., Baghdadi, H.H., 2004. Effect of isoflavones on reproductive performance, testosterone levels, lipid peroxidation, and seminal plasma biochemistry of male rabbits. J. Environ. Sci. Health B39, B819–833. Zhishen, J., Mengcheng, T., Jianming, W., 1999. The determination of flavonoid in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64, 555–559.
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
The onset of puberty of Pelibuey male hair sheep is not delayed by the short term consumption of Morus alba or Hibiscus rosa-sinensis foliage E. Aguilar-Urquizo a,b, J.R. Sanginés-García b, J.A. Delgadillo c, C. Capetillo-Leal a, J.F.J. Torres-Acosta a,n a
FMVZ, Universidad Autónoma de Yucatán, Mérida, Yucatán, Km 15.5, Carretera Mérida-Xmatkuil, Mérida, México División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Conkal, Km 16.3, Antigua Carretera Mérida-Motul, Conkal, Yucatán, México c Centro de Investigación en Reproducción Caprina, Universidad Autónoma Agraria Antonio Narro, Periférico Raúl López Sánchez y Carretera a Santa Fe, C.P. 27054 Torreón, Coahuila, México b
a r t i c l e in f o
abstract
Article history: Received 16 May 2012 Received in revised form 26 April 2013 Accepted 31 May 2013
The present trial evaluated the effect of phytoestrogens consumed from Mulberry (Morus alba) or Hibiscus (Hibiscus rosa-sinensis) foliage, on the onset of puberty of Pelibuey male lambs. The design of the study aimed at identifying possible acute changes on the onset of puberty. Eighteen male lambs were used (10779 days of age, 1673 cm scrotal circumference (SC), 3.470.1 body condition score (BCS) and 1771 kg bodyweight (BW)). At weaning, the lambs were distributed in three groups (n¼6 each): (i) Control group (balanced feed based on grains +star grass), (ii) Mulberry Group (Mulberry foliage+balanced feed) and (iii) Hibiscus Group (Hibiscus foliage+balanced feed). BW, SC and body condition score (BCS) were determined every 14 days. An estrogenized adult female was used to attempt the collection of semen samples from the males two times per week. The onset of puberty was determined as the presence of sperm showing progressive movement in the ejaculate. From then, semen collections continued in the males (once a week) until the first normal ejaculate was obtained. The nutritional value of the foliages, as well as the content of total phenols, total tannins and condensed tannins, was determined. The phytoestrogen content (Equol, 4′7-dimetoxyisoflavone, Genistein, Rutin and Ononin) was determined from the methanolic extract of both plants. Total dry matter consumptions were similar between the Control and Mulberry groups and both were higher than the Hibiscus group (Po0.05). The total consumption of phytoestrogens and tannins was similar in the Mulberry and the Hibiscus group. The short-term consumption of phytoestrogens from Mulberry or Hibiscus foliage failed to delay the onset of puberty. The mean age at the onset of puberty was similar in the three groups (146 days in the control and Mulberry groups and 154 days in the Hibiscus group; P40.05). The mean age at the first normal ejaculate was also similar between groups (167 days for Mulberry and Hibiscus groups and 169 days in the control group; P40.05). The BW (Control: 21.673.1; Mulberry: 20.771.9; Hibiscus: 19.872.8 kg), the BCS and the SC were similar between groups at the onset of puberty. The present trial showed that a short exposure to M. Alba or H. rosa-sinensis foliage through diet did not influence the onset of puberty of Pelibuey male lambs. & 2013 Published by Elsevier B.V.
Keywords: Puberty Male sheep Phytoestrogens Morus alba Hibiscus rosa-sinensis Tropics Tannins
n Corresponding author. Tel.: +52 999 942 3200; fax: +52 999 942 3205. E-mail addresses: [email protected], [email protected] (J.F.J. Torres-Acosta).
1871-1413/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.livsci.2013.05.031
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
1. Introduction The foliage of Morus alba (Mulberry) and Hibiscus rosasinensis (Hibiscus) may become viable substitutes for the imported feed-stuffs used in the diets of growing sheep due to its high foliage productivity under tropical conditions. However, both plants contain phytoestrogens (flavonoids and isoflavonoids) that may affect the development of the reproductive tract of different mammals (Vasudeva and Sharma, 2008; You et al., 2002). Some flavonoids and isoflavonoids may act as antagonists or agonists of the steroidal hormones, because these metabolites may bind to estrogenic receptors (ER α and/or β) (Weihua et al., 2001; Whitten et al., 2002). The devastating effects of phytoestrogens on the reproduction of female ruminants have been recognized. However, the subclinical effects of low concentrations of phytoestrogens are considered now more important than the occasional outbreaks of clinical estrogenism (Adams, 1995). The effect of phytoestrogens consumption on the reproduction of male ruminants has been considered “little” (Adams, 1995), but the magnitude of those effects has been poorly studied for ruminants consuming forages containing phytoestrogens (Retana-Márquez et al., 2012). The effects of phytoestrogens on a given population can only be detected by measuring concentrations of phytoestrogens in the forage, or their effect on exposed animal populations (Adams, 1995). The use of Mulberry or Hibiscus foliages in the diet of weaned male lambs represents an opportunity to study the effect of the consumption of foliages containing low concentrations of phytoestrogens on their reproductive performance. Such effect should be evaluated on (a) the acute phase, by measuring the effect of short term consumption of these forages on the onset of puberty, and (b) the chronic phase by measuring the effect on the reproductive performance/ subclinical signs on adult males. These aspects should be explored before Mulberry and Hibiscus foliages are used extensively in the diets of growing male lambs, especially if those foliages are suggested for the nutrition of young
379
males intended for breeding purposes. We hypothesize that the short-term consumption of phytoestrogens contained in Mulberry and Hibiscus foliages could delay the onset of puberty in Pelibuey male lambs. Therefore, the objective of the study was to determine the effect of phytoestrogen consumption from the foliage of Mulberry or Hibiscus on the onset of puberty of Pelibuey male lambs. 2. Materials and methods This trial was performed in the Instituto Tecnológico de Conkal (ITC), Yucatán, México (21105′ N and 89132′ W) at 7 m above sea level. The predominant climatic conditions are hot, sub-humid with summer rainfall (Aw0(x′)(f)gi) with a mean ambient temperature of 26.5 1C and 900 mm of rainfall (Flores-Guido and Espejel-Carvajal, 1994). 2.1. Experimental animals and groups The experimental protocol was approved by the Academic Committee of the Posgraduate Unit (FMVZ-Universidad Autónoma de Yucatán) and the respective funding body (2547.09-P DGEST). A total of 18 male Pelibuey hair sheep lambs were used (from the same parturition group). Animals were born in June and were all managed under similar conditions in terms of feeding and housing. The animals were randomly assigned to the following three experimental groups (n ¼6): (i) Control Group ¼ Balanced feed, (ii) Mulberry Group¼Mulberry foliage ad-libitum+balanced feed and (iii) Hibiscus Group ¼Hibiscus foliage ad-libitum+balanced feed. The balanced feed was prepared with a mixture of grains that were confirmed to be free of phytoestrogens as described below (Table 1). At the beginning of the study, males of the Control, Mulberry and Hibiscus groups had similar BW (17 72, 18 71 and 17 71 kg, respectively), age (108 79, 106 711 and 106710 days, respectively), body condition score (BCS) (3.4 70.2, 3.570.0 and 3.4 70.2, respectively) and
Table 1 Ingredients used for the experimental diets and the chemical composition including ash, crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF). Experimental groups
Ingredients Sorghum (g kg−1 DM)
Corn (g kg−1 DM)
Meat meal (g kg−1 DM)
Star grass hay (g kg−1 DM)
Mulberry fodder (g kg−1 DM)
Hibiscus fodder (g kg−1 DM)
Control group Mulberry group Hibiscus group
280 140 140
310 140 140
150 0 0
260 120 120
0 600 0
0 0 600
Diet composition
Ash (g kg−1 DM)
Crude protein (g kg−1 DM)
NDF (g kg−1 DM)
ADF (g kg−1 DM)
87.2 40.1 40.1
115.7 89.2 89.2
618.8 490.5 490.5
315.3 186.0 186.0
108.7 100.7
131.8 99.4
375.1 478.7
237.5 281.7
Balanced feed Control group Mulberry group Hibiscus group Foliages Mulberry foliage Hibiscus foliage
380
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
scrotal circumference (SC) (1673, 15 71 and 17 73 cm, respectively). The trial started with a 10-day adaptation period starting on October (beginning of autumn). After that, the average length of time animals were exposed to the diets was 40 days at the onset of puberty, and 60 days at the first normal ejaculate.
glass slide (previously heated at 401) and a cover-slip was placed on top of the sample. (f) The semen was observed under a microscope (40 ) to confirm the presence of spermatozoa with progressive movement.
2.2. Animal management
From the moment when the male sheep were positive to the presence of viable spermatozoa, the individual males were taken every week (Saturday morning only) to perform further semen collections. The same protocol described above was used in further semen collections. These semen samples were obtained to determine the time when the animals were producing their first normal ejaculate. All the semen samples were analyzed to determine the semen volume and the percentage of sperm abnormalities (as described by Baril et al. (1993)). The latter was determined from semen smears stained with eosin–nigrosin (evaluating 200 spermatozoa). The sperm concentration was determined using a computer-assisted semen analysis (CASAs, ISASPROISIER, Spain). The following features were considered in semen samples to be declared normal: at least 0.35 mL of volume, a sperm concentration above 3100 million/mL and at least 65% of normal spermatozoa (Baril et al., 1993).
The Mulberry and Hibiscus group received their balanced feed individually (2% of their body weight (BW) in dry matter (DM)) as well as the respective foliage (2.5% of their BW in DM). The control group received only the balanced feed (4.5% of their BW in DM). The adjustment of the quantity of feed offered was performed every second week (when the BW was determined). Foliage refusal was collected daily. Concentrate feed was refused at a very low level for the duration of the study (nearly 40 g per animal per day). Animals received water and a mineral supplement (Fosforisals, Purina México) ad libitum for the duration of the study (Calcium 4%, Phosphorus 1%, Manganeso 3%, Selenio 3 ppm, Cobalto 5 ppm, Magnesio 1000 ppm, Cobre 2 ppm, Iodo 25 ppm, Zinc1000 ppm, Vit A 60 U.I/kg, Vit E 120 U.I./kg y Vit D 90 U.I./kg). The different groups were offered a diet to achieve a theoretical growth rate of 100 g/day. The composition of the diets is presented in Table 1. 2.3. Animal bodyweight, scrotal circumference, body condition score and average daily gain (ADG) The weight, SC and body condition score (BCS) were measured every 14 days from the beginning of the study. Animals were refrained from feed for 16 h prior to weighing. The SC was determined as described by Fuenmayor et al. (1990) and the BCS as reported by Russel (1984). Animals were also weighed on the day when the puberty was declared. The individual ADG was calculated by subtracting the initial BW to that recorded at the onset of puberty and the difference was divided by the number of days needed to reach puberty. 2.4. Onset of puberty The onset of puberty was declared when spermatozoa with progressive movement were found in the ejaculate of experimental lambs (Valencia et al., 1977). The collection of semen was performed using an artificial vagina and a standing female sheep. For that purpose, the following protocol was performed: (a) an adult female sheep (nonpregnant) was estrogenized. (b) All the experimental lambs were taken individually to the pen of the estrogenized female to establish direct individual contact for 5 min (Tuesdays and Thursdays every week). Males were observed during the 5 min they spent with the female to identify the mating attempts. When a lamb was observed to perform penetration (copula), it was then scheduled for a semen collection on the following morning. (c) The semen collections were performed with an artificial vagina. (d) The respective semen samples were collected in conical glass tubes. (e) A semen sample was placed in a
2.5. First normal ejaculate
2.6. Feed intake and feed analyses The voluntary feed intake (total dry matter intake) was determined three times per week for the duration of the trial. Samples of the different portions of the diet were collected every second day. The samples were used to prepare a bulk-sample per week. The analyses performed were dry matter, ashes, crude protein, neutral detergent fiber and acid detergent fiber (FAO, 2000). The content of total phenols (TP) and total tannins (TT) was determined using the Folin–Ciocalteu method (Makkar, 2003). The quantification of tannins was made using a diode array spectrophotometer (Spectrophotometer UV–vis Genesys 10S, Thermo Scientifics). The quantification of TT and TP was made at 725 nm. The condensed tannin (CT) content was quantified as described by Price et al. (1978). The quantification of CT was made by spectrophotometry at 520 nm. 2.7. Phytoestrogen identification and quantification A qualitative screening technique (Galisteo, 1997) was used to determine the presence/absence of phytoestrogens in every ingredient of the feeds used for the experimental groups. The only ingredients positive to phytoestrogens were the Mulberry and Hibiscus foliages. The phytoestrogens contained in them were identified and quantified. This was performed on five samples obtained from weekly bulk samples of the foliages (managed according to Zhishen et al. (1999)). Phytoestrogen standards evaluated were Equol, 4′7-dimetoxyisoflavone, Genistein, Rutin and Ononin. Firstly, plant methanolic extracts for each of the five weekly bulk samples were prepared (Klump et al., 2001). Extracts were injected in an HPLC system (ClarityChrom-Chromatographic SW. Knauer, Darmstad, Germany) with a C18 reversed-phase column 5 mm (250 4.6 mm) for
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
separation of the metabolites. Mobile phase was a gradient of water/acetic acid (96:4 V/V) (A) and Acetonitrile (B) (0 min 90:10; 1 min 80:20; 7 min 35:65; 15 min 23:77; 23 min 90:10; v/v). Flow rate was set at 1 mL/min. Column was used at a temperature of 25 1C and the readings were performed with a UV detector (wave length of 260 nm). Solutions of standards at various concentrations from 0.5 to 4 mg/mL were injected into the HPLC system and the calibration curves were established for each standard compound with a correlation coefficient of at least 0.998 for each standard.
381
3. Results The total DM daily intake of the Control (786.5 719.8) and Mulberry group (837.4727.2 g DM) were similar and both were higher than the Hibiscus group (701.6727.2 g DM) (P o0.05). This was caused by the low daily foliage intake recorded for the Hibiscus group (348.8 721.9 g DM) compared to the Mulberry group (476.8 721.9 g DM) (P o0.05). The TP content of the two types of foliages (Table 2) was similar (Mulberry: 18.8 72.6 and Hibiscus: 13.1 72.6 g kg−1 DM; P 40.05) and so it was for the TT content (Mulberry: 11.372.0 and. Hibiscus: 8.0 7 2.0 g kg−1 DM; P 40.05) and the CT (Mulberry: 0.3 70.1 and Hibiscus: 0.270.1 g kg−1 DM; P4 0.05). In spite of the difference in foliage intake between the Mulberry and Hibiscus group, the total phytoestrogen consumption was similar in the Mulberry and Hibiscus groups (P40.05). Such similarity in the consumption of phytoestrogens was mainly due to the higher consumption of the flavonoids (rutin and 4′7-dimetoxyisoflavons) in the Hibiscus foliage compared to Mulberry. The isoflavonoid genistein was consumed more by sheep fed Mulberry, and the ononin was consumed more in the animals fed Hibiscus foliage (Po0.05) (Table 3). Age at the onset of puberty in Control Pelibuey lambs and in those fed M. alba or H. rosa-sinensis foliage did not differ (P 40.05; Table 4). Furthermore, the first normal ejaculate was also obtained at the same age in the three experimental groups (P40.05). There was no difference in the BW at the onset of puberty or at the first normal
2.8. Data analyses The mean age and BW at the onset of puberty, as well as the mean feed intake (DM), foliage intake (DM), ADG, BCS and SC were compared between the three groups using the GLM procedure and the post-hoc Tukey test of SAS 9.1 for Windows. The same analyses were performed on the data obtained when the first normal ejaculate was obtained from the males. The GLM procedure was also used to compare the contents of TP, TT and CT of the two foliage types (Mulberry and Hibiscus). The difference in the quantity of the different phytoestrogens consumed and the total quantity of phytoestrogens consumed in the foliage of the two plants were compared with respective non-parametric NPAR1WAY procedures for two samples (SAS 9.1 for Windows) using the Wilcoxon rank sum test.
Table 2 Total phenols (TP), total tannins (TT) and condensed tannins (CT) contained in the foliage of Morus alba and Hibiscus rosa-sinensis. Morus alba foliage
TPn TTn CTT1
Hibiscus rosa-sinensis foliage
Minimum
Maximum
Mean
S.E.
Minimum
Maximum
Mean
S.E.
11.5 6.7 0.2
23.6 13.8 0.5
18.8a 11.3a 0.3a
2.6 2.0 0.1
7.3 3.0 0.1
23.6 16.5 0.3
13.1a 8.1a 0.2a
2.6 2.0 0.1
S.E.¼ Standard error. a Different letters between columns indicate differences (P o0.05). n Expressed as tannic acid equivalent (g kg−1 DM). 1 Expressed as catechin equivalent (g kg−1 DM).
Table 3 Consumption (mg/day) of phytoestrogens, flavonids and isoflavonoids of Pelibuey sheep fed foliage of Morus alba (Mulberry) and Hibiscus rosa-sinensis (Hibiscus). Mulberry group
Phytostrogen Consumption (Total) Flavonoids (Total) Rutin 4′7-Dimetoxyisoflavone Isoflavonoids (Total) Genistein Ononin Equol a,b
Hibiscus group
Min
Max
Median
Min
Max
Median
170.1 39.4 9.4 170.2 109.2 102.7 0.0 0.0
271.2 90.3 6.2 271.3 228.8 181.0 10.3 37.6
209.2a 63.5 41.6a 24.0a 142.1 142.1a 1.5a 3.0a
64.7 34.0 34.0 0.0 29.1 8.3 5.4 1.5
480.1 210.3 210.4 15.0 298.3 85.4 229 31.8
223.3a 134.3 126.8b 2.1b 73.0 43.3b 18.0b 13.5a
Different letters in the same row indicate differences (Po 0.05).
382
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
Table 4 Mean age, body weight, scrotal circumference, body condition score and average daily gain ( 7 standard error) at the onset of puberty and at the moment of the first normal ejaculate in male Pelibuey lambs fed Mulberry and Hibiscus foliage compared to lambs fed a control diet. Variables
Age (days) Body weight (kg) Scrotal circumference (cm) Body condition score (1–5) Average daily gain (g/day) a,b
Onset of puberty
First normal ejaculate
Mulberry
Hibiscus
Control
Mulberry
Hibiscus
Control
146.2 7 8.6a 20.7 7 1.9a 21.6 7 1.3a 3.5 7 0.0a 71.5 7 37.2ab
1547 22.4a 19.87 2.8a 22.57 2.4a 3.57 0.3a 49.07 24.0b
1467 14.4a 21.6 7 3.1a 23.3 7 3.5a 3.5 7 0.0a 102.4 7 36.2a
167.17 10.9a 21.3 7 2.9a 20.87 2.7a 3.5 7 0.0a 60.17 39.0ab
167.0 7 9.8a 19.5 7 2.4a 21.3 7 2.8a 3.5 7 0.0a 34.3 7 16.9b
169.1 78.7a 22.3 71.5a 21.9 72.2a 3.5 70.0a 85.1 728.3a
Different letters in the same row at the respective reproduction stage indicate differences (Po 0.05).
ejaculate in the three groups (Table 4). Likewise, the SC and the BCS were similar throughout the trial in the three experimental groups. Finally, the ADG was similar (P40.05) between the Control and Mulberry groups, and both grew faster than the Hibiscus group during the experimental period (P o0.05; Table 4). 4. Discussion The onset of puberty of Pelibuey lambs was not delayed by the phytoestrogens consumed in the two types of foliage from weaning. Although the Mulberry group consumed more foliage than the Hibiscus group, the total quantity of phytoestrogens consumed was similar in both groups. Individual phytoestrogens consumed with each foliage type were different in composition. The Mulberry group consumed more 4′7dimetoxyisoflavone and Genistein than the Hibiscus group. Meanwhile, the Hibiscus group consumed more Rutin and Ononin than the Mulberry group. However, this difference in composition of phytoestrogens consumed by the Pelibuey lambs did not delay the onset of puberty. Whitten et al. (1992) showed that phytoestrogens cause biological effects in the animals when: (i) they consume material containing high quantities of some specific phytoestrogens (i.e. coumestrol, genisteina), or (ii) they consume low concentrations for a long period of time. Indeed, long periods of consumption of phytoestrogenic materials may decrease sperm concentration in humans (Chavarro et al., 2008). Similarly, high amounts of phytoestrogens from soy foods, have been related to lower testicular weight, germ cell number and Sertoli cell function in Asian men compared with Caucasian and Hispanic individuals (Johnson et al., 1998). Studies performed with rodent models have confirmed that the exposure to phytoestrogen early in life can be associated to decreased testicular weight or size, decreased spermatogenesis and lower FSH levels (Atanassova et al., 2000). However, the effect of exposure of adult males to phytoestrogens varies with different animal species. While some studies performed in male rodents showed negative effects on reproductive activity (Glover and Assinder, 2006), other studies reported a positive effect of phytoestrogen consumption on semen quality (Yousef et al., 2004). Thus, the susceptibility to phytoestrogens on male animals seems to be species-specific (Chavarro et al., 2008). The latter highlights the importance of producing specific evidence for male sheep rather than trying to use data from other animal species. In our study, lack of any negative effect of phytoestrogens on the onset puberty could be explained by at least two non-
exclusive hypotheses. Firstly, the period of consumption of the phytoestrogenic materials might have been too short to delay the onset puberty (40 days from the start of the study) or to first normal ejaculate (60 days from the start of the study). Secondly, all the experimental males of the experiment were in a constant contact with an estrogenized female used in the process of training for semen collection of males using an artificial vagina. Males received a constant stimulus that consisted of visual, olfactory and physical contact twice a week until the moment when they performed their first complete copula with an ejaculate. Thereafter, males were exposed to the same female once a week until the moment when they got their first normal ejaculate. The presence of a female (either receptive or not) can modify the pattern of behavior of male sheep affecting plasmatic testosterone concentrations as well as the frequency and pulsatility of LH especially in males with sexual experience (González et al., 1991a, 1991b). All those aspects may have had stronger influence on the onset of puberty than the consumption of phytoestrogens in the foliage. Changes in sexual behavior have been already described in male rats without sexual experience when exposed to females in estrus (Portillo and Paredes, 2004). Although the animals fed the Hibiscus fodder grew slowest than the group fed Mulberry or the Control group (Po0.05), the onset of puberty was similar in the three groups. This suggested that age was more important than BW or ADG to reach puberty in Pelibuey males. Animals in the Hibiscus group showed a low ingestion of Hibiscus foliage (348.8 g/day) compared to other trials where it was used dry meal of Hibiscus (Mata et al., 2006; Ruiz-Sesma et al., 2006). It is unlikely that the animals suffered any reduction in foliage consumption due to the low TT or CT content of the plants. The mucilage present in the chopped foliage could have caused the low ingestion of Hibiscus fresh foliage (Shimizu et al., 1993). Lack of any acute effect of foliage consumption on puberty should be considered only as a first step and cannot be extrapolated to other stages of the reproductive life of male sheep. 5. Conclusion The present study showed that the short-term consumption of Mulberry or Hibiscus foliages did not affect the onset of puberty of Pelibuey lambs. Also, the first normal ejaculate was obtained at the same age in all the experimental groups. The total consumption of
E. Aguilar-Urquizo et al. / Livestock Science 157 (2013) 378–383
phytoestrogens and tannins was similar in the Mulberry and the Hibiscus group. Thus, the consumption of phytoestrogens from the foliage of Mulberry or Hibiscus for a short time did not affect the reproductive function of Pelibuey males early in their reproductive life. Conflict of interest The authors confirm that they have no conflict of interest that could inappropriately influence, or be perceived to influence the work included in this paper.
Acknowledgments The financial support for the present trial was obtained from the Dirección General de Educación Superior Tecnológica (DGEST) (Project no. 2547.09-P). Aguilar-Urquizo E., received a Doctoral scholarship from CONACYT, Mexico. References Adams, N.R., 1995. Detection of the effects of phytoestrogens on sheep and cattle. J. Anim. Sci. 73, 1509–1515. Atanassova, N., McKinnell, C., Turner, K.J., Walker, M., Fisher, J.S., Morley, M., Millar, M.R., Groome, N.P., Sharpe, R.M., 2000. Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility: evidence for stimulatory effects of low estrogen levels. Endocrinology 141, 3898–3907. Baril, G., Chemineau, P., Cognie, Y., Guérin, Y., Leboeuf, B., Orgeur, P. Vallet, J., 1993. Manuel de formation pour L'insemination artificielle chez les ovins et les caprins. Organisation des Nations Unies pour L'alimentation et L'agriculture, Rome, Italie, 125. Chavarro, J.E., Toth, T.L., Sadio, S.M., Hauser, R., 2008. Soy food and isoflavone intake in relation to semen quality parameters among men from an infertility clinic. Hum. Reprod. 23, 2584–2590. FAO, 2000. Quantification of Tannnins in Tree Foliage. A Laboratory Manual for the FAO/IAEA Coordinated Research Project on “Use of Nuclear and Related Techniques to Develop Simple Tannin Assays for Predicting and Improving the Safety and Efficiency of Feeding Ruminants on Tanniniferous Tree Foliage”. FAO/IAEA Working Document IEA, Vienna. Flores-Guido, J.S., Espejel-Carvajal, I., 1994. Tipos de vegetación de la Península de Yucatán. Etnoflora yucatanense. Universidad Autónoma de Yucatán. Mérida, Yucatán, México. Fuenmayor, F.C., Martínez, A.M., Valle, A., Fuentes, A., Quintana, H., Regueiro, C., 1990. Observaciones sobre el desarrollo testicular y desprendimiento del prepucio en corderos West African, Berbados y Persa Cabeza Negra. Zootec. Trop. 8, 73–84. Galisteo, M.M., 1997. Rosmarinus tomentosus Huber-Morath & Maire: Estudio Farmacognóstico. Tesis Doctoral Inéd. Facultad de Farmacia, Universidad de Granada España. Glover, A., Assinder, S.J., 2006. Acute exposure of adult male rats to dietary phytoestrogens reduces fecundity and alters epididymal steroid hormone receptor expression. J. Endocrinol. 189, 565–573. González, R., Orgeur, P., Poindron, P., Signoret, J.P., 1991a. Female effect in sheep. I. The effects of sexual receptivity of females and sexual experience of rams. Reprod. Nutr. Dev. 31, 97–102.
383
González, R., Levy, F., Orgeur, P., Poindron, P., Signoret, J.P., 1991b. Female effect in sheep. II. Role of volatile substances from the sexually receptive female; implication of the sense of smell. Reprod. Nutr. Dev. 31, 103–109. Johnson, L., Barnard, J.J., Rodriguez, L., Smith, E.C., Swerdloff, R.S., Wand, X.H., Wang, C., 1998. Ethnic difference in testicular structure and spermatogenic potential may predispose testes of Asian men to a heightened sensitivity to steroidal contraceptives. J. Androl. 19, 348–357. Klump, P.S., Allred, C.M., MacDonald, L.J., Ballam, M.J., 2001. Determination of isoflavonen in soy and selected foots containing soy by extraction, saponification, and liquid chromatography: collaborative study. J. AOAC Int. 84, 1865–1883. Makkar, H.P.S., 2003. Quantification of Tannins in Tree and Shrub Foliage. A Laboratory Manual. Food and Agriculture Organization of the United Nations/International Atomic Energy Agency (FAO/IAEA), Vienna, Austria 49–53. Mata, E.M.A., Hernández, S.D., Cobos, P.M.A., Ortega, C.M.E., Mendoza, M.G.D., Arcos-García, J.L., 2006. Comportamiento productivo y fermentación ruminal de corderos suplementados con harina de cocoíte (Gliricidia sepium), morera (Morus alba) y tulipán (Hibiscus rosa-sinensis). Rev. Cient. XVI, 249–256. Portillo, W., Paredes, R.G., 2004. Sexual incentive motivation, olfactory preference, and activation of the vomeronasal protection pathway by sexually relevant cues in non-copulating and naïve male rats. Horm. Behav. 46, 330–340. Price, M.L., Van Scoyoc, S., Butler, L.G., 1978. A critical evaluation of the vanillin reaction assay for tannin in sorghum grain. J. Agric. Food Chem. 26, 1214–1218. Retana-Márquez, S., Hernández, H., Flores, J.A., Muñoz-Gutiérrez, M., Duarte, G., Vielma, J., Fitz-Rodríguez, G., Fernández, I.G., Keller, M., Delgadillo, J.A., 2012. Review. Effects of phytoestrogens on mammalian reproductive physiology. Trop. Subtrop. Agroecosyst. 15, 129–145. Ruiz-Sesma, D.L., Lara-Lara, P.E., Sierra-Vázquez, A.C., Aguilar-Urquizo, E., Magaña-Magaña, M.A., Sanginés-García, J.R., 2006. Evaluación nutritiva y productiva de ovinos alimentados con heno de Hibiscus rosasinensis. Zootec. Trop. 24, 467–482. Russel, A., 1984. Body condition scoring of sheep. Farm Pract. 6, 91–93. Shimizu, N., Tomodo, M., Suzuki, I., Takada, K., 1993. Plant mucilages. XLIII. A representative mucilage with biological activity from the leaves of Hibiscus rosa-sinensis. Biol. Pharm. Bull. 16, 735–739. Valencia, J., Barrón, C., Fernández-Baca, S., 1977. Pubertad en corderos Tabasco x Dorset. Vet. Méx. 8, 127–130. Vasudeva, N., Sharma, S.K., 2008. Post-Coital antifertility activity of Hibiscus rosa-sinensis Linn roots. eCAM 5, 91–94. Weihua, Z., Mäkelä, S., Andersson, L., Salmi, S., Saji, S., Webster, J.I., Jensen, E.V., Nilsson, S., Warner, M., Gustafsson, J., 2001. A role for estrogen receptor B in the regulation of growth of the ventral prostate, 98. 〈www.pnas.org/cgl/dol/10.1073/pnas.111150898〉. Whitten, P.L., Russell, E., Naftolin, F., 1992. Effects of a normal, humanconcentration, phytoestrogen diet on rat uterine growth. Steroids 57, 98–106. Whitten, P.L., Patisaul, H.B., Young, L.J., 2002. Neurobehavioral actions of coumestrol and related isoflavonoids in rodents. Neurotoxicol. Teratol. 24, 47–54. You, L., Sar, M., Bartolucci, E.J., McIntyre, B.S., Sriperumbudur, R., 2002. Modulation of mammary gland development in prepubertal male rats exposed to genistein and methoxychlor. Toxicol. Sci. 66, 216–225. Yousef, M.I., Esmail, A.M., Baghdadi, H.H., 2004. Effect of isoflavones on reproductive performance, testosterone levels, lipid peroxidation, and seminal plasma biochemistry of male rabbits. J. Environ. Sci. Health B39, B819–833. Zhishen, J., Mengcheng, T., Jianming, W., 1999. The determination of flavonoid in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64, 555–559.