Effect of complement C3 genotypes on milk production in sheep L. SOTIROV*1, I. DIMITROV2, M. DJORBINEVA2 and E. JELIAZKOV3 1 Department of Genetics; Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria 2 Institute of Agricultural Science, 6000 Stara Zagora, Bulgaria. 3 Department of Genetics, Agrarian Faculty, Trakia University, 6000 Stara Zagora, Bulgaria

* Corresponding author : Assoc. Prof. Dr. L. Sotirov, Department of Genetics; Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria, e-mail: HYPERLINK "mailto:[email protected]" [email protected]

SUMMARY

RÉSUMÉ

The aim of the present study was to investigate the influence of the complement genotype affiliation (determined via the C3 complement component polymorphism) upon the milk productivity in sheep. The sheep included in this experiment were milk type crossings (n = 118 sheep) - Stara Zagora-East-Friesian and (Stara Zagora-East-Friesian) - Blackhead Pleven breed. Compared to the other C3 genotypes (SS, FF, FS and F5S), the F7S genotype gave the highest milk productiveness for the whole and the milking lactation (p < 0.01). This high productivity was associated with a high daily milk yield and with the longer lactation duration. The lowest scores (milk productiveness, daily milk yield, numbers of lactation days) were observed in the F5S genotype sheep. Albeit the lactation duration was low in the FF genotype sheep, the milk productiveness was comparable to values observed with the FS and SS genotypes and this was probably due to a high daily milk yield. Moreover, the lifelong milk production was enhanced in this particular genotype. The calculations of the point serial coefficients evidenced a strong correlation between C3 genotype affiliation and milk productivity in sheep. These results suggest that a selection based on C3 genotype would improve lactation performances in sheep.

Influence du profil génotypique du complément (globine C3) sur la production laitière de la brebis. Par L. SOTIROV, I. DIMITROV, M. DJORBINEVA and E. JELIAZKOV

Keywords : sheep - C3 genotype - milk production

L'objectif de cette étude est d'analyser I'influence du profil génotypique du complément (défini par le polymorphisme de la globine C3) sur la production laitière des brebis. Au total, 118 brebis de type laitier (croisements Stara Zagora X Frisonne de l'Est et (Stara Zagora X Frisonne de I'Est) x Tête noire de Pleven) dont l'appartenance aux différents génotypes en C3 a été identifiée ont été utilisées. Comparé aux autres génotypes (SS, FF, FS et F5S), le génotype de type F7S a conduit à la production de lait la plus élevée (mesurée sur l'ensemble de la lactation ou pendant la période de lactation proprement dite). Cette forte productivité a été associée à un fort rendement laitier journalier et à un allongement de la durée de lactation. Les scores les plus faibles (production laitière, nombre de jours de lactation, rendement laitier journalier) ont été observés chez les brebis de type FS. Bien que la durée de lactation ait été faible chez les brebis du génotype FF, la production laitière a été comparable à celle obtenue chez les animaux FS ou SS en raison d'un rendement laitier journalier élevé. En outre, la durée de production (cumulée sur plusieurs lactations) a été augmentée chez ce génotype particulier. La détermination des coefficients de corrélation par doubles séries a mis en évidence une forte corrélation entre le profil génotypique du C3 et la productivité laitière chez la brebis. Ces résultats suggèrent qu'une sélection fondée sur le génotype C3 pourrait améliorer les performances laitières des brebis.

Mots-clés : brebis - génotype C3 - production laitière.

Introduction The selection of sheep in order to increase their milk production is a principal goal of animal breeding specialists. In order to fulfil this aim, they are looking for a relationship between various genetic markers and this primary parameter of productivity. PIRISI et al. [13] tested the effects of sheep alpha(s1)-casein CC, CD and DD genotypes on milk composition and cheese yield. Cheesemaking trials, carried out in a pilot plan, showed that CC milk had better cheesemaking characteristics than DD milk, while CD milk was intermediate. Both one day old and fully ripened DD cheeses presented the lower fat: dry matter ratios and alpha (s1)-I-casein electrophoretic mobility. According to the authors these genotypes could be considered as markers of milk and/or cheese quality. DIEZ-TASCON et al. [7] studied Churra sheep for the presence of quantitative trait loci (QTL) influencing milk production traits on the chromosome 6. Eight half-sib families were genotyped for 11 microsatellites and marker-QTL effects analysed using yield deviations (YD) as quantitative measurements for the following traits: milk yield, protein yield, and protein percentage. The results suggest a region on ovine chromosome 6, close to the casein Revue Méd. Vét., 2006, 157, 7, 391-395

cluster, with an influence on milk traits and particularly on protein percentage. These results, the first ones, reported that QTL affected milk traits in sheep. HORSTICK et al. [10] studied the milk protein variants of beta-casein and showed a significant relationship to protein content and the milk protein variants of alpha (S2)-casein to milk yield. For the milk protein variants of beta-lactoglobulin milk performance traits were not significantly correlated. The association analysis for the blood protein variants of transferrin revealed a significant effect on milk and protein yield and for haemoglobin a significant association to protein content. The variants of the vitamin-D-binding protein showed no effect on the analysed milk performance traits. DARCAN and GUNEY [4] confirmed that haemoglobin and transferrin polymorphisms were found to be effective on the performances of Awassi and Cukurova Assaf breeds. On the basis of comparisons between cattle and sheep genome mapping information, CALVO et al. [2] examined the ovine alpha-amylase gene as a possible genetic marker for milk traits in sheep. The authors isolated two DNA fragments (832 and 2360 bp) corresponding to two different AMY genes, and one SNP in intron 3 and one GTG deletion in exon 3 were found in the 2360 bp DNA fragment. The

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2360bp ovine AMY DNA fragment was located on chromosome 1 by linkage mapping using the International Mapping Flock. No association was found between estimated breeding values for milk yield, protein and fat contents and AMY genotypes in a daughter design comprising 13 Manchega families with an average of 29 daughters (12-62) per sire. Some authors [7, 10, 14-18] have already reported the influence of C3 genotypes on lysozyme concentrations and complement activity in sheep and horses, and DIMITROV et al. [8] demonstrated that C3 genotypes influence some productive traits in sheep. The aim of the present study was to investigate the influence of genotype affiliation (determined via the C3 complement component polymorphism) upon the milk productivity in sheep.

Materials and Methods Animals The sheep included in this study were milk type crossings (n = 118 sheep) - Stara Zagora x East-Friesian and (Stara Zagora x East-Friesian) x Blackhead Pleven breed. At the beginning of the experiment, the sheep were 2-3 years old. Blood for analysis was sampled in 10 ml tubes from v. jugularis. The blood was allowed to clot for one hour at room temperature (25°C) and the samples were centrifuged at 2000g for 10 min. Milk production characterization For each sheep, the milk production traits (number of lactation days, milk productiveness (in kg), daily milk yield (in kg/day)) were established for the whole lactation (period from lambing to the end of lactation) and for the milking lactation (period from weaning to the end of lactation). The lifelong milk production (average milk production for the whole life of the animal) and the corresponding number of lactation days were also recorded. Determinations of C3 complement component polymorphism The polymorphism of C3 complement component was determined by the method of TEISBERG [21]:

SOTIROV (L.) AND COLLABORATORS

1. Buffers a. Stock solutions for gels - 5,5-diethylbarbituric acid sodium salt (Diemal Na, Loba-Chemie, Austria) 23 mM; 5,5-diethylbarbituric acid (Reanal, Hungary) 3.7 mM; Calcium-L(+)- lactate (Fluka AG, Switzerland) 0.9 mM; pH 8.6. b. Stock solution for tray buffer - 5,5-diethylbarbituric acid sodium salt (Diemal Na, Loba-Chemie, Austria) 61 mM; 5,5-diethylbarbituric acid (Reanal, Hungary) 10 mM; Calcium-L(+)- lactate (Fluka AG, Switzerland) 1.8 mM; pH 8.6. 2. Electrophoresis The first buffer was used for preparation of agarose gel (1%) where serum samples were applied. After their absorption, the electrophoresis was carried out at 20 V/cm for about 2.5 h using the second buffer in the trays. Gels were stained for 1 min with 1% Amido Black 10 B and destained for overnight (destaining solution: ethanol, distilled water and glacial acetic acid (5:5:1) and 2 tablespoons of active charcoal powder were added in order to allow the manifold use of the destaining solution). Statistical analysis Data were analysed using the fixed effect MANOVA model (Program STATISTICA, StatSoft, Inc., USA) and Point biserial correlation coefficient (VassarStat, Vassar College Poughkeepsie, NY, USA). Differences between genotype means were tested using t-test and differences were considered as significant when p values were less than 0.05.

Results The influence of observed C3 genotypes upon the milk productiveness in milk type sheep is presented in Table 1. It was observed that the highest milk productiveness for the whole lactation and for the milking lactation was associated with the F7S genotype. Indeed, F7S genotype sheep presented significantly higher values with a low dispersion (17.25% and 25.16% for the whole lactation and the milking lactation respectively) than sheep of the other C3 genotypes (SS, FF and FS - p < 0.05), whereas the F5S genotype was

TABLE I. — Effects of C3 genotype affiliation on milk productiveness and numbers of lactation days in milk type sheep for the whole lactation and for the milking lactation. Results are expressed as means ± standard errors. Revue Méd. Vét., 2006, 157, 7, 391-395

EFFECT OF COMPLEMENT C3 GENOTYPES ON MILK PRODUCTION IN SHEEP

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TABLE II. — Correlations (rpb) between the different C3 genotypes and milk productiveness in milk type sheep for the whole lactation and for the milking lactation.

associated with the lowest values (p < 0.01), and the dispersion of the results was also relatively reduced in this group (20.14% and 28.20% for the whole lactation and the milking lactation respectively). Intermediate milk productiveness was observed in the SS, FS and FF genotypes, and in this latter group, the values were greatly dispersed (44.13% and 47.07% for the whole lactation and the milking lactation respectively). The calculations of the strength of the relationship between the genotype affiliation and the milk productivity in sheep using the point biserial coefficient are showed in Table 2. For the whole lactation period, these values have considerably depended on the animal genotype and were ranged from rpb: 0.21 (genotypes F5S – FF) to rpb: 0.60 (genotypes F5S – F7S). The low values of this parameter (rpb: 0.21 to 0.25, genotypes F5S – FF and F5S – SS, respectively) were associated with a low degree of significance whereas moderate (rpb: 0.35, genotypes F5S - FS) and high values (rpb: 0.60, genotypes F5S – F7S) were highly significant. The coefficients calculated also for the milking period varied also from low (rpb: 0.25, genotypes F5S - SS) to considerable values (rpb: 0.61, genotypes F5S – F7S). Again, as for the whole lactation, the highest rpb coefficient was significantly associated with the genotype F7S. Again, the numbers of lactation days for the whole lactation and for the milking lactation were the highest in the F7S genotype group (p < 0.05). For the SS and FS genotypes, the durations of the whole lactations were slightly but not significantly reduced compared to the F7S genotype, whereas milking lactation periods were markedly decreased (p < 0.05).Moreover, the numbers of lactation days for the whole and the milking periods were dramatically lowered in the FF and F5S genotypes compared to the F7S genotype (p