Seasonal variations of the serum proteins in sheep and goats ( Short Communication )

The aim of this study was to assess the seasonal trend of the serum protein content of sheep and goats. The tested animals were six female goats (Maltese breed) and six female sheep (Valle del Belice breed). All animals were clinically healthy and not pregnant or lactating before or during the study. On blood samples, collected through an external jugular venipuncture every 30 days for 12 months, electrophoresis was performed using a semiautomated AGE system and then electrophoretic curves with the relative protein concentrations were analyzed. One way for repeated measure analysis of variance (ANOVA) was applied to determine the effect of time and by means of cosinor rythmometry, mesor (mean level), amplitude (half the range of oscillation) and acrophase (Φ, time of peak) were determined. The results showed a seasonal rhythm on Albumin and Alumin/Globulin ratio for sheep and goats, with different acrophases, winter for goats and spring for sheep. A seasonal rhythm was shown also in Alpha 2 globulins by sheep and in Beta globulins by goats. The difference in the acrophase can be attributed to a different production pattern of melatonin in goat, so the liver production of albumin is major during the winter that has a longer scotophase.


Introduction
Most long-lived species exhibit seasonal cycles of physiological functions to cope with seasonal fluctuations in climate and food availability (Patkowski et al. 2006, Duarte et al. 2010).The light initiates a cascade of physiological events mediating the input and interpretation of day length to the output of specific hormones that ultimately determine whether animals prepare to develop, reproduce, hibernate, enter dormancy, or migrate (Schneider & Rehbock 2003, Bradshaw et al. 2010).These seasonal rhythms reflect the endogenous adaptive mechanism to react in advance to the regular environmental changes associated with the seasons (Piccione et al. 2009).It is well known that in mammals, seasonal timekeeping depends on the generation of a signal that reflects daylenght (Felska-Blaszczyk & Brzozowski, Hazlerigg et al. 2004, Pala & Savas 2005).Limited information is provided about the seasonal rhythms of small ruminants (Alila-Johansson et al. 2001, Alila-Johansson et al. 2004, Piccione et al. 2007, Piccione et al. 2009a).Growing interest is rising about the serum proteins, which concentration reflects the physiological conditions of an animal and results as an important diagnostic tool to assess animal health (Castillo et al. 1997, Castillo et al. 1999, Antunovic et al. 2004, Alberghina et al. 2010).Different studies investigated the more frequent pathologies and their relationship with the serum protein variations (Yarim et al. 2009, Braun et al. 2010).In ruminants, the serum protein contents, which differ between sheep and goat, were assessed as necessary to study metabolic disorders (Alberghina et al. 2010, Waziri et al. 2010).In fact, as observed in goats, sensitivity to several diseases appears to be higher during the winter season (Al- Busaidi et al. 2008).On the basis of these considerations, it was opportune to assess the trend of seasonal variations of serum proteins in sheep and goats.

Materials and methods
The experimental subjects were six female goats (Maltese breed, aged 3±1 years, mean weight 45±3 kg) and six female sheep (Valle del Belice breed, aged 4±1 years, mean weight 68±6 kg).All subjects were clinically healthy and not pregnant or lactating before or during the study.They were kept free from internal and external parasites.Their health status was evaluated every month, before the blood sampling, based on behaviour, rectal temperature, heart rate, respiratory profile, cough, nasal discharge, ocular discharge, appetite, faecal consistency and haematological profile.Animals were housed in Sicily, Italy (Latitude 38° 6' N, Longitude 13° 20' E, Altitude 50 m above sea level) into two different pens for species under natural photoperiod and environmental conditions (Figure 1).Natural photoperiod in this region varies from L/D (light dark ratio) 15/9 at the summer solstice to L/D 10/14 at the winter solstice.The area is characterized by an annual rainfall of 51.5 mm (range: 5-98) generally occurring in autumn and winter.Mean annual maximum and minimum temperatures are 33 °C in August and 10 °C between January and February, respectively; with relative humidity between 69 and 73 % through the year.Ambient temperature and relative humidity were continuously recorded, during all the experimental period, with a data logger (Gemini, Chichester, West Sussex, UK).All the animals had free access to water and to goodquality alfalfa hay.Concentrate (oats 23 %, corn 36 %, barley 38 % and mineral and vitamin supplement 3 %) was provided once daily (200 g per animal per day).Protocols of animal husbandry and experimentation followed applicable regulation in Italy.
Blood samples were collected through an external jugular venipuncture, using vacutainer tubes (Terumo Corporation, Japan) with no additive, every 30 days at the same hour (09:00) for one year (12 months).
The total protein concentrations were determined by the biuret method using an automated analyzer (Konelab 20, Dasit, Helsinki, Finland).The protein standard was albumin (5.00 g/ dL; Dasit Milano, Italy).Electrophoresis was performed using a semiautomated AGE system (Helena Laboratories, Helena Biosciences, Gatheshade, UK) according to the procedures described by the manufacturer.For each serum sample, 10 μL were applied to numbered sample wells containing Agarose Gel previously prepared.Each gel could accommodate up to 24 samples.Films were electrophoresed for 28 min at 450 V.After electrophoresis, films were simultaneously fixed using an automated system (SAS2, Helena Biosciences), stained in blue stain acid solution (Coomassie Blue Brilliant R250, Helena Biosciences) for 10 min, and then dried at 37 °C.After destaining in acetic acid and drying completely for 15 min films were scanned on a densitometer (Ez-Scan, Helena Biosciences), electrophoretic curves plus related quantitative specific protein concentrations for each sample were displayed.Relative protein concentrations within each fraction were determined as the optical absorbance percentage, and absolute concentrations (g/dL) were calculated using the total protein concentration.
All the results were expressed as mean±standard deviation (SD).Data were normally distributed (P<0.05,Kolmogorov-Smirnov test).One way for repeated measure Analysis of Variance (ANOVA) was applied to determine the effect of time during the experimental period (12 months) and P-values <0.05 were considered statistically significant.Data were analyzed using the software STATISTICA 7.0 (StatSoft 2004).Using single cosinor rhythmometry procedure (Nelson et al. 1979), three rhythmic parameters were determined: Mesor (middle value of the fitted cosine representing a rhythm-adjusted mean), expressed in the same conventional unit of the relative parameter, with the confidence interval (C.I.) at 95 %, Amplitude (the distance from the Mesor to the minimum and maximum of the fitted cosine function), expressed in the same unit as the relative Mesor, and Acrophase (Φ.Time of peak value in fitted cosine function), expressed in months with C.I. at 95 %.For each parameter, the mean level of each rhythm was computed as the arithmetic mean of all values in the data set (12 data points).The amplitude of a rhythm was calculated as half the range of oscillation, which on its turn was computed as the difference between peak and trough.The acrophase of a rhythm was determined by an iterative curve-fitting procedure based on the single Cosinor procedure.For each variable for each animal, a cosine wave was fitted to the data points according to the function: where Y t denotes each data pointing the time series, M is the mean level of the rhythm, A is the amplitude, θ t is the trigonometric angle (in degrees) corresponding to time t, and φ is the angle displacement for the acrophase.The value of φ was determined by iteration: the true value was considered to be the one that produced the smallest sum of squares of the deviations between iterated cosine functions and the raw data.

Results
The application of ANOVA showed a significant effect of time during the experimental period on all the variables in sheep, and it showed a significant effect of time on albumin, alpha 2 and beta globulin and albumin/globulin ratio (alb/glob) in goats.
The application of the periodic model and statistical analysis of the cosinor procedure demonstrated seasonal rhythms of Albumin and Alb/Glob both in sheep and goats (Table 1).Alpha 2 globulins showed a seasonal rhythmicity in sheep, whereas beta globulins showed seasonal rhythmicity in goats (Figure 1).

Discussion
A seasonal rhythm was found for the serum proteins in albumin and alb/glob both for sheep and goats with acrophases between July and September for sheep and December for goats.These changes are connected to the changes of light and temperature throughout the year (Alila-Johansson et al. 2004).In fact, the hypothalamic pacemaker, directly innervated from retina, results as the principal factor involved in determining circadian rhythms (Moore Ede et al. 1986).In normal conditions, albumin variations are affected by numerous factors such as heat haemoconcentration that can take to an increase of its value (Braun et al. 2010).In this case, the opposite trend shown for the two small ruminant species is attributable only to a difference, found by other authors, in melatonin pattern.The nocturnal production of melatonin is higher in sheep than in goats (Todini et al. 2010).As already demonstrated by other studies in mouse and goat liver, there is a circadian clock in this organ that is not strictly dependent from feeding (Oishi et al. 2003, Piccione et al. 2008).This peacemaker regulates transcriptional proteins process also, and, in particular, makes this organ like an autonomous peacemaker but its relationship with the effect of melatonin in its regulation is not still clear.On the basis of the present results, it is conceivable that albumin production from liver is strictly dependent on this circadian clock and, maybe, on melatonin release (Alila-Johansson 2001, Piccione et al. 2008).This theory is widely demonstrated in the human species, in which transcription factors, like »albumin site D-binding proteins« and others, are present in peripheral tissues and participate as »cell-clock« in controlling regulation of other proteins (Allaman-Pillet et al. 2004).
The goat is a diurnal animal with a nocturnal acrophase of melatonin so, the longer scotoperiod (winter) corresponds with the major amount of melatonin production (Refinetti 1999).In sheep, circulating melatonin concentrations seems instead to be independent from photoperiod length and this is due to withdrawal of inhibitory effect of light on melatonin production by the pineal gland in this species (Andresson et al. 2005, Molik et al. 2006).
The same explanation could be given for the albumin/globulin ratio, because this ratio is strictly dependent from the albumin serum content and so demonstrated the same seasonal pattern in the studied species.
Relatively to the other protein fractions, alpha 2 globulins for sheep and beta globulins for goats, which showed a seasonal trend, these were different from those of albumin and alb/ glob ratio and had both the acrophase during the spring-summer season.It is conceivable that these two fractions have a different liver production despite the other fractions and the major serum release corresponds with the hot season, as these are widely within the range of the relative species and that a dehydration is excluded.
This study shed light on the effective seasonal characteristic of the most common small ruminant species, and indicates that an important difference exists between goat and sheep, just dependent on the seasonal »clock«.Further investigations should be conduct to evaluate how the serum proteins synthesis is subjected to change in other small ruminants species and if these changes interest in some manner other liver products.

Figure 1
Figure 1 Mean (±SD) serum concentration of Albumin (a), Alpha 2 globulins (b) Beta globulins (c) and Alb/Glob ratio (d), expressed in their conventional units of measurement, obtained in six sheep and six goats, with the acrophase (Φ), during 12 months of study, with the mean light/dark cycle duration (L/D, expressed in hours).