Dairy sheep often exhibit sequences of under- and over-feeding due to seasonal changes in forages, by-products availability and climatic uncertainty (Chilliard et al. Reference Chilliard, Bocquier and Doreau1998). In particular, high producing dairy ewes, especially in early lactation or when suckling two or more lambs experience negative energy balance. (Bocquier & Caja, Reference Bocquier and Caja1993; Sorensen et al. Reference Sorensen, Adam, Findlay, Marie, Thomas, Travers and Vernon2002). Moreover, group fed animals can be either under- or over-fed when food is given according to average group requirements and when dominance hierarchies are formed at the food bunk (Estevez et al. Reference Estevez, Andersen and Nævdal2007). The ability of ruminants to cope with periods of under- and over-feeding is the result of a long evolutionary process in natural conditions where food availability fluctuates with season (Chilliard et al. Reference Chilliard, Bocquier and Doreau1998).
Ruminants undergo different adaptations to cope with changes in feeding level and nutrient availability allowing the maintenance of energy homeostasis. The energy homeostasis is regulated by the central nervous system through signals which are produced peripherally. One of the main category of the peripheral signals is hormones whose secretion is in proportion to the status of adipose tissue (AT) reserves. These adiposity signals, which includes insulin from the pancreatic islets, and leptin, mainly from AT, are transported from the circulation into the hypothalamus (Woods et al. Reference Woods, Lutz, Geary and Langhans2006; Roche et al. Reference Roche, Blache, Kay, Miller, Sheaham and Miller2008).
Over the last 15 years AT has been recognized as a true and complex endocrine tissue, but further it is the major site of fatty acid (FA) metabolism. It is well known that overfed ruminants preferentially use carbohydrates to generate ATP, and surplus carbohydrates are converted into FA, which are stored as tricylglycerides in the AT. During under-feeding the stored FA in AT are hydrolyzed to free FA which play significant role in energy production (Palou et al. Reference Palou, Priego, Sánchez, Villegas, Rodríguez, Palou and Picó2008). During lactation, the major portion of this energy derived from FA is used by the mammary gland. The shuttling of FA from AT to mammary epithelium represents a very critical source of triglycerides precursors during lactation, under conditions of negative energy balance. Adipose tissue mobilization accounts for less than 10% preformed FA in milk fat, except during periods of negative energy balance when their proportion increases substantially (Bauman & Griinari, Reference Bauman and Griinari2001).
Although the milk FA have been extensively examined in high producing dairy cows, especially during early lactation when they are in negative energy balance (Kay et al. Reference Kay, Kolver, Thomson, Roche and Baumgard2005; Van Knegsel et al. Reference Van Knegsel, van den Brand, Dijkstra, Tamminga and Kemp2005, Reference Van Knegsel, van den Brand, Dijkstra, van Straalen, Heetkamp, Tamminga and Kemp2007; Garnsworthy et al. Reference Garnsworthy, Masson, Lock and Mottram2006; Stoop et al. Reference Stoop, Bovenhuis, Heck and van Arendonk2009), data on sheep are lacking. Since sheep milk is mainly used for cheese making and its chemical composition and its FA profile affects cheese production and quality the aim of the present study was to determine the effects of long term under- and over-feeding on milk chemical composition and FA profile and on energy homeostasis, through plasma insulin and leptin concentration in sheep.
Materials and Methods
Twenty-four 3-year-old Friesian crossbred dairy sheep at 90–98 d in milk which gave birth to twins were maintained at the Agricultural University of Athens. Mean initial body weight (BW) of all animals was 59±3·5 kg. Housing and care of animals conformed to Ethical Committee guidelines of Faculty of Animal Science. The experiment started three months post-partum, when sheep were assigned to three homogeneous sub-groups (n=8) according to their BW and milk yield (Table 2). Throughout the experimental period each sheep of each group was fed individually. The animals of each group were fed with a diet which covered 70% (under-feeding), 100% (control) and 130% (over-feeding) of their individual energy and crude protein requirements respectively. The quantities of food offered to the animals were adjusted at the 0, 12, 24, 31, 39 and 52 experimental day according to their individual requirements based on their BW and milk yield. The average daily feed intake of each group under the three dietary treatments throughout the experimental period is presented in Table 1. The whole experimental period lasted 60 d.
Table 1. Average daily feed intake (kg/animal) by sheep under the three dietary treatments throughout the experimental period
† Figures in brackets are shown the days in milk
‡ 70%,
§ 100%,
¶ 130%=percentage of the animals energy and crude protein requirements
The diet consisted of alfalfa hay and concentrates with a forage/concentrate ratio=50/50. The concentrate diet (g/kg) consisted of: maize grain, 360; barley grain, 360; soybean meal, 160; wheat middlings, 110; calcium phosphate, 15; common salt, 3; mineral and vitamins premix, 2.
Samples collection
All animals were milked twice a day at 8 am and 6 pm with a milking machine. Individual milk samples were collected from sheep at 0, 12, 24, 31, 39 and 52 d for chemical analysis, and at day 39 and day 60 for FA determination after mixing the yield from the evening and the morning milking on a percent volume (5%). Blood samples were collected at days 24, 31, 39, 45, 52 and 60 of the experiment from the jugular vein into EDTA-containing tubes and subsequently centrifuged at 2700 g for 15 min to separate plasma from the cells. Samples from day 39 and 60 were used for FA and free FA (FFA) determination, while those of days 24, 31, 39, 45, 52 and 60 were used for hormone detection. The milk and the plasma samples were stored at −80°C until the FA and hormones analyses.
Table 2. Body weight (kg) of sheep at the three dietary treatments throughout the experimental period
† 70%,
‡ 100%,
§ 130%=percentage of the animals energy and crude protein requirements
¶ sem=pooled sem
†† Figures in brackets are shown the days in milk
Means with different superscripts (a, b) in each row between dietary treatments differ significantly (P⩽0·05)
Milk and plasma fatty acid analyses
The milk samples were analysed for FA according to the method of Nourooz-Zadeh & Appelqvist (Reference Nourooz-Zadeh and Appelqvist1998) as described by Tsiplakou et al. (Reference Tsiplakou, Chadio, Papadomichelakis and Zervas2011). The plasma FA analysis was carried out with the method of Bondia-Pons et al. (Reference Bondia-Pons, Castellote and Lopez-Sabater2004). For the determination of FA concentration an Agilent 6890 N gas chromatograph equipped (Agilent Technologies 2850, Centerville Road, Wilmington, USA) with an HP-88 capillary column (60 m×0·25 mm i.d. with 0·20 μm film thickness, Agilent) and a flame ionization detector, was used. Plasma non-esterified fatty acids (NEFA) concentrations were determined by spectrophotometric assays using kit C, WAKO, Unipath S.A., Dardilly, France.
Hormones determination
Plasma insulin concentrations were measured using a double antibody radioimmunoassay kit (Linco Research Inc. St. Charles. MO, USA). The sensitivity of the method was 0·1 ng/ml and the intra- and inter-assay coefficients of variation were 4·6 and 9·4%, respectively. Plasma leptin levels were determined using a commercial radioimmunoassay kit (Multi-Species Leptin RIA Kit, Linco Research, St. Charles, MO, USA), that is suitable for sheep plasma (Delavaud et al. Reference Delavaud, Bocquier, Chilliard, Keisler, Gertler and Kann2000). The intra- and inter-assay coefficients of variation were 4·3 and 5·1%, respectively.
Calculations
Short Chain Saturated Fatty Acids (SCFA)=C6:0 +C8:0+C10:0+C11:0,
Medium Chain Saturated Fatty Acids (MCFA)=C12:0+C13:0 +C14:0 +C15:0+C16:0,
Long Chain Saturated Fatty Acids (LCFA)=C18:0+C20:0+C22:0+C23:0+C24:0,
Poly-Unsaturated Fatty Acids (PUFA)=cis-9, trans-11CLA C18:2+C18:2n-6+C18:n-3+C18:n-6+C20:2+C20:n-3+C20:n-6+C20:n-4+C20:5n-3+C22:2,
Mono-Unsaturated Fatty Acids (MUFA)=C14:1+C15:1+C16:1+C17:1+C18:1+trans-11 C18:1+C20:1,
Saturated/Unsaturated ratio=S/U: (SCFA+MCFA+LCFA)/(PUFA+MUFA) and
The Atherogenicity index (AI) was defined as described by Ulbricht & Southgate (Reference Ulbricht and Southgate1991).
Statistical analysis
Data are presented ±sem. Experimental data were analysed using the SPSS statistical package (version 16.0). The BW, the milk chemical composition, the FA profile of milk and blood plasma and the hormone concentrations in blood plasma were analysed using a general linear model (GLM) for repeated measures analysis of variance (ANOVA) with dietary treatments (T=under-feeding=70%; control=100%; over-feeding=130%) and sampling time (S) as fixed effects and their interactions (T×S) according to the model:
where y ijk is the dependent variable, μ the overall mean, T i the effect of dietary treatment, S j the effect of sampling time (j=6 for milk chemical composition, 2 for milk and plasma FA profile, 6 for hormones concentrations) (T×S)ij the interaction between dietary treatments and sampling time and e ijk the residual error.
Further to that the treatment effect (T=under-feeding=70%; control=100%; over-feeding=130%) on sheep body weight (BW) and milk chemical composition was tested on a day basis (sampling time at days 0, 12, 24, 31, 39 and 52) by one way ANOVA with the same statistical package.
Post hoc analyses were performed when appropriate using Duncan's multiple range test and significance was set at 0·05.
Results and Discussion
Positive energy balance and increased milk production have been reported to result in a decrease in milk fat content in sheep (Bocquier & Caja, Reference Bocquier and Caja2001). The reduction in milk yield observed in the underfed sheep, compared with overfed, of this study maybe due to the fact that restricted feed intake reduces cardiac output and decreases blood flow and nutrient supply to the mammary gland (Guinard-Flament et al. Reference Guinard-Flament, Delamaire, Lemosquet, Boutinaud and David2006) (Table 3). Furthermore, there is a concomitant reduction in mammary glucose uptake per unit blood flow following a feed restriction which induces down regulation of the glucose transporter-1 and sodium glucose contransporter-1 genes (Boutinaud et al. Reference Boutinaud, Ben Chedly, Delamaire and Guinard-Flament2008). Because glucose is the primary precursor for lactose synthesis and lactose is the major osmotic agent in milk, reduced mammary glucose uptake has a major-limiting effect on milk synthesis (Burke et al. Reference Burke, Williams, Hofman, Kay, Phyn and Meier2010), as evidenced also by the lower lactose content detected in the underfed compared to overfed sheep (Table 3). From the results of Table 3 it also becomes apparent that the overfed sheep had higher daily milk fat and protein yield. Total protein and fat are the two main criteria applied to sheep milk payment in many countries (Pirisi et al. Reference Pirisi, Lauret and Dubeuf2007). Further to that, most of the sheep milk is used for cheese making and it is well known that the cheese yield depends on milk's protein content (Zervas & Tsiplakou, Reference Zervas and Tsiplakou2011). In addition the cheese quality is closely related to milk lipids and consequently to milk fat content. From the above we can conclude that the milk from the overfed sheep is superior compared with the control and the underfed sheep milk as far as the daily fat and protein yields are concerned.
Table 3. Mean (±sem†) of fat corrected milk yield‡ and milk composition in sheep at the three dietary treatments throughout the experimental period
† sem=pooled se of means
‡ Fat corrected milk in 6% according to the equation Y6%=(0·28+0·12F)M where, F=fat%, and M=milk yield in kg
§ 70%,
¶ 100%,
†† 130%=percentage of the animals energy and crude protein requirements
‡‡ Figures in brackets are shown the days in milk
Means with different superscript (a, b) in each row (between dietary treatments) for each component differ significantly (P⩽0·05)
The milk fat content, further to the farmer income improvement, also affects, the texture and the firmness of cheese while its FA profile is very important for consumer's health. Taking into account that diet can modify the milk FA profile in this study three different feeding levels (under-feeding, control, over-feeding) were chosen which are the usually found under practical conditions at a farm level, in order to examine the effects on milk FA profile. In a recent study of Tsiplakou et al. (Reference Tsiplakou, Chadio, Papadomichelakis and Zervas2011) with goats over- and under- feeding was shown to exert a significant effect on milk's FA profile with an increase in C18:0 and cis-9 C18:1 content in milk of the underfed animals, which is in good agreement with the results of the present study (Table 4). This increase could be partly attributed to the mobilization of fat from AT, since AT in ruminants has been reported to be rich in both C18:0 and cis-9 C18:1 FA (Chilliard et al. Reference Chilliard, Ferlay, Rouel and Lamberet2003). Furthermore, C18:0 is the main FA which is released from AT during lipolysis (Rukkwamsuk et al. Reference Rukkwamsuk, Geelen, Druip and Wensing2000; Gillis et al. Reference Gillis, Duckett and Sackmann2004; Kay et al. Reference Kay, Kolver, Thomson, Roche and Baumgard2005). A decrease in the production of milk LCFA from early to mid lactation has been reported in dairy cows (Garnsworthy et al. Reference Garnsworthy, Masson, Lock and Mottram2006; van Knegsel et al. Reference Van Knegsel, van den Brand, Dijkstra, van Straalen, Heetkamp, Tamminga and Kemp2007), in line with the present results, implying that when positive energy balance is increased the production of LCFA in milk of overfed sheep is decreased (Table 4). Further to that, it could be suggested that the mobilization of body fat stores of the underfed sheep contributes to the increased secretion of LCFA in milk (Van Knegsel et al. Reference Van Knegsel, van den Brand, Dijkstra, van Straalen, Heetkamp, Tamminga and Kemp2007). It has been clearly shown that C18:0 (although it is a LCFA and is classified as SFA) and cis-9 C18:1 have a powerful affect in lowering LPL and triglycerides and probably in increasing the level of high density lipoprotein (HDL) (Molkentin Reference Molkentin2000; Kris-Etherton et al. Reference Kris-Etherton, Griel, Psota, Gebauer, Zhang and Etherton2005) which makes the milk of the underfed sheep better than the controls and the overfed in terms of these two FA.
Table 4. The mean individual FA concentrations (% of total FA), FA groups, S/U ratio and the AI index of sheep milk at the three dietary treatments and the two sampling times
† 70%,
‡ 100%,
§ 130%=percentage of the animals energy and crude protein requirements
¶ sem=pooled sem
†† Figures in brackets are shown the days in milk
‡‡ SCFA: Short Chain Saturated Fatty Acids=C6:0+C8:0+C10:0+C11:0
§§ MCFA: Medium Chain Saturated Fatty Acids=C12:0+C13:0+C14:0+C15:0
¶¶ LCFA: Long Chain Saturated Fatty Acids=C16:0+C18:0+C20:0+C22:0+C23:0+C24:0
††† SFA: Saturated Fatty Acids=SCFA+MCFA+LCFA
‡‡‡ PUFA: Poly-Unsaturated Fatty Acids=cis-9, trans-11CLA C18:2 +C18:2n-6+C18:3n-3+C18:3n-6+C20:2+C20:3n-3+C20:3n-6+C20:4+C20:5n-3+C22:2
§§§ MUFA: Mono-Unsaturated Fatty Acids=C14:1+C15:1+C16:1+C17:1+C18:1+trans-11C18:1+C20:1
¶¶¶ S/U: Saturated/Unsaturated=(SCFA+MCFA+LCFA)/(PUFA+MUFA) and
†††† AI: Atherogenicity index=(C12:0+4×C14:0+C16:0)/(PUFA+MUFA)
Means with different superscript (a, b, c) in each row (between dietary treatments, and between sampling time) for each fatty acid differ significantly (P⩽0·05)
Apart from the increase in C18:0 content in milk, a numerical increase in C16:0 was also found in underfed sheep, compared with controls and overfed. Interestingly, the opposite has been observed in dairy cows during negative energy balance, with the C16:0 being the most affected FA, followed by the C18:0 (Stoop et al. Reference Stoop, van Arendonk, Heck and Bovenhuis2008, Reference Stoop, Bovenhuis, Heck and van Arendonk2009), implying species differences in lipid mobilization.
The results concerning the concentrations of C6:0, C8:0, C10:0, C11:0, C12:0, C14:0, SCFA and MCFA, which were significantly lower in milk fat of the underfed sheep compared with overfed (Table 4), are in agreement with those of Tsiplakou et al. (Reference Tsiplakou, Chadio, Papadomichelakis and Zervas2011) for under- and over-fed goats. Accordingly, Eknæs et al. (Reference Eknæs, Kolstad, Volden and Hove2006) in goats and Kay et al. (Reference Kay, Mackle, Auldist, Thomson and Bauman2004) in cows reported that milk concentrations of FA which are synthesised de novo are increased from early to mid lactation, probably as a result of increased energy available for de novo FA synthesis (Bauman & Griinari, Reference Bauman and Griinari2003). On the other hand, the concentration of C4:0, which is also produced endogenously, was shown to be higher in the milk of underfed compared with control sheep. Butyrate can arise from two pathways independently of the acetyl coenzyme A carboxylase pathway and may also not be inhibited by the high NEFA concentrations associated with underfeeding (Palmquist et al. Reference Palmquist, Beaulieu and Barbano1993). Palmquist is correct Ed. Thus, the milk from the overfed sheep is more beneficial to C6:0, C8:0 and C10:0 FA compared with the underfed, as it has been proved that these FA are associated with characteristic flavor of cheeses and can also be used to detect admixtures of milk from different species.
In the milk of the overfed sheep, a higher PUFA concentration was observed (Table 4). This suggests that during weight gain (over-feeding) the incorporation of PUFA into milk is more predominant than during either weight balance (control) or weight loss (under-feeding), as dietary FA are less diluted by the FA released from AT (Beynen et al. Reference Beynen, Hermus and Hautvast1980; Yeom et al. Reference Yeom, Schonewille and Beynen2005). In the recent years the PUFA have received much attention from the nutritionist, consumers and researchers because of its several beneficial and bioactive function on human health including antiatherogenic, immune system stimulation etc. (Ruxton et al. Reference Ruxton, Calder, Reed and Simpson2005). The PUFA concentration in sheep milk comprise mainly of linoleic and α-linolenic acid as well as their positional and geometric isomers like cis-9, trans-11CLA C18:2. In the present study, no differences were observed in cis-9, trans-11CLA C18:2 milk fat content of sheep between dietary treatments, despite the higher trans-11 C18:1 concentration in the milk of overfed sheep compared with underfed and controls. In cows, alterations in feed intake has been reported to have variable effects on milk fat content of CLA, ranging from increase, after restricting feed intake by approximately 30% (Jiang et al. Reference Jiang, Fonden Bjoerck and Emanuelson1996) to decrease (Stanton et al. Reference Stanton, Lawless, Kjellmer, Harrington, Devery, Connolly and Murphy1997). More recently, Nozière et al. (Reference Nozière, Grolier, Durand, Ferlay, Pradel and Martin2006) reported that the low feeding level, compared with high, caused an increase in CLA cow's milk. Alterations in feed intake would obviously affect substrate supply and change rumen environment. Both these factors may have contributed to a change in ruminant biohydrogenation process and consequently to CLA and trans-11 C18:1 production. In addition, under-feeding would increase the supply of CLA and trans-11 C18:1 from mobilized body fat stores and the magnitude of this increase would be related to the extent and duration of the negative energy balance. Recent data suggest that consumption of trans-11 C18:1 may impart health benefits beyond those associated with CLA (Field et al. Reference Field, Blewett, Proctor and Vine2009). Overall, as concerns the milk FA profile, the milk of the underfed sheep (has higher concentrations of C18:0,cis-9 C18:1, LCFA, MUFA and lower concentration of SFA, AI and S/U ratio) is superior compared with overfed despite the fact that the milk of the overfed animals is richer in some individual FA (C6:0, C8:0, C10:0 and trans-11 C18:1) within each broad classification which have unique biological properties and health effects.
Metabolic changes due to underfeeding include alterations in the energy balance leading to increased lipid mobilization with consequent elevation of plasma NEFA concentrations (Contreras et al. Reference Contreras, O'Boyle, Herdt and Sordillo2010). In the present study, underfed sheep exhibited a significant increase in blood NEFA concentrations compared with control or overfed (Table 5). Similar results have been reported by Rezapour & Taghinejad-Roudbaneh (Reference Rezapour and Taghinejad-Roudbaneh2011) in sheep fed with a restricted diet compared with controls.
Table 5. The mean FA concentrations (% of total FA) in total lipids of sheep plasma between dietary treatments and sampling time
† 70%,
‡ 100%,
§ 130%=percentage of the animals energy and crude protein requirements
¶ sem=pooled sem
†† Figures in brackets are shown the days in milk
Means with different superscript (a, b, c) in each row (between dietary treatments, and between sampling time) for each fatty acid differ significantly (P⩽0·05)
It has been demonstrated that AT under adequate feeding level, is firstly enriched with dietary PUFA and MUFA and to a lesser extent with LCFA. The results of the present study, which showed an increase in plasma C20:5n-3, C22:6n-3, C20:3n-3 and C18:3n-6 concentrations of underfed sheep compared with controls (Table 5), are in good agreement with previous data reported that C20:5n-3 is readily and C18:3n-3 moderately mobilized (Raclot, Reference Raclot2003). Moreover, the higher C18:0 and cis-9 C18:1 concentrations detected in the plasma of the underfed animals, may also be due to the mobilization of AT, since AT in ruminants is rich in these particular FA (Chilliard et al. Reference Chilliard, Ferlay, Rouel and Lamberet2003). On the other hand, the significantly lower C18:2n-6 concentration in the plasma of underfed sheep, compared with controls and overfed, could possibly be explained by the fact that this FA has dietary origin and is taken up by the mammary gland from plasma. Similar results have been reported for cows (Contreras et al. Reference Contreras, O'Boyle, Herdt and Sordillo2010) and starved sheep (Jackson & Winkler, Reference Jackson and Winkler1970).
Nutrition has a significant impact on numerous metabolic functions including hormones production. There is a direct relationship between insulin concentrations and feeding level as insulin responds directly to nutrients absorbed from the gastrointestinal tract (Ouellet et al. Reference Ouellet, Seoane, Bernier and Lapierre2001). In this study significantly higher plasma insulin concentrations were observed in overfed, compared with underfed sheep (Table 6). Similarly, increased blood insulin concentrations have been demonstrated in sheep fed a high energy diet for 3 months (Caldeira et al. Reference Caldeira, Belo, Santos, Vazques and Portugal2007), for 8 weeks (Grazul-Bilska et al. Reference Grazul-Bilska, Caton, Borowczyk, Arndt, Bilski, Weigl, Kirsch, Redmer, Reynolds and Vonnahme2007) and for 6 d (Vinoles et al. Reference Vinoles, Forsberg, Martin, Cajarville, Repetto and Meikle2005), or when the sheep were fed with a diet covering twice their maintenance requirements (Zhang et al. Reference Zhang, Blache, Blackberry and Martin2005). On the other hand decreased plasma insulin levels in sheep, being in negative energy balance, have been reported by Scaramuzzi et al. (Reference Scaramuzzi, Campbell, Downing, Kendall, Khalid, Munoz-Gutierrez and Somchit2006) and by Henry et al. (Reference Henry, Goding, Tilbrook, Dunshea, Blache and Clarke2004) after short-term fasting (32 h).
Table 6. The mean (±sem†) concentrations (ng/ml) of leptin and insulin of sheep blood plasma at the three dietary treatments throughout the experimental period
† sem=pooled sem
‡ 70%,
§ 100%,
¶ 130%=percentage of the animals energy and crude protein requirements
†† Figures in brackets are shown the days in milk
Means with different superscript (a, b) in each row (between dietary treatments) for each hormone differ significantly (P⩽0·05)
Leptin concentrations in sheep are positively related to both the daily ingested energy in the short term, and the degree of adiposity and the level of nutrition in the long term (Chilliard et al. Reference Chilliard, Bonnet, Delavaud, Faulconnier, Leroux, Djiane and Bocquier2001). Leptin plays a key role in regulating energy intake and energy expenditure (Roche et al. Reference Roche, Blache, Kay, Miller, Sheaham and Miller2008). Leptin's plasma concentrations have been reported to decrease when sheep were fed with a diet covering 50% (Nørgaard et al. 2008) or 39% (Delavaud et al. Reference Delavaud, Bocquier, Chilliard, Keisler, Gertler and Kann2000) of their energy and protein requirements respectively, while higher blood leptin concentrations have been found in sheep fed with a diet covering 1·8 times compared with a diet covering 1·2 times the maintenance energy requirements (Tokuda et al. Reference Tokuda, Delavaud and Chilliard2002). The significantly higher plasma leptin concentrations found in the overfed sheep of this study, compared with underfed ones, are in good agreement with the previous studies (Table 6). Thus both hormones, insulin and leptin, are related to sheep feeding level and consequently correspond to their energy balance and homeostasis.
Conclusions
The results of the present study clearly demonstrate that both under- and over-feeding of sheep caused considerable alterations in milk chemical composition and in FA profile. More specifically overfeeding had a positive impact on daily milk fat and protein yield which affects the cheese making and the milk payment in many countries. Concerning the milk FA profile, the milk of the underfed animals is superior, compared with overfed, based on health aspects, while the opposite happens when taking into account the FA which are associated with the characteristic flavor of cheeses. Additionally the significant increase of C18:0 in the milk fat and blood plasma of the underfed animals, compared with overfed, could possibly be a good indicator of the underfeeding in small ruminants and underlines the species differences compared with cows where C16:0 is the main FA which mobilized during under feeding conditions. Finally the results concerning the plasma insulin and leptin concentrations indicate that these two hormones are associated with the animals energy balance and homeostasis.
The financial support from the State Scholarship Foundation of Greece as postdoctoral fellowship is acknowledged. The authors also express their thanks to Miss Stavroula Papaefstathiou for her contribution to the experimental work.
