Study of polymorphisms in the promoter region of ovine β-lactoglobulin gene and phylogenetic analysis among the Valle del Belice breed and other sheep breeds considered as ancestors

The aim of this work was to sequence the promoter region of β-lactoglobulin (BLG) gene in four sheep breeds, in order to identify polymorphisms, infer and analyze haplotypes, and phylogenetic relationship among the Valle del Belice breed and the other three breeds considered as ancestors. Sequencing analysis and alignment of the obtained sequences showed the presence of 36 single nucleotide polymorphisms (SNPs) and one deletion. A total of 22 haplotypes found in “best” reconstruction were inferred considering the 37 polymorphic sites identified. Haplotypes were used for the reconstruction of a phylogenetic tree using the Neighbor-Joining algorithm. The number of polymorphisms identified showed high variability within breeds. Analysis of genetic diversity indexes showed that the Sarda breed presented the lowest nucleotide diversity, whereas the Comisana breed presented the highest one. Comparing the nucleotide diversity among breeds, the highest value was obtained between Valle del Belice and Pinzirita breeds, whereas the lowest one was between Valle del Belice and Sarda breeds. Considering that polymorphisms in the promoter region of BLG gene could have a functional role associated with milk composition, the lowest value of nucleotide diversity between Valle del Belice and Sarda breeds may be related to a higher similarity of milk composition of these two breeds compared to the others. Further analyses will be conducted in order to evaluate the possible correlation between the genetic diversity indexes and the BLG content in milk of our breeds.     

Jennet milk production during the lactation in a Sicilian farming system

In Italy, the interest for jennet milk production has recently developed. An 18-month-long experiment was carried out on a jennet farm near Milo (CT), where 24 jennets, which derived from the Ragusana breed, were tested for milk yield and composition over an entire lactation period. The jennets were fed with hay and concentrate in a large paddock. From the 28th post-foaling day to the end of the lactation, the jennets were machine-milked twice a day with an in-between milking interval of 5 h. The milk amount from each jennet was recorded every 3 weeks and individual samples were collected and analyzed for fat, protein, casein, non-proteic nitrogen, lactose and somatic cell count. This study showed that jennets at Sicilian latitudes are not seasonal polyestrous. The daily milk yield, the length of lactation and the milk characteristics varied depending on the foaling season. The total average milk production was 490 ± 36 kg in 295 ± 12 post-foaling days, considering two milking records per day. During the lactation, milk yield decreased constantly from 1.98 to 1.28 kg/jennet per day. When looking at the jennet milk quality during lactation, the percentage of fat and protein decreased, while the lactose percentage increased, according to a tendency apparently unique for equines when compared to the ruminants. When looking at the productive season, spring generally gave the best qualitative and quantitative results. Based on these results, jennet milk yield and quality could be improved; furthermore, jennet milk production may turn out to be a profitable business.     

Consequences of the inscription of local breeds in protected designation of origin cow cheese specifications for the genetic management of the herds

In the mountainous areas of Europe with a humid climate, dairy cattle production is a major agricultural activity, and the milk is often processed into cheese according to protected designation of origin (PDO) specifications. We analyzed the extent to which PDO specifications and/or a mountain environment influence the spatial distribution of estimated breeding values (EBVs) of cows and the herd-year effects (HYEs) for milk yield (kg/lactation) and protein and fat contents (g/kg), as well as lactation ranks and calving months. The study focused on the northern French Alps. A total of 37 023 lactations, recorded in 2006, in 1153 herds were analyzed. The cows belonged to the Montbéliarde (21 516 lactations), Abondance (10 346 lactations) and Tarentaise (5161 lactations) breeds. The two factors of variation considered were the status of the commune where the farm was located in relation to PDO (three categories: area with no PDO, area with a PDO with no milk yield limit, area with a PDO with a milk yield limit) and 'mountain' environment (four categories based on the European regulation: plain, piedmont, mountain and high mountain). In the Abondance breed, the average lactation rank increased with an increase in production constraints due to the PDO or to a mountain environment. In the Abondance and Tarentaise breeds, grouping of calving in winter was most marked in the 'PDO with a milk yield limit' and 'high-mountain' categories. In the Tarentaise breed, no significant effect on any trait and any variable was found in the 'PDO' or 'mountain' categories. In the other two breeds, the average EBV for milk yield decreased with an increase in the constraints due to PDO, with differences of 226 and 93 kg between extreme values in the Abondance and Montbéliarde breeds, respectively. The average HYE for milk yield was higher in the Abondance breed in the 'PDO with no milk yield limit' category than in the other categories (+740 and +1110 kg, respectively); HYE was not affected by the 'PDO' factor in the Montbéliarde breed or by the 'mountain' factor in either breed. Concerning the protein and fat contents, the effect of the 'PDO' and 'mountain' factors depended on the trait, the variable and the breed. The proportion of individual decisions (the farmer makes the decision) v. collective decisions (breed management) concerning herd dynamics in the face of existing constraints is discussed.     

Estimating myostatin gene effect on milk performance traits using estimated gene content for a large number of non-genotyped cows

The objective of this study was to estimate the myostatin (mh) gene's effect on milk, protein and fat yield in a large heterogeneous cow population, of which only a small portion was genotyped. For this purpose, a total of 13 992 889 test-day records derived from 799 778 cows were available. The mh gene effect was estimated via BLUP using a multi-lactation, multi-trait random regression test-day model with an additional fixed regression on mh gene content. As only 1416 animals, (of which 1183 cows had test-day records) were genotyped, more animals of additional breeds with assumed known genotype were added to estimate the genotype (gene content) of the remaining cows more reliably. This was carried out using the conventional pedigree information between genotyped animals and their non-genotyped relatives. Applying this rule, mean estimated gene content over all cows with test-day records was 0.104, showing that most cows were homozygous +/+. In contrast, when gene content estimation was only based on genotyped animals, mean estimated gene content over all cows with test-day records was with 1.349 overestimated. Therefore, the applied method for gene content estimation in large populations needs additional genotype assumptions about additional animals representing genetic diversity when the breed composition in the complete population is heterogeneous and only a few animals from predominantly one breed are genotyped. Concerning allele substitution effects for one copy of the 'mh' gene variant, significant decreases of -76.1 kg milk, -3.6 kg fat and -2.8 kg protein/lactation were obtained on average when gene content estimation was additionally based on animals with assumed known genotype. Based on this result, knowledge of the mh genotypes and their effects has the potential to improve milk performance traits in cattle.     

Candidate genes for milk production traits in Portuguese dairy sheep

In this study, the influence of the β-lactoglobulin, prolactin and α_S1-casein variants on milk production traits of three Portuguese sheep breeds, including Serra da Estrela, White Merino and Black Merino, was analyzed. The genetic variants of each marker were identified using PCR-RFLP. β-Lactoglobulin genotype AA was associated with lower milk yield in Serra da Estrela and Merino ewes. This marker also affected milk fat content in Serra da Estrela and protein content in Merino. First results regarding the influence of the sheep prolactin gene on milk production traits are reported. In the Serra da Estrela breed, ewes carrying prolactin genotype AA were significantly associated with lower milk yield, but this influence of prolactin genotypes on milk yield was not detected in Merino. Prolactin genotypes were also significantly associated with milk fat and protein contents in the Serra da Estrela breed. A suggestive effect of the α_S1-casein locus on milk yield was detected in Serra da Estrela, but no associations were found between the variants of this marker with milk fat and protein content.     

Evaluation of reference lactation length in Chios dairy sheep

Definition and establishment of a fixed reference lactation length could provide useful tools for on-farm comparison of ewes and flock management as well as genetic evaluations for the breeding programme. The objectives of this study were to (i) evaluate different reference lactation lengths for the Chios dairy sheep and (ii) define the most suitable reference length for the breed. A total of 260 042 test-day milk records from 24 474 ewes in 130 flocks collected between 2003 and 2014 were used; 15 different lactation lengths were evaluated ranging from 120 to 260 days, defined at 10-day intervals as reference for the Chios sheep. The evaluation criteria included: (a) heritability and repeatability of milk yield in each reference lactation, (b) genetic correlation of reference lactation milk yield with actual lactation milk yield and yield at first test-day record and (c) correlated response in reference lactation milk yield from selection based on first test-day milk yield. The latter emulates genetic gains achieved for milk yield based on early lactation selection. Heritability and repeatability estimates of reference lactation milk yield and genetic correlation with actual lactation yield favoured long reference lactations (180 to 230 days). On the contrary, correlation with first test-day record milk yield was higher for short lactations (120 to 170 days). Moreover, selection on first test-day record milk yield would lead to a correlated response in reference yield in 220 days equal to 85% of the highest estimate achieved in the maximum reference length of 260 days (190 days when only considering first lactation milk yield). Based on the results of the present study, an overall reference lactation length for the Chios breed of 220 days post-lambing and a first lactation reference length of 190 days post-lambing are recommended.     

Analysis of polymorphism within <Emphasis Type="Italic">POU1F1</Emphasis> gene in relation to milk production traits in dairy Sarda sheep breed

The ovine POU1F1 gene is localized on chromosome 1 and it contains five introns and six exons. In different mammalian species some mutations in different exons are associated with different production traits. The aim of our research was to study the POU1F1 gene nucleotide sequence to detect possible polymorphisms and their relationships with milk productive traits in Sarda breed sheep. The study had been conducted on 140 ewes, 4 or 5 years old coming from a farm located in Sardinia. All the animals were multiparous, lactating and in their third to fifth lactation. Individual milk yield had been recorded monthly and for each sample fat, protein, casein, lactose, and somatic cell count values were analysed. A jugular blood sample was collected from each ewe to perform genomic DNA extraction. PCR, SSCP and sequencing analysis were carried out to examine the six exons to highlight possible SNPs. One-way ANOVA was used to analyse association of variants with milk yield and/or its composition. Two novel SNP were found: 121 C>T in the 5′UTR of the fourth intron fragment and 249 G>A in the 3′UTR of the sixth exon fragment. The statistical analysis did not shown association between milk productive traits and the found polymorphisms. However, further investigations about the promoter region or the prophet genes, like the PROP-1, could clarify its exact role in regulating the productive traits in sheep.     

Cheesemaking aptitude of two Spanish dairy ewe breeds: Changes during lactation and relationship between physico-chemical and technological properties

Individual milk samples from two Spanish ewe breeds (Guirra and Manchega) were collected and evaluated during the lactation. Changes in the physico-chemical and technological characteristics during the lactation period were studied, and correlations between variables were estimated. Bulk milks from both ewe breeds were sampled three times during lactation, and physico-chemical and technological properties studied by using mini cheese productions. Fat, protein, casein, lactose, total solids, gel firmness and rate of curd aggregation were the physico-chemical and coagulation variables significantly influenced throughout lactation. With the advance of the lactation, milk took more time to coagulate, produced a less strong gel and showed a lower rate of curd aggregation. Milk total solids and estimated cheese yield increased, while curd draining capacity decreased during lactation. Correlation study showed that rennet clotting time had a positive correlation with pH and protein components. However, gel firmness and rate of curd aggregation were negatively correlated with fat and protein, while these two last parameters correlated positively with cheese yield. Curd draining and somatic cell counts did not show a significant correlation with any factor. The physico-chemical evaluation of bulk milks and their technological parameters studied by means of mini cheese productions did not show differences between the breeds evaluated.     

Identification of novel SNPs in the Sarda breed goats POU1F1 gene and their association with milk productive performance

The aim of the study was to detect polymorphism in the POU1F1 gene in Sarda breed goat, as well as to establish if SNPs could be associated with milk productive traits. The research was conducted on 129 Sarda breed goats from 4 to 5 years old, multiparous, lactating and in their third to fifth lactation. We report nine exonic and seven non-coding regions SNPs within the Sarda goat POU1F1 gene, namely, Ex 1 61 G>C; Ex 1 108 G>A; Ex 3 C>T; Ex 3 92 C>T; Ex 4 110 A>G; Ex 5 34 G>A resulting in Arg213Lys change; IVS4 641 G>A, IVS4 643 A>C, IVS4 659 G>A, IVS4 677 A>C, IVS4 G699Del, IVS4 709 C>G, Ex 6 17 G>A resulting in Arg228Ser change, Ex 6 58 G>T, Ex 6 172 T>C, 3′UTR 110 T>C. A statistically significant association was found between genotype TT, in position 17 of the exon 6 (3.1 % of frequency), and increased milk yield (P < 0.01) while genotype GT (25.6 % of frequency) was associated with a higher fat content. Genotype TT in position 58 of the exon 6 (3.9 % of frequency) was found to be associated with a higher fat (P < 0.01) and protein content (P < 0.05). Twenty-eight haplotypes were detected, but no significant association between the haplotypes and the milk production traits have been found. Our data, as well as providing new SNPs extending the POU1F1 gene characterization, evidence a relationship between polymorphism and milk production traits in Sarda goat breed.     

Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep

The aim of the study was to develop a reliable method for the RNA extraction from milk of Sarda sheep breed and to highlight if the extracted RNA can be used for expression study on mammary genes involved in milk fat synthesis using RT-qPCR. The main result is that a sample of 150 ml of milk provides an optimal amount of RNA (73.5 μg/ml). The highest RNA concentration has been found in the samples analysed within 4 h after collection. The RNA extracted was positively correlated to the number of somatic cells (P < 0.001). The efficiency of the extraction method was confirmed by the results obtained from qPCR which showed a Ct value, for SREBPF1 gene of 26.8 ± 0.15. This research demonstrated that the high-quality of the RNA obtained is suited to use for studies of mammary genes expression in sheep, avoiding any damage caused by mammary gland biopsy.     

Once-a-day milking of Holstein and Montbéliarde cows for 7 weeks in mid-lactation

This study aimed at comparing the effects of once-a-day (OAD) milking during the descending phase of lactation between cows from the two most common breeds in France (Holstein and Montbéliarde). This study was carried on during two successive summers on a total of 50 Holstein and 38 Montbéliarde cows. During 7 weeks, half of the cows from each breed was milked OAD while the other half was milked twice a day (TAD). The animals were also followed for the next 3 or 5 weeks when they were all milked TAD, to check for any residual effect of OAD milking. The behaviour of OAD cows was observed around milking time. The incidence of diseases, the main performance variables (milk production, milk flow rate, live weight and body condition score), the detailed composition of milk (fat, protein, lactose, somatic cells, minerals, pH, free fatty acid (FFA), nitrogen fractions and enzymes) and some technological variables (clotting time and curd firmness) were measured on all cows.Some signs of disturbance were observed in the OAD cows at the time when milking was omitted: some cows mooed, some went close to the exit of the paddock, some leaked milk prior to milking. However, these signs disappeared after 2 days. After the experimental period, the live weight and the body condition score of TAD and OAD cows did not differ significantly. OAD cows produced 4.5 kg/day less than TAD (P < 0.001), this being more marked in Holstein (5.7 kg/day, P < 0.001) than Montbéliarde (3.3 kg/day, P < 0.001) cows. The milk contents of fat, whey protein, casein, total protein and phosphorus, and its plasminogen activity, were higher with OAD cows while lactose and FFA contents, and lipoprotein lipase activity were lower, with no interaction observed with breed. During the subsequent 3 weeks, when all cows were again milked TAD, OAD cows still produced 1.7 kg/day less milk (P < 0.01) with slightly higher fat and protein content.OAD milking for 7 weeks during the descending phase of lactation decreased milk production but increased milk content of most components, with a low residual effect. Montbéliarde cows were less affected by OAD milking than Holstein cows.     

Confirmation and refinement of a QTL on BTA5 affecting milk production traits in the Fleckvieh dual purpose cattle breed

We analysed a QTL affecting milk yield (MY), milk protein yield (PY) and milk fat yield (FY) in the dual purpose cattle breed Fleckvieh on BTA5. Twenty-six microsatellite markers covering 135 cM were selected to analyse nine half-sib families containing 605 sons in a granddaughter design. We thereby assigned two new markers to the public linkage map using the CRI-MAP program. Phenotypic records were daughter yield deviations (DYD) originating from the routinely performed genetic evaluations of breeding animals. To determine the position of the QTL, three different approaches were applied: interval mapping (IM), linkage analysis by variance component analysis (LAVC), and combined linkage disequilibrium (LD) and linkage (LDL) analysis. All three methods mapped the QTL in the same marker interval ( BM2830-ETH152 ) with the greatest test-statistic value at 118, 119.33 and 119.33 cM respectively. The positive QTL allele simultaneously increases DYD in the first lactation by 272 kg milk, 7.1 kg milk protein and 7.0 kg milk fat. Although the mapping accuracy and the significance of a QTL effect increased from IM over LAVC to LDL, the confidence interval was large (13, 20 and 24 cM for FY, MY and PY respectively) for the positional cloning of the causal gene. The estimated averages of pair wise marker LD with a distance <5 cM were low (0.107) and reflect the large effective population size of the Fleckvieh subpopulation analysed. This low level of LD suggests a need for increase in marker density in following fine mapping steps.     

Alterations in the milk yield and composition during different stages of lactation cycle in elite and non-elite Karan-Fries cross-bred cows (Holstein Fresian x Tharparkar)

Alterations in the milk constituents throughout the lactation cycle have been studied in 20 Karan-Fries cross-bred cows (Holstein Fresian x Tharparkar) divided into two groups, viz. elite (n = 10) and non-elite (n = 10). About 100 ml of composite milk samples (from all the quarters) were collected through hand milking in sterile tubes from the day of calving at 15-day interval till 300 days of lactation cycle. Different milk constituents (viz. fat, protein, lactose, Solid not fat) were estimated by automatic milk analyzer. The daily milk yield varied significantly (p < 0.01) between group and different days of lactation cycle. Milk fat percentage of milk did not differ significantly between groups and different days of lactation cycle. Milk protein and lactose percentage did not differ significantly between groups but differed significantly (p < 0.001) between days. Solid not fat (SNF) percentage of milk was significantly (p < 0.01) higher in elite cows compared to non-elite cows and also varied significantly (p < 0.05) during different days of lactation cycle. In both the group of cows, milk protein, fat, and SNF percentage was highest and lactose percentage was lowest up to 1st week of lactation cycle and rapidly declined thereafter due to the transition of the colostrum into milk. After 2nd week of lactation, all the milk constituents under study were almost remained unaltered till the end of the cycle. The above stated investigation not only substantiates the already existing information of lactation stage-specific alteration in milk constituents but also depict the exact point of transition of these constituents during the lactation cycle so the nutritional and managemental interventions could be carried out in proper time.     

Modeling homeorhetic trajectories of milk component yields,body composition and dry-matter intake in dairy cows: Influence of parity,milk production potential and breed

The control of nutrient partitioning is complex and affected by many factors, among them physiological state and production potential. Therefore, the current model aims to provide for dairy cows a dynamic framework to predict a consistent set of reference performance patterns (milk component yields, body composition change, dry-matter intake) sensitive to physiological status across a range of milk production potentials (within and between breeds). Flows and partition of net energy toward maintenance, growth, gestation, body reserves and milk components are described in the model. The structure of the model is characterized by two sub-models, a regulating sub-model of homeorhetic control which sets dynamic partitioning rules along the lactation, and an operating sub-model that translates this into animal performance. The regulating sub-model describes lactation as the result of three driving forces: (1) use of previously acquired resources through mobilization, (2) acquisition of new resources with a priority of partition towards milk and (3) subsequent use of resources towards body reserves gain. The dynamics of these three driving forces were adjusted separately for fat (milk and body), protein (milk and body) and lactose (milk). Milk yield is predicted from lactose and protein yields with an empirical equation developed from literature data. The model predicts desired dry-matter intake as an outcome of net energy requirements for a given dietary net energy content. The parameters controlling milk component yields and body composition changes were calibrated using two data sets in which the diet was the same for all animals. Weekly data from Holstein dairy cows was used to calibrate the model within-breed across milk production potentials. A second data set was used to evaluate the model and to calibrate it for breed differences (Holstein, Danish Red and Jersey) on the mobilization/reconstitution of body composition and on the yield of individual milk components. These calibrations showed that the model framework was able to adequately simulate milk yield, milk component yields, body composition changes and dry-matter intake throughout lactation for primiparous and multiparous cows differing in their production level.     

Differential expression of genes in milk of dairy cattle during lactation

The milk fat globule (MFG) is one of the most representative of mammary gland tissues and can be utilized to study gene expression of lactating cows during lactation. In this study, RNA‐seq technology was employed to detect differential expression of genes in MFGs at day 10 and day 70 after calving between two groups of cows with extremely high (H group) and low (L group) 305‐day milk yield, milk fat yield and milk protein yield. In total, 1232, 81, 429 and 178 significantly differentially expressed genes (false discovery rate q < 0.05) were detected between H10 and L10, H70 and L70, H10 and H70, and L10 and L70 respectively. Gene Ontology enrichment and pathway analysis revealed that these differentially expressed genes were enriched in biological processes involved in mammary gland development, protein and lipid metabolism process, signal transduction, cellular process, differentiation and immune function. Among these differentially expressed genes, 178 (H10 vs. L10), 4 (H70 vs. L70), 68 (H10 vs. H70) and 22 (L10 vs. L70) were found to be located within previously reported QTL regions for milk production traits. Based on these results, some promising candidate genes for milk production traits in dairy cattle were suggested.     

Association of the <Emphasis Type="Italic">DGAT1</Emphasis> gene polymorphism in bulls with cow milk performance

The substitution of lysine for alanine (K232A) in the acyl-CoA:diacylglycerol acyltransferase, which is encoded by the DGAT1 gene, was tested for the significance for breeding evaluation of bulls of the holsteinized Black-and-White breed. The breeding value was estimated by the DYDC (daughter yield deviation to contemporaries) method with modification. The frequency of allele 232K in the bulls examined was 0.28, lower than in Holstein bulls (0.4–0.6). The greatest effect of the A232K substitution was observed for the percent fat milk yield (1.4σ) and milk yield (0.76σ), and a lower effect was established for the milk protein yield (0.47σ) and percent protein milk yield (0.44σ). In the case of milk fat yield, the effect was nonsignificant. A method was proposed for converting the data on fat yield in order to obtain significant results in this case as well. The effect of the A232K substitution was estimated at 154 kg for milk yield, 2.8 kg for milk protein yield, 0.079% for percent milk fat, and 0.015% for percent milk protein yield. The results are discussed in the context of multiple pleiotropic effects of the K232A substitution in the DGAT1 gene. It is proposed that the K232A substitution of the DGAT1 gene may be used as a golden standard in comparisons of the effect on milk production traits for the total gene set. This approach will allow a meta-analysis of the gene effects in spite of the different dairy cattle breeds and methods used to analyze their breeding value. In view of more than 30-year experience of using sperm of Holstein bulls, including those breeds in North America, it was noted that the effect of the A232K substitution on milk production traits agreed well with the data reported for the North American commercial population of Holstein cattle.     

Lactation influences the serum level of leptin and growth hormone during the daily bathyphase in ewes

The aim of the study was to investigate the influence of early lactation on leptin and growth hormone (GH) during bathyphase. Forty lactating Sarda ewes were divided into two equal groups on the basis of their milk production levels: HIGH (>1100 g/day) and LOW (<900 g/day). From the 5th to the 110th day after lambing, every 15 days, body condition score (BCS) was recorded and milk samples were collected. At the same data point, blood sampling was performed and leptin, GH and, Non-Esterified Fatty Acids (NEFA) were assessed. On milk, fat and proteins were determined. Statistical differences were observed in BCS, leptin, GH, NEFA and fat concentration in milk between the two groups. BCS was lower in high group and decreased from the 20th to the 90th day of lactation. Leptin was higher in low group and increased from the 50th and the 65th day of lactation, in low and high groups, respectively. GH and NEFA were higher in high group and decreased from the mild lactation. In high group, BCS and milk yield were negatively correlated and leptin was negatively correlated with GH and NEFA. In low group, leptin was positively correlated with BCS and negatively correlated with the all studied parameters. GH and NEFA were positively correlated with both groups. In conclusion, milk yield had an effect on the leptin and GH plasma values recorded during their bathyphase.