Alpaca semen quality in relation to different diets

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1 CSIRO PUBLISHING Reproduction, Fertility and Development Alpaca semen quality in relation to different diets N. S. Juyena A, J. Vencato A, G. Pasini B, I. Vazzana C and C. Stelletta A,D A Department of Animal Medicine, Productions and Health, University of Padova, Viale dell Università 16, 35020, Legnaro (Padova), Italy. B Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Viale dell Università 16, 35020, Legnaro (Padova), Italy. C Istituto Zooprofilattico Sperimentale della Sicilia A. Mirri, Via Rocco Dicillo 4, 90129, Palermo, Italy. D Corresponding author. calogero.stelletta@unipd.it Abstract. The aim of the present study was to evaluate the biochemical composition of seminal plasma, along with semen quality, of alpacas maintained on different diets (hay; hay þ pasture grazing; pasture grazing þ sheep concentrate; pasture grazing þ horse concentrate; 1 4, respectively). Alpacas (n ¼ 5) were fed the four different diets for a period of 6 weeks each. During the period of feeding of each diet, semen was collected using an artificial vagina to determine its volume, viscosity, sperm concentration and sperm motility. Moreover, testicular volume and body condition score were evaluated. Seminal plasma was analysed biochemically to measure total protein, triglyceride, cholesterol, g-glutamyl transferase, alanine aminotransferase (ALT) and alkaline phosphatase levels. Protein profiles were investigated using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was high variability in semen parameters between different males maintained on the same diet. Semen volume increased significantly (P, 0.05) when alpacas were fed diets containing commercial sheep and horse concentrates. In contrast, sperm concentration and motility decreased significantly (P, 0.05) from Period 1 to Period 4. Dietary changes had no effect on viscosity. Significant reductions were seen in triglyceride and cholesterol content, as well as g-glutamyl transferase, ALT and alkaline phosphatase concentrations, from Period 1 to Period 4. Regardless of experimental period, a wide variation was seen in seminal plasma enzyme concentrations between alpacas, whereas diet had no effect on glucose and total protein concentrations in the seminal plasma. Eight protein bands, with molecular weights ranging from 200 to 14 kda, were considered in electrophoresis gel after image analysis. Proteins fractions of the 14-kDa (total protein express in md dl 1 with a molecular weight of 14-kDa, TP8) and 21-kDa (total protein express in md dl 1 with a molecular weight of 21-kDa, TP7) bands were not present in all samples of alpaca seminal plasma. There were no significant changes in the concentration of any protein fractions during the four periods. Moreover, the protein fraction of the 60-kDa (total protein express in md dl 1 with a molecular weight of 60-kDa, TP3) band was the most prevalent in all periods. These results demonstrate that there are marked changes in semen quality, as well as some parameters related to the composition of alpaca seminal plasma, that are dependent on diet, which may indicate the need for specific diet formulation to improve reproductive performance. We hypothesise that, in alpacas, the mechanisms underlying the changes in some reproductive traits in response to feeding regimens could be related to changes in the endocrine gonadal system. Additional keywords: alpacas, diet, energy excess, semen quality. Received 22 February 2012, accepted 26 May 2012, published online 17 July 2012 Introduction New world camelids (llamas, alpacas) are important animal resources in the Altiplanic region of South America because they provide fibre and meat for high Andean people. In Italy, there has been an increase in the number of alpaca farms recently, with farmers interested in keeping alpacas for cottage fibre production and breeding for economic purposes. Alpacas are reared using semi-intensive systems and feed concentrates specified for other animals because of the absence of alpacaspecifics product. Under such conditions and management Journal compilation Ó CSIRO 2012 practices, it is necessary to understand the effects of the intake of different foods to optimise the supply of nutrients to alpacas. In addition, the reproductive physiology of alpacas is considerably different from that of other domestic animals, with some peculiar characteristics of alpaca semen (e.g. low sperm concentration, motility and viability, as well as the viscose nature of semen) revealing the need for improvements in semen quality and providing a powerful stimulus for morphological and biochemical investigations into seminal plasma. Reproductive performance is the culmination of many complex and integrated

2 B Reproduction, Fertility and Development N. S. Juyena et al. anatomical, developmental, physiological and behavioural processes, and there is apparently a strong relationship between nutritive status and reproductive performance (Van Saun 2008). Interactions between nutrition and reproduction have been the subject of numerous studies in large ruminants and the effect of nutrition on milk, growth and reproduction has been investigated extensively in conventional farming species (Hammadi et al. 2001). It is well documented that decreased nutrient intake, especially a low-protein diet, delays the onset of puberty in bulls and decreases testosterone levels (Nolan et al. 1990), testicular size and sperm concentration in the ejaculate (Alkass et al. 1982). The beneficial effects of a mixed diet (pasture grazing and feeding of concentrates) have also been reported in cattle (Perry et al. 1991; Tegegne et al. 1992). It has also been demonstrated that spermatogenesis in rams is influenced by protein intake (San Martin and Bryant 1989; Bravo 2002). However, there is little, and only fragmented, information regarding the effects of diet on reproductive function in male alpacas. Therefore, the aim of the present study was to evaluate the effects of different diets on the composition of seminal plasma and semen quality. Materials and methods Animals Five male alpacas (5 8 years old) were used in the present study. The alpacas were maintained in an animal stable in the Department of Animal Medicine, Productions and Health, University of Padova (Padova, Italy) and were housed separately from female alpacas. Of the five male alpacas, four were Huacaya alpacas and one was a Suri alpaca. Before the experiment was started, the male alpacas were trained to copulate with an artificial vagina in the presence of a teaser female. Experimental design The study was divided into four experimental periods of 6 weeks each, during which the alpacas were maintained on one of four different diets (hay; hay þ pasture grazing; pasture grazing þ sheep concentrate; pasture grazing þ horse concentrate; 1 4, respectively; Table 1). During each experimental period, semen was collected once a week for 4 weeks. To mitigate the effects of the previous diet, Table 1. Time Timing of the experimental periods and dietary composition Diet 1 2 Mar Apr 2009 Hay 2 13 Apr May 2009 Hay þ pasture grazing 3 25 May Jul 2009 Pasture grazing þ sheep concentrate A 4 6 Jul Aug 2009 Pasture grazing þ horse concentrate B A The sheep concentrate was comprised of wheat flour, corn flour, corn seed, wheat bran, soya bean, calcium carbonate and sugar molasses. Feed integrity: crude protein 16%; crude fibre 10%; crude lipids 2.7%; ash 7%. B The horse concentrate was comprised of wheat bran, maize bran, wheat straw, wheat flour, rice polish, calcium carbonate, maize, sugar molasses, sodium chloride and crushed soya beans. Feed integrity: crude protein 12%; crude lipids 3%; crude fibre 11%; ash 7.5%. semen was not collected for the first 2 weeks of the new diet. Before semen collection, testicular volume and the body condition score (BCS) were determined. After the length, width and thickness of both testes were measured using callipers, testicular volume was calculated using the following formula (Gouletsou et al. 2008): Testicular volume ¼ length width thickness 0:5236 The BCS of alpacas (on a scale of 1 5, where 1 is emaciated and 5 is obese) was measured by palpation of the lumbar region, as described for small ruminants (Russel 1991). Semen collection and evaluation Semen was collected using a modified ovine artificial vagina in presence of a teaser female. The artificial vagina was prepared by placing foam in the anterior inner latex to make a cervix-like structure and a glass tube was attached to this to collect the semen. During semen collection, the artificial vagina was wrapped in an electric heating pad to maintain the internal temperature of the artificial vagina at 388C. Copulation time was recorded (starting from when male alpaca mounted the female and finishing when the male stood up and did not show any interest in the female). Semen was retrieved from the artificial vagina by repeated sharp downward thrusts to dislodge the viscous semen and then collected in a graduated test tube. Immediately after collection, the semen tube was placed in a water bath at 378C. Classical semen parameters, such as volume, consistency (viscosity), concentration and percentage of motile spermatozoa, were evaluated. Ejaculate volume was recorded directly from the collecting tube. The viscosity of the semen was graded according to Bravo et al. (1997) as follows: (1) viscous, when the semen did not drop from a Pasteur pipette; (2) semiviscous, when some semen dropped from the Pasteur pipette to a glass slide; and (3) liquid, when semen was fluid and dropped readily from the Pasteur pipette. Sperm concentration was determined using a Cell VU Sperm counting chamber (Millenium Sciences, New York, NY, USA) and is expressed as the number of spermatozoa per ml. The total number of spermatozoa was determined by multiplying the sperm concentration by ejaculate volume. To evaluate motility, 10 ml semen was placed on prewarmed glass slides, coverslipped and examined under a phase contrast microscope at 40 magnification. Sperm motility, detected as an oscillatory motion of the flagellum, is given as the percentage of motile spermatozoa. After evaluation, semen samples were centrifuged at 2400g for 40 min at 208C, and the seminal plasma was separated and stored at 208C until biochemical analysis and gel electrophoresis. Biochemical analysis and gel electrophoresis Biochemical analysis was performed using an automatic analyser using specific kits (912 Automatic Analyzer; Hitachi Boehering Mannheim, Mannheim, Germany). Seminal plasma concentrations of alanine aminotransferase (ALT), g-glutamyl transferase and alkaline phosphatase (ALP) were determined, as

3 Alpaca semen in relation to diet Reproduction, Fertility and Development C were glucose, total protein, triglyceride, cholesterol and calcium concentrations. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to identify and quantify protein profiles in alpaca seminal plasma. Polyacrylamide gel electrophoresis was performed using 16% polyacrylamide gels according to the methods of Laemmli (1970). Samples were diluted 1 : 20 with sample buffer. Each sample contained 15 mg total protein. Protein standards (Broad Molecular Weight Standards; Bio-Rad, Hercules, CA, USA) contained protein bands of different molecular weights: 200, , 97.4, 66.2, 45, 31, 21.5, 14.4 and 6.5. Electrophoresis was performed at 48 ma for 2 h using Power Pac Basic Power Supply (Bio-Rad). Gels were stained with Coomassie Brilliant Blue. Gel images were processed using the Sante Dicom Viewer Image J (Santesoft, Athens, Greece) program. The density of the bands was measured to determined total protein percentage (TP%) and the relative concentration of protein fractions (TP). Statistical analysis Data are given as the mean s.e.m. Data were analysed by twoway repeated-measures ANOVA, with period and animals considered as independent variables, and semen quality parameters and results of biochemical and semiquantitative analyses considered as dependent variables. P, 0.05 was considered significant. All data were analysed using SIGMASTAT Results Effects of diets on alpaca semen quality and other reproductive parameters There was high variability in different parameters between male alpacas fed the same diet, as indicated in Fig. 1a d. Semen volume increased slightly in 3 and 4, with the increase reaching statistical significance in Alpaca 1 in Period 4 (P, 0.05). Significant declines (P, 0.05) in both sperm motility and concentration were noted in all alpacas when they were fed sheep concentrate during Period 3, with sperm concentration increasing significantly thereafter in Period 4, although motility remained at low levels. Ejaculates collected from Alpaca 5 did not contain any spermatozoa at any time during the study. There were no significant changes in semen collection time (Fig. 1e) or viscosity (Fig. 1d) in any of the males at any time in the study. In addition, a foam layer was found in most viscid ejaculates. The type of diet had no effect on either right or left testicular volume (Fig. g, h). The BCS increased significantly (P, 0.05) from Period 1 to Period 4 (Fig. 1f). Effects of diets on the biochemical composition of alpaca seminal plasma Changes in energy parameters, total protein and glucose levels of alpaca seminal plasma are given in Table 2. Triglyceride and cholesterol concentrations decreased significantly from Period 1 to Period 4 in four alpacas, whereas significant increases (P, 0.05) were seen in these parameters in the seminal plasma of Alpaca 5. Conversely, the type of diet had no effect on glucose or total protein concentrations in the seminal plasma. Among the five males, energy profiles were higher in the seminal plasma of Alpaca 2 throughout the study period. The type of diet had a significant effect (P, 0.05) on enzyme concentrations in the seminal plasma (Table 3). g-glutamyl transferase concentrations were higher in the seminal plasma from all alpacas during Period 2 and then declined significantly (P, 0.05). Seminal plasma concentrations of ALT and ALP decreased from Period 1 to Period 4, except in Alpaca 3, in which the concentrations of these enzymes increased in the seminal plasma collected during 3 and 4. Moreover, there was wide variation in enzyme concentrations between alpacas, regardless of the type of diet being fed. The highest ALP concentration was detected in Alpaca 1, whereas the lowest was in Alpaca 4. Assessment of the electrophoretic profile of seminal plasma proteins revealed eight bands in total between 200 and kda. Surprisingly, not all the bands were present in samples from all alpacas (Fig. 2a d). The protein profile of seminal plasma from Alpaca 2 (Fig. 2b) differed from that of the other four alpacas: protein fractions in the range kda were present in the seminal plasma of Alpaca 2, whereas a (a) (b) (c) (d) Volume (ml) Concentration ( 10 6 ) Motility % Viscosity (e) (f ) (g) (h) Semen collection time (min) BCS Right testicular volume (cm 3 ) Left testicular volume (cm 3 ) Fig. 1. Changes in (a) semen volume, (b) sperm concentration, (c) sperm motility, (d ) semen viscosity, (e) time required for semen collection, ( f) body condition score (BCS) and (g) right and (h) left testicular volume in the five male alpacas maintained on different diets: hay (Period 1); hay þ pasture grazing (Period 2); pasture grazing þ sheep concentrate (Period 3); pasture grazing þ horse concentrate (Period 4). Significant changes (P, 0.05) were seen in semen volume, sperm concentration, sperm motility and BCS only. Data are the mean s.e.m.

4 D Reproduction, Fertility and Development N. S. Juyena et al. Table 2. Energy profiles and total protein concentrations in alpaca seminal plasma during dietary 1]4 Data are the mean s.e.m. Different symbols indicate significant differences between male alpacas, whereas different superscript letters indicate significant differences between periods (P, 0.05). Period 1, hay; Period 2, hay þ pasture grazing; Period 3, pasture grazing þ sheep concentrate; Period 4, pasture grazing þ horse concentrate Period Alpaca Glucose (mg dl 1 ) Triglyceride (mg dl 1 ) * ya y * * y * a * a y * * y * yb * b y * * y * c b b Cholesterol (mg dl 1 ) a a a b Total protein (g L 1 ) * y * * * a * y * * * a a b Table 3. Enzyme concentrations in alpaca seminal plasma collected during dietary 1]4 Data are the mean s.e.m. Different symbols indicate significant differences between male alpacas, whereas different superscript letters indicate significant differences between periods (P, 0.05). Period 1, hay; Period 2, hay þ pasture grazing; Period 3, pasture grazing þ sheep concentrate; Period 4, pasture grazing þ horse concentrate Period Alpacas g-glutamyl transferase (IU L 1 ) ya y a a ya * yb * b y yb * yb * b y yb * yb ya y Alkaline phosphatase (IU L 1 ) * a * z y yz yz yab * z y z yb * y y y yb y y * * Alanine aminotransferase (IU L 1 ) * ya * * * * ya * * * b y * b * y y (a) (b) (c) (d) (e) Fig. 2. Quantitative protein composition of the seminal plasma of (a) Alpaca 1, (b) Alpaca 2, (c) Alpaca 3, (d ) Alpaca 4 and (e) Alpaca 5, as determined by onedimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis after feeding of the different diets: hay (Period 1); hay þ pasture grazing (Period 2); pasture grazing þ sheep concentrate (Period 3); pasture grazing þ horse concentrate (Period 4).

5 Alpaca semen in relation to diet Reproduction, Fertility and Development E Table 4. Effects of diet on protein profiles of seminal plasma from alpacas during dietary 1]4 Data are the mean s.e.m. Period 1, hay; Period 2, hay þ pasture grazing; Period 3, pasture grazing þ sheep concentrate; Period 4, pasture grazing þ horse concentrate Protein profile Relative protein concentration (mg dl 1 ) protein fraction around TP7 (21 kda) was absent during the experimental periods. Changes in plasma profiles are given in Table 4. There were no significant changes in the relative quantity of all protein profiles during the four feeding periods, although marked variations were observed within individual animals. The TP3 protein fractions (,60 kda) were the most abundant in all samples. Discussion Period 1 Period 2 Period 3 Period 4 TP1 (200 kda) TP2 (97 kda) TP3 (60 kda) TP4 (45 kda) TP5 (40 kda) TP6 A (31 kda) TP7 A (21 kda) TP8 (14 kda) A These protein fractions were not present in alpaca seminal plasma. The results of the present study reveal considerable animal-toanimal and week-to-week variations with regard to the attributes of alpaca seminal plasma. Considerable variation in semen quality parameters in individual male alpacas was observed in the present study, which is in agreement with results from previous studies investigating male variation (Buendía et al. 2002; Flores et al. 2002; Vaughan et al. 2003; Giuliano et al. 2008). In the present study, the average time for semen collection using the artificial vagina was 9 20 min and is in accordance with the time of min reported by Brown (2000) for semen collection from alpacas. There were no significant changes in collection time among males and mating length also remained constant. Similar findings were reported by Tibary and Memon (1999), who stated that the duration of copulation is determined mainly by the male and may be affected by breed, age, season and frequency of the collection. The effect of extended mating time on semen quality cannot be ignored in the alpaca. An extended collection time results in the semen being exposed to latex, high temperatures and atmospheric conditions, leading to changes in the ratio of semen constituents and altering semen osmolarity and ph (Morton et al. 2010). The type of rubber in the artificial vagina and the length of time the semen stays in contact with the liner have been reported to have a considerable effect on the motility of individual spermatozoa (Deen and Sahani 2000). In the present study, ejaculate volume ranged from 1 to 7 ml across the experimental periods. This confirms findings in a previous study into the volume of alpaca semen, which was reported to range from 0.4 to 12.5 ml (Garnica et al. 1993). Regarding the consistency of the semen, the viscid nature of semen was specific for individual alpacas and did not change with the type of diet. The degree of viscosity varies between males (Tibary and Memon 1999) and decreases with an increasing number of ejaculates (Bravo et al. 1997). The viscous nature of seminal plasma is a result of the presence of mucopolysaccharides from secretions of the bulbourethral glands or the prostate (Garnica et al. 1993), referred to as glycosaminoglycans, which are made up of 95% long-chain polysaccharides and 5% protein (Morton et al. 2009). Recent studies with human semen have demonstrated that the existence of a highly organised network of disulfide bonds, oligosaccharide and peptide chains in seminal plasma is responsible for the rheological characteristics of ejaculates with high viscosity and these molecules, which are responsible for the hyperviscous rheological behaviour, could be a key factor in sperm physiology and sperm motility (Mendeluk et al. 2000). Viscous seminal plasma acts as a reservoir for spermatozoa until ovulation, which occurs 36 h after copulation (Garnica et al. 1993). Foam formation in viscid seminal plasma during ejaculation is a well-known characteristic of camelids, particularly when semen is collected using an artificial vagina (Vaughan et al. 2003; Giuliano et al. 2008), and may result from the retrieval and reinsertion of the penis into the collection container (Ratto 2005). In the present study, the sperm concentration varied between 20 and 240 million spermatozoa ml 1, which is slightly higher than that reported previously in this species (from to 150 million spermatozoa ml 1 ; Bravo 1995; Garnica et al. 1995). Such wide variations have been attributed to differences between male alpacas, semen collection methods and the number of ejaculates (Tibary and Memon 1999). In the present study, there was a marked decrease in sperm concentration during 3 and 4, when the alpacas were allowed to graze on pasture and were supplemented with sheep and horse concentrate, respectively. During the same period of time, the change in testicular size was less marked. This finding appears to contradict previous findings, whereby a correlation between sperm production and testicular size was found in camelids (Galloway 2000). In the present study, sperm motility ranged between 5% and 80%, again demonstrating high levels of variation between males, whereas mean motility was low (,35%). Mean sperm motility in the present study is within the range reported for alpaca semen collected using an artificial vagina (15.3% 63.7%; Bravo et al. 1997; Vaughan et al. 2003). Percentage motility decreased significantly in 3 and 4. Moreover, only oscillatory motility was observed in samples collected during all periods. This is in agreement with previous studies, which also described oscillatory motion (Garnica et al. 1993; Bravo et al. 2000; Giuliano et al. 2002; Vaughan et al. 2003). In contrast, Morton et al. (2010) observed both oscillatory and progressive motility depending on the viscid nature of the semen. Tibary and Memon (1999) stated that sperm could gain progressive motility upon liquefaction of the semen. Results from the biochemical analyses revealed that there were no changes in total protein and glucose concentrations in the seminal plasma with changes in diet, whereas cholesterol, triglyceride, g-glutamyl transferase, ALP and ALT concentrations did change significantly (P, 0.05). The values of the

6 F Reproduction, Fertility and Development N. S. Juyena et al. paremeters obtained in the present study were higher than those reported previously (El-Manna et al. 1986; Garnicaet al. 1993; Agarwal et al. 2004). In the seminal plasma of camelids, glucose is the principle sugar and it is converted into fructose by either phosphorylation or monophosphorylation through sorbitol dehydrogenase and aldose reductase (Agarwal et al. 2004). The sugar composition of seminal plasma is correlated with fertility, mainly due to its importance for sperm energy production (Garner et al. 2001). Seminal lipids, specifically phospholipids and cholesterol, have particular relevance in the structure and function of the plasma membrane of spermatozoa (Cross 1998) and may play significant roles in sperm structure, metabolism, capacitation and the fertilisation of female gametes (Hafez 1987). The presence of lipids in the seminal plasma is important because of the ability of spermatozoa to take up lipid components or fatty acids from the surrounding environment under some circumstances (Cerolini et al. 2001). Studies with ram semen indicate that reductions in sperm concentration and motility are associated with a decreased seminal plasma lipid content (Taha et al. 2000). Although total lipids have been quantified in alpaca seminal plasma (Garnica et al. 1993), their functions have not yet been studied in camelids; they may play a role in the maturation of the sperm plasma membrane, as well as in maintaining its integrity. Research in large ruminants has revealed that seminal plasma enzymes such as ALT and ALP are essential for metabolic processes that provide the energy for viability, motility and the fertility of spermatozoa. The concentration of these enzymes in seminal plasma is considered as a sensitive indicator of plasma membrane damage and altered membrane function, which may occur due to inadequate epididymal maturation associated with an increased frequency of semen collection. A possible source of these enzymes is thought to be the testes or epididymides because the enzymes show a positive correlation with sperm concentration and a negative correlation with semen volume (Kareskoski and Katila 2008). Transaminases are located primarily in the midpiece of spermatozoa (Mann and Lutwak-Mann 1981), whereas ALP activity is found on the sperm head, midpiece and tail fragments, and it is known that ALP regulates the phosphorylation of proteins by a camp-dependent protein kinase necessary for sperm motility (Tang and Hoskins 1975). g-glutamyl transferase plays an important role in protecting spermatozoa against oxidative stress (Hinton et al. 1998). In the present study, one-dimensional SDS-PAGE revealed eight protein bands, but not all bands were present in all samples from all male alpacas. Of the different proteins, TP3 (,60 kda) was most abundant. During the study period, there were no significant changes in protein profiles. In camelids, research into the identification and quantification of proteins has concentrated mainly on a specific protein fraction of high molecular weight that has gonadotrophin-releasing hormone (GnRH)-like activity (Paolicchi et al. 1999; Pan et al. 2001), named ovulation-inducing factor. Studies on proteins of smaller molecular weight are very limited. Moreover, the seminal vesicle, which is known to be the main source of most of the seminal plasma protein fractions described for other small ruminants, is absent in alpacas. Therefore, the sources of these proteins remain unknown. Homologous proteins of ram seminal plasma proteins (RSPs) or bull seminal plasma proteins (BSPs) have not been confirmed in alpaca seminal plasma. Results of one-dimensional SDS-PAGE demonstrated the presence of TP8 (14 15 kda) in alpaca seminal plasma, which is a protein known to protect ram spermatozoa against cold shock (Pérez-Pé et al. 2001). In a previous study on alpaca seminal plasma proteins, we found that this protein fraction is related to the freezability of alpaca semen (Marion et al. 2010). In the present study, we found that feeding of sheep or horse concentrates had a negative effect on semen quality, particularly sperm concentration and motility, which were significantly changed in Period 4. The only positive effect of these concentrates was on semen volume, which increased after the feeding of these concentrates to male alpacas. There is a lack of information regarding the effects of diet on alpaca reproduction. Perry et al. (1991) and Tegegne et al. (1992) reported that young bulls kept on grazing plus concentrate feed exhibited a better growth rate than those kept on grazing alone, with an earlier age at puberty and a greater scrota1 circumference. This variation in response may result from inherent differences in the metabolic systems of alpaca and may force us to consider developing diets specific for the alpaca. However, there is very little information in the literature regarding nutritional effects on the reproductive performance of male alpacas and the mechanism by which diet can affect reproductive capacity remains unclear. Diet-induced changes may result from variations in gonadotrophin levels, which would affect endocrine gonadal function and secretions from accessory glands. The mechanisms underlying the changes in some reproductive traits in response to feeding regimens would be related to changes in the secretion of sex hormones, such as testosterone (Brown 1993). The protein part of the diet could be responsible for the effects of the diet and would act via a GnRH-independent pathway (Hötzel et al. 1998). Research in other ruminants shows that both low-protein and excess-protein diets diminish pulsatile LH secretion by altering body metabolism (for a review, see Kaur and Arora 1995). Presumably similar mechanisms may be responsible for the changes in the reproductive performance of alpacas, in addition to changes in the composition of the seminal plasma, seen with the different diets in the present study. However, this issue requires further clarification in future studies. Energy intake has a greater effect on sexual activity than protein supply because sexual activity involves significant physical effort and, therefore, energy expenditure (Brown 1994). A high-energy diet could have a negative effect on sperm quality by increasing scrotal surface temperature, leading to decreased sperm motility (Coulter and Kozub 1984). Coulter et al. (1997) studied the effect of dietary energy and concluded that semen quality decreases when the level of energy supply is high. These effects are perhaps related to excess fat in the neck of the scrotum and/or insulation of the testes by scrotal tissues, which increases scrotal/testicular temperature and thereby decreases sperm production and seminal quality. In addition to proteins and their interaction among proteins, many minerals and vitamin present in diets can also affect reproductive performance (Smith and Akinbamijo 2000). In alpaca, in addition to dietary effects, the timing of the study period and environmental temperature cannot be ignored. Seasonal variations in seminal

7 Alpaca semen in relation to diet Reproduction, Fertility and Development G plasma composition, especially proteins, is well documented in small ruminants (Cardozo et al. 2006). These seasonal changes in protein profiles could result from seasonal variations in gonadotrophin levels and their receptors in the testes (Xu et al. 1991), which would affect endocrine gonadal function and the secretions of the epididymides and seminal vesicles. Many previous studies (Gauly et al. 1995; Lichtenwalner et al. 1996; Giuliano et al. 2008) have noted variations in semen parameters within and between South American male camelids, although none has specifically investigated these variations. The findings of the present study provide baseline information regarding the formulation of alpaca diets with a view to improving reproductive performance. Future research should be undertaken to investigate the carryover effects of different nutritional regimens on reproductive performance in the male alpaca. More work is needed to understand the reasons for the different reproductive performance responses in the male alpaca to different diets. Acknowledgements The authors thank all colleagues and students who contributed to this study and a special thanks to the owners Renè & Esther Steiger for their support. References Agarwal, V. K., Ram, L., Rai, A. K., Khanna, N. D., and Agarwal, S. P. (2004). Physical and biochemical attributes of camel semen. J. Cam. Res 1, 25. Alkass, J. E., Bryant, M. J., and Walton, J. S. (1982). Some effects of level of feeding and body condition upon sperm production and gonadotropin concentrations in the ram. Anim. Prod. 34, doi: / S Russel, A. (1991). Body condition scoring of sheep. In: Sheep and Goat Practice. (Ed. E. Boden.) p. 3. (Bailliere Tindall: Philadelphia.) Bravo, P. W. (1995). Physiology of reproduction and fertility evaluation in the male alpaca. Proc. Postgrad. Found. Vet. Sci. 257, Bravo, P. W. (2002). The Reproductive Process of South American Camelids. (Seagull Printing: Salt Lake City, UT.) Bravo, P. W., Flores, D., and Ordonez, C. (1997). Effect of repeated collection on semen characteristics of alpacas. Biol. Reprod. 57, doi: /biolreprod Bravo, P. W., Callo, M., and Garnica, J. (2000). The effect of enzymes on semen viscosity in llamas and alpacas. Small Rumin. Res. 38, doi: /s (00) Brown, B. W. (1993). Gonadotrophin and testosterone concentrations and testicular growth in rams supplemented with lupines from birth to puberty. Proc. Aust. Soc. Reprod. Biol. 25, 34. Brown, B. W. (1994). A review of nutritional influences on reproduction in boars, bulls and rams. Reprod. Nutr. Dev. 34, doi: / RND: Brown, B. W. (2000). A review on reproduction in South American camelid. Anim. Reprod. Sci. 58, doi: /s (99) Buendía, P., Soler, C., Paolicchi, F., Gago, G., Urquieta, B., Pérez-Sánchez, F., and Bustos-Obregón, E. (2002). Morphometric characterization and classification of alpaca sperm heads using the Sperm-Class Analyzer(R) computer-assisted system. Theriogenology 57, doi: / S X(01) Cardozo, J. A., Fernández-Juan, M., Forcada, F., Abecia,, A., Muiño- Blanco, T., and Cebrián-Pérez, J. A. (2006). Monthly variations in ovine seminal plasma proteins analyzed by two-dimensional polyacrylamide gel electrophoresis. Theriogenology 66, doi: /j.ther IOGENOLOGY Cerolini, S., Maldjian, A., Pizzi, F., and Gliozzi, T. M. (2001). Changes in sperm quality and lipid composition during cryopreservation of boar semen. Reproduction 121, doi: /rep Coulter, G. H., and Kozub, G. C. (1984). Testicular development, epididymal sperm reserves and seminal quality in two-year-old Hereford and Angus bulls: effect of two levels of dietary energy. J. Anim. Sci. 59, Coulter, G. H., Cook, R. B., and Kastelic, J. P. (1997). Effects of dietary energy on scrotal surface temperature, seminal quality, and sperm production in young beef bulls. J. Anim. Sci. 75, Cross, N. L. (1998). Role of cholesterol in sperm capacitation. Biol. Reprod. 59, doi: /biolreprod Deen, A., and Sahani, M. S. (2000). Preliminary attempts to collect and cryopreserve camel semen. J. Camel. Prac. Res. 7, El-Manna, M. M., Tingari, M. D., and Ahmed, A. K. (1986). Studies on camel semen. II. Biochemical characteristics. Anim. Reprod. Sci. 12, doi: / (86) Flores, P., Garcia-Huidobro, J., Munoz, C., Bustos-Obregon, E., and Urquieta, B. (2002). Alpaca semen characteristics previous to a mating period. Anim. Reprod. Sci. 72, doi: /s (02) Galloway, D. B. (2000). The development of the testicles in alpacas in Australia. In Proceedings of the Australian Alpaca Association Conference, Canberra. pp Garner, D. L., Thomas, C. A., Gravance, C. G., Marshall, C. E., DeJarnette, J. M., and Allen, C. H. (2001). Seminal plasma addition attenuates the dilution effect in bovine sperm. Theriogenology 56, doi: / S X(01) Garnica, J., Achata, R., and Bravo, P. W. (1993). Physiological and biochemical characteristics of alpaca semen. Anim. Reprod. Sci. 32, doi: / (93)90059-z Garnica, J., Flores, E., and Bravo, P. W. (1995). Citric acid and fructose concentrations in seminal plasma of the alpaca. Small Rumin. Res. 18, doi: / (95) Gauly, M., and Leidinger, H. (1995). Semen quality, characteristics volume distribution and hypoosmotic sensitivity of spermatozoa of Lama glama and Lama guanicoe. In: Proceedings of the 2nd European Symposium on South American Camelids. (Eds M. Gerken, C. Renieri.) pp (Publ. Universita degli Studi di Camerino.) Giuliano, S. M., Spirito, S. E., and Maragaya, M. H. (2002). Electroejaculation and seminal parameters in vicuña(vicugna vicugna). Theriogenology 57, 583. Giuliano, S., Director, A., Gambarotta, M., Trasorras, V., and Miragaya, M. (2008). Collection method, season and individual variation on seminal characteristics in the llama (Lama glama). Anim. Reprod. Sci. 104, doi: /j.anireprosci Gouletsou, P. G., Galatos, A. D., and Leontides, L. S. (2008). Comparison between ultrasonographic and caliper measurements of testicular volume in the dog. Anim. Reprod. Sci. 108, doi: /j.anire PROSCI Hafez, E. S. E. (1987). Semen evaluation. In Reproduction in Farm Animals. 5th edn. (Ed. E. S. E. Hafez.) pp Hammadi, M., Khorchani, T., Khaldi, G., Majdoub, A., Abdouli, H., Slimane, N., Portetelle, D., and Renaville, R. (2001). Effect of diet supplementation on growth and reproduction in camels under arid range conditions. Biotechnol. Agron. Soc. Environ. 5, Hinton, B. T., Lan, Z. J., Rudolph, D. B., Labus, J. C., and Lye, R. J. (1998). Testicular regulation of epididymal gene expression. J. Reprod. Fertil. Suppl. 53, Hötzel, M. J., Markey, C. M., Walken-Brown, S. W., Blackberry, M. A., and Martin, G. B. (1998). Morphometric and endocrine analyses of the

8 H Reproduction, Fertility and Development N. S. Juyena et al. effects of nutrition on the testis of mature merino rams. J. Reprod. Fertil. 113, doi: /jrf Kareskoski, M., and Katila, T. (2008). Components of stallion seminal plasma and the effects of seminal plasma on sperm longevity. Anim. Reprod. Sci. 107, doi: /j.anireprosci Kaur, H., and Arora, S. P. (1995). Dietary effects on ruminant livestock reproduction with particular reference to protein. Nutr. Res. Rev. 8, doi: /nrr Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, doi: / A0 Lichtenwalner, A. B., Woods, G. L., and Weber, J. A. (1996). Seminal collection, seminal characteristics and pattern of ejaculation in llamas. Theriogenology 46, Mann, T., and Lutwak-Mann, C. (1981). Male reproductive function and semen. In Physiology, Biochemistry and Investigative Andrology. (Springer-Verlag: Berlin.) Marion, I., Juyena, N. S., Pasini, G., and Stelletta, C. (2010). Relazione tra le proteine del liquido seminale e la congelabilità del seme nell alpaca (Lama pacos): uno studio preliminare. (SIRA: Bologna.) Mendeluk, G. R., González, L., Flecha, L. G., Castello, P., Blanco, A. M., and Bregni, C. (2000). Factors involved in the biochemical etiology of human seminal plasma hyperviscosity. J. Androl. 21, Morton, K. M., Gibb, Z., Bertoldo, M., and Chis Maxwell, W. M. (2009). Effect of diluent, dilution rate and storage temperature on longevity and functional integrity of liquid stored alpaca (Vicugna pacos) semen. J. Cam. Sci. 2, Morton, K. M., Thomson, P. C., Bailey, K., Evans, G., and Maxwell, W. M. C. (2010). Quality parameters for alpaca (Vicugna pacos) semen are affected by semen collection procedure. Reprod. Domest. Anim. 45, Nolan, C. J., Neuendorff, D. A., Godfrey, R. W., Hams, P. G., Welsh, T. H., McArthur, N. H., and Randel, R. D. (1990). Influence of dietary energy intake on prepubertal development of Brahman bulls. J. Anim. Sci. 68, Pan, G., Chen, Z., Liu, X., Li, D., Xie, Q., Ling, F., and Fang, L. (2001). Isolation and purification of the ovulation-inducing factor from semlnal plasma in the Bactrian camel (Camelus bactrianus). Theriogenology 55, doi: /s x(01) Paolicchi, F., Urquieta, B., Del Valle, L., and Bustos-Obregón, E. (1999). Biological activity of the seminal plasma of alpacas: stimulus for the production of LH by pituitary cells. Anim. Reprod. Sci. 54, doi: /s (98)00150-x Pérez-Pé, R., Cebrián-Pérez, J. A., and Muiño-Blanco, T. (2001). Semen plasma proteins prevent cold-shock membrane damage to ram spermatozoa. Theriogenology 56, doi: /s x(01) X Perry, V. E. A., Chenoweth, P. J., Post, T. B., and Munro, R. K. (1991). Patterns of development of gonads, sex drive and hormonal responses in tropical beef bulls. Theriogenology 35, doi: / x(91)90297-q Ratto, M. H. (2005). Ovarian follicular synchronization, ovulation and oocyte development in llamas and alpacas. Ph.D. Thesis, University of Saskatchewan, Saskatoon. San Martin, F. A., and Bryant, F. C. (1989). Nutrition of domesticated South American llamas and alpacas. Small Rumin. Res. 2, doi: / (89) Smith, O. B., and Akinbamijo, O. O. (2000). Micronutrients and reproduction in farm animals. Anim. Reprod. Sci. 60]61, doi: / S (00) Taha, T. A., Abdel-Gawad, E. I., and Ayoub, M. A. (2000). Monthly variations in some reproductive parameters of Barki and Awassi rams throughout 1 year under subtropical conditions. 2: biochemical and enzymatic properties of seminal plasma. J. Anim. Sci. 71, Tang, F. Y., and Hoskins, D. D. (1975). Phosphoprotein phosphatase of bovine epididymal spermatozoa. Biochem. Biophys. Res. Commun. 62, doi: /s x(75) Tegegne, A., Entwistle, K. W., and Mukasawa-Mugerwa, E. (1992). Nutritional influences on growth and onset of puberty in Boran and Boran Friesian bulls in Ethiopia. Theriogenology 37, doi: / x(92)90099-d Tibary, A., and Memon, M. A. (1999). Reproduction in the male South American Camelidae. J. Camel. Prac. Res. 6, Van Saun, R. J. (2008). Effect of nutrition on reproduction in llamas and alpacas. Theriogenology 70, doi: /j.theriogenol OGY Vaughan, J. L., Macmillan, K. L., Anderson, G. A., and D Occhio, M. J. (2003). Effects of mating behaviour and the ovarian follicular state of female alpacas on conception. Aust. Vet. J. 81, doi: / J TB11442.X Xu, Z. Z., McDonald, M. F., McCutcheon, S. N., and Blair, H. T. (1991). Seasonal-variation in testis size, gonadotropin-secretion and pituitaryresponsiveness to GnRH in rams of 2 breeds differing in time of onset of the breeding-season. Anim. Reprod. Sci. 26, doi: / (91)