Protein in culture and endogenous lipid interact with embryonic stages in vitro to alter calf birthweight after embryo vitrification and warming.

Short-term protein removal in vitro improves long-term blastocyst competence to survive vitrification. We investigated the mechanisms and effects underlying protein removal. Day-6 morulae and early blastocysts were cultured individually with and without protein for 24h. Development and lipid content were analysed in expanded blastocysts derived from morulae (M-XB) and from early blastocysts (EB-XB). Expression of genes involved in lipid metabolism, stress responses and apoptosis was analysed in fresh and vitrified-warmed M-XB produced with and without protein. Pregnancy rates, birth rates and birthweight (BW) were recorded after transfer of embryos. Day-7 EB-XB production rates (with, 66.9±6.2 and without, 68.8±6.0 protein) were higher than M-XB rates (with, 21.4±4.6 and without, 9.4±4.6 protein; P<0.005). EB-XB showed fewer lipids than M-XB (P=0.03). In fresh M-XB, expression of sterol regulatory element binding protein (SREBP1) was lower with (4.1±2.2) than without (13.6±2.2) protein, contrary to results obtained for Patatin-like phospholipase domain containing 2, Hormone-sensitive lipase and Bcl-2-associated X protein (P<0.05). Protein did not affect pregnancy rates and birth phenotypes (P>0.05). However, BW was higher (P<0.01) in calves born from vitrified M-XB (48.6±3.4kg) than from EB-XB (39.8±2.9kg). Such effects were more pronounced in females (P<0.001). Calves from fresh embryos did not show BW differences. These results indicate that embryonic kinetics and vitrification impact birth phenotypes, at least in females. Alterations might involve exogenous protein and mobilisation of lipid stocks.

Differences in genetic structure assessed using Y-chromosome and mitochondrial DNA markers do not shape the contributions to diversity in African sires.

Up to 173 African sires belonging to 11 different subpopulations representative of four cattle groups were analysed for six Y-specific microsatellite loci and a mitochondrial DNA fragment. Differences in Y-chromosome and mtDNA haplotype structuring were assessed. In addition, the effect of such structuring on contributions to total genetic diversity was assessed. Thirty-five Y-chromosome and 71 mtDNA haplotypes were identified. Most Y-chromosomes analysed (73.4%) were of zebu origin (11 haplotypes). Twenty-two Y-haplotypes (44 samples) belonged to the African taurine subfamily Y2a. All mtDNA haplotypes belonged to the "African" taurine T1 haplogroup with 16 samples and nine haplotypes belonging to a recently identified subhaplogroup (T1e). Median-joining networks showed that Y-chromosome phylogenies were highly reticulated with clear separation between zebu and taurine clusters. Mitochondrial haplotypes showed a clear star-like shape with small number of mutations separating haplotypes. Mitochondrial-based FST -statistics computed between cattle groups tended to be statistically non-significant (p > .05). Most FST values computed among groups and subpopulations using Y-chromosome markers were statistically significant. AMOVA confirmed that divergence between cattle groups was only significant for Y-chromosome markers (ΦCT  = 0.209). At the mitochondrial level, African sires resembled an undifferentiated population with individuals explaining 94.3% of the total variance. Whatever the markers considered, the highest contributions to total Nei's gene diversity and allelic richness were found in West African cattle. Genetic structuring had no effect on patterns of contributions to diversity.

Lack of haplotype structuring for two candidate genes for trypanotolerance in cattle.

Bovine trypanotolerance is a heritable trait associated to the ability of the individuals to control parasitaemia and anaemia. The INHBA (BTA4) and TICAM1 (BTA7) genes are strong candidates for trypanotolerance-related traits. The coding sequence of both genes (3951 bp in total) were analysed in a panel including 79 Asian, African and European cattle (Bos taurus and B. indicus) to identify naturally occurring polymorphisms on both genes. In general, the genetic diversity was low. Nineteen of the 33 mutations identified were found just one time. Seventeen different haplotypes were defined for the TICAM1 gene, and 9 and 12 were defined for the exon 1 and the exon 2 of the INHBA gene, respectively. There was no clear separation between cattle groups. The most frequent haplotypes identified in West African taurine samples were also identified in other cattle groups including Asian zebu and European cattle. Phylogenetic trees and principal component analysis confirmed that divergence among the cattle groups analysed was poor, particularly for the INHBA sequences. The European cattle subset had the lowest values of haplotype diversity for both the exon1 (monomorphic) and the exon2 (0.077 ± 0.066) of the INHBA gene. Neutrality tests, in general, did not suggest that the analysed genes were under positive selection. The assessed scenario would be consistent with the identification of recent mutations in evolutionary terms.

Genetic relationships among American donkey populations: insights into the process of colonization.

This study presents the first insights into the genetic diversity and structure of the American donkey metapopulation. The primary objectives were to detect the main structural features underlying variability among American donkey populations, identify boundaries between differentiated gene pools, and draw the main colonization pathways since the introduction of donkeys into America in the 15th century. A panel of 14 microsatellite markers was applied for genotyping 350 American donkeys from 13 countries. The genetic structure of this metapopulation was analysed using descriptive statistics and Bayesian model-based methods. These populations were then compared to a database containing information on 476 individuals from 11 European breeds to identify the most likely ancestral donor populations. Results showed the presence of two distinct genetic pools, with confluence of the two in Colombia. The southern pool showed a unique genetic signature subsequent to an older founder event, but lacked any significant influence of modern gene flow from Europe. The northern pool, conversely, may have retained more ancestral polymorphisms and/or have experienced modern gene flow from Spanish breeds. The Andalusian and, to a lesser extent, the Catalan breeds have left a more pronounced footprint in some of the American donkey populations analysed.

Assessing introgression of Sahelian zebu genes into native Bos taurus breeds in Burkina Faso.

A total of 350 samples were analyzed to estimate zebu gene proportions into two different taurine cattle breeds of Burkina Faso (Lobi and N'Dama) using 38 microsatellites and various statistical methodologies. West African and East African zebu samples were sequentially used as reference parental populations. Furthermore, N'Dama cattle from Congo, the composite South African Bonsmara cattle breed and a pool of European cattle were used successively as second parental populations. Independently of the methodology applied: (a) the use of West African zebu samples gave higher admixture coefficients than the East African zebu; (b) the higher zebu proportions were estimated when the European cattle was used as parental population 2; and (c) the use of the N'Dama population from Congo as parental population 2 gave the more consistent zebu proportion estimates for both the Lobi and the N'Dama breeds. In any case, the zebu admixture proportions estimated were not negligible and were always higher in the N'Dama cattle than in the Lobi cattle of Burkina Faso. This suggested that the introgression of Sahelian zebu genes into the taurine cattle of Southern West Africa can follow a complex pattern that can depend on local agro-ecological features. The current research pointed out that the estimation of admixture coefficients is highly dependent on both the assumptions underlying the methodologies applied and the selection of parental populations. Our analyses suggest that either too high or nil genetic identity between the parental and the expectedly derived populations must be avoided.

Elements of functional genital asymmetry in the cow.

Asymmetry in the cow affects ovarian function and pregnancy. In this work we studied ovarian and uterine asymmetry. Synchronised animals, in which in vitro-produced embryos (n=30-60) had been transferred on Day 5 to the uterine horn ipsilateral to the corpus luteum (CL), were flushed on Day 8. Ovulatory follicle diameter, oestrus response and total protein flushed did not differ between sides. However, a corpus luteum in the right ovary led to plasma progesterone concentrations that were higher than when it was present in the left ovary. Fewer embryos were recovered from the left than the right horn. Among 60 uterine proteins identified by difference gel electrophoresis, relative abundance of nine (acyl-CoA dehydrogenase, very long chain; twinfilin, actin-binding protein, homologue 1; enolase 1; pyruvate kinase isozymes M1/M2 (rabbit); complement factor B Bb fragment ; albumin; fibrinogen gamma-B chain; and ezrin differed (P<0.05) between horns. Glucose concentration was higher, and fructose concentration lower, in the left horn. In a subsequent field trial, pregnancy rates after embryo transfer did not differ between horns (51.0±3.6, right vs 53.2±4.7, left). However, Day 7 blood progesterone concentrations differed (P=0.018) between pregnant and open animals in the left (15.9±1.7 vs 8.3±1.2) but not in the right horn (12.4±1.3 vs 12.4±1.2). Progesterone effects were independent of CL quality (P=0.55). Bilateral genital tract asymmetry in the cow affects progesterone, proteins and hexoses without altering pregnancy rates.

Lack of mitochondrial DNA structure in Balkan donkey is consistent with a quick spread of the species after domestication.

A total of 132 mtDNA sequences from 10 Balkan donkey populations were analysed to ascertain their regional genetic structure and to contribute to the knowledge of the spreading of the species after domestication. The Balkan donkey sequences were compared with those from 40 Burkina Faso donkeys as an African outgroup to account for possible local Balkan scenarios. The 172 sequences gave 62 different haplotypes (55 in Balkan donkey). Virtually all the analysed populations had haplotypes assigned to either Clade 1 or Clade 2 even though the relative proportion of Clade 1 or 2 haplotypes differed across populations. Geographical maps constructed using factors computed via principal component analysis showed that the Balkan donkey populations are not spatially structured. AMOVA confirmed a lack of genetic structure in Balkan donkey mtDNA. Balkan populations were poorly differentiated (ΦST  = 0.071). Differentiation between the Balkan donkey and the African outgroup also was low. The lack of correspondence between geographical areas and maternal genetic structure is consistent with the hypothesis suggesting a very quick spread of the species after domestication. The current research illustrates the difficulties to trace routes of expansion in donkey, as the species has no geographical structure.

Mitochondrial analysis sheds light on the origin of hair sheep.

A total of 180 mtDNA sequences from hair Caribbean (93), West African (73) and Canarian-wooled (14) sheep were analysed to shed light on the origin of hair sheep. A comparison of 360 Iberian sheep sequences retrieved from GenBank was performed to assess a possible European origin of the Caribbean hair sheep. These 180 sequences gave 48 different haplotypes (16 in Caribbean sheep). All Caribbean and Canarian-wooled sequences and 91.8% of the West African samples belonged to haplogroup B. The sheep analysed showed wide haplotypic identity. Caribbean sheep shared roughly two-thirds of their samples with Canarian-wooled and West African samples, respectively. Principal component analysis showed that the Caribbean and the Canarian-wooled sheep clustered together. Additional analyses showed that hair and Iberian sheep had wide genetic identity. It was not possible to ascertain a single Canarian, African or European origin of the Caribbean hair sheep using mtDNA markers only. European, African and Caribbean hair sheep maternal genetic backgrounds likely result from related domestication events.

Mitochondrial DNA and Y-chromosome diversity in East Adriatic sheep.

Variation in mitochondrial DNA (mtDNA) and Y-chromosome haplotypes was analysed in nine domestic sheep breeds (159 rams) and 21 mouflon (Ovis musimon) sampled in the East Adriatic. Mitochondrial DNA analyses revealed a high frequency of type B haplotypes, predominantly in European breeds, and a very low frequency of type A haplotypes, which are more frequent in some Asian breeds. Mitochondrial haplotype Hmt-3 was the most frequent (26.4%), and 37.1%, 20.8% and 7.6% of rams had haplotypes one, two and three mutations remote from Hmt-3 respectively. In contrast, Y-chromosome analyses revealed extraordinary paternal allelic richness: HY-6, 89.3%; HY-8, 5.0%; HY-18, 3.1%; HY-7, 1.3%; and HY-5, 1.3%. In fact, the number of haplotypes observed is comparable to the number found in Turkish breeds and greater than the number found in European breeds so far. Haplotype HY-18 (A-oY1/135-SRYM18), identified here for the first time, provides a link between the haplotype HY-12 (A-oY1/139-SRYM18) found in a few rams in Turkey and haplotype HY-9 (A-oY1/131-SRYM18) found in one ram in Ethiopia. All mouflons had type B mtDNA haplotypes, including the private haplotype (Hmt-55), and all were paternally monomorphic for haplotype HY-6. Our data support a quite homogeneous maternal origin of East Adriatic sheep, which is a characteristic of European breeds. At the same time, the high number of haplotypes found was surprising and intriguing, and it begs for further analysis. Simultaneous analysis of mtDNA and Y-chromosome information allowed us to detect a large discrepancy between maternal and paternal lineages in some populations. This is most likely the result of breeder efforts to 'upgrade' local populations using rams with different paternal origins.

Founder and present maternal diversity in two endangered Spanish horse breeds assessed via pedigree and mitochondrial DNA information.

Pedigree information and 179 mtDNA sequences from two endangered Spanish horse breeds, the Asturcón pony (143) and the Mallorquí horse (36), were analysed to asses: (i) the pedigree and molecular maternal genetic diversity of the two breeds; (ii) the concordance between the dam lines recorded in the corresponding studbooks and the mtDNA haplotypes identified; and (iii) to assess the losses of maternal genetic variability occurred from the foundation of the studbooks to present. Up to 50 Asturcón and 18 Mallorquí founder dam lines were identified in the studbooks analysed. Up to 315 Asturcón mares and 51 Mallorquí mares that foaled in the last 5 years of recording formed a reference population. Only 35 Asturcón and 13 Mallorquí founder dam lines were represented in their reference populations. Sequences from a total of 38 Asturcón and 12 Mallorquí dam lines could be obtained. The 179 sequences obtained gave 15 different haplotypes, 11 and 9 of them being identified, respectively, in the Asturcón pony and in the Mallorquí horse. Five different haplotypes (roughly two-thirds of the sequences) were shared by the two horse breeds. Most dam lines analysed had a single mtDNA haplotype. However, more than one haplotype was detected within eight of the dam lines in Asturcón pony. The found inconsistencies are likely to result from deficiencies in genebank management. The maternal N(e) (mN(e)) computed using the dam line information was higher in the Asturcón pony (20.5) than in the Mallorquí horse (15.9), while these figures were on the opposite direction for the haplotypic line information (6.4 and 9.4, respectively). The ratio of the computed mN(e) values to the actual number of founder dam lines were always higher in the Mallorquí horse probably due to a more balanced distribution of individuals kept for reproduction among studs. Consequences for the conservation programmes of the analysed breeds are discussed.

Estimation of effective population size from the rate of coancestry in pedigreed populations.

We introduce a simple method to estimate effective population size from increase in coancestry (Δc(jk)) for all pairs of individuals j and k in a reference subpopulation. An increase in pairwise coancestry for any pair of individuals j and k can be defined assuming that a hypothetical mating between them would give an individual with an inbreeding coefficient equal to c(jk), where c(jk) is the coancestry coefficient between the individuals j and k. The equivalent measure to discrete generations value (g(jk)) corresponding to the individual jk can be computed by averaging discrete equivalents generations of its parents (g(j) and g(k)). The mean increase in coancestry for all pairs of individuals in a reference subpopulation can be used to estimate a realized effective population size based on coancestries that would provide information on the effective size of a population under random mating. Performance of the new parameter was tested on simulated and empirical (horse) populations with different mating strategies and population structures. The routines needed to compute the introduced parameters have been included in a new version of the program ENDOG.

Multiple paternal origins of domestic cattle revealed by Y-specific interspersed multilocus microsatellites.

In this study, we show how Y-specific interspersed multilocus microsatellites, which are loci that yield several amplified bands differing in size from the same male individual and PCR reaction, are a powerful source of information for tracing the history of cattle. Our results confirm the existence of three main groups of sires, which are separated by evolutionary time and clearly predate domestication. These three groups are consistent with the haplogroups previously identified by Götherström et al. (2005) using five Y-specific segregating sites: Y1 and Y2 in taurine (Bos taurus) cattle and Y3 in zebu (Bos indicus) cattle. The zebu cattle cluster clearly originates from a domestication process that was geographically and temporally separated from that of taurine clusters. Our analyses further suggest that: (i) introgression of wild sire genetic material into domesticated herds may have a significant role in the formation of modern cattle, including the formation of the Y1 haplogroup; (ii) a putative domestication event in Africa probably included local Y2-like wild sires; (iii) the West African zebu cattle Y-chromosome may have partially originated from an ancient introgression of humped cattle into Africa; and (iv) the high genetic similarity among Asian zebu sires is consistent with a single domestication process.

Y-specific microsatellites reveal an African subfamily in taurine (Bos taurus) cattle.

Five cattle Y-specific microsatellites, totalling six loci, were selected from a set of 44 markers and genotyped on 608 Bos taurus males belonging to 45 cattle populations from Europe and Africa. A total of 38 haplotypes were identified. Haplogroups (Y1 and Y2) previously defined using single nucleotide polymorphisms did not share haplotypes. Nine of the 27 Y2-haplotypes were only present in African cattle. Network and correspondence analyses showed that this African-specific subfamily clustered separately from the main Y2-subfamily and the Y1 haplotypes. Within-breed genetic variability was generally low, with most breeds (78%) showing haplotypes belonging to a single haplogroup. AMOVA analysis showed that partitioning of genetic variation among breeds can be mainly explained by their geographical and haplogroup assignment. Between-breed genetic variability summarized via Principal Component Analysis allowed the identification of three principal components explaining 94.2% of the available information. Projection of principal components on geographical maps illustrated that cattle populations located in mainland Europe, the three European Peninsulas and Mediterranean Africa presented similar genetic variation, whereas those breeds from Atlantic Europe and British Islands (mainly carrying Y1 haplotypes) and those from Sub-Saharan Africa (belonging to Y2-haplogroup) showed genetic variation of a different origin. Our study confirmed the existence of two large Y-chromosome lineages (Y1 and Y2) in taurine cattle. However, Y-specific microsatellites increased analytical resolution and allowed at least two different Y2-haplotypic subfamilies to be distinguished, one of them restricted to the African continent.

Metabolomic Profiling of Bos taurus Beef, Dairy, and Crossbred Cattle: A Between-Breeds Meta-Analysis.

Cattle breeds may differ substantially in their metabolism. However, the metabolomes of dairy and beef cattle are not well-known. Knowledge of breed-specific metabolic features is essential for biomarker identification and to adopt specific nutritional strategies. The muscle hypertrophy (mh), a beef cattle phenotype present in Asturiana de los Valles (AV) but absent in Asturiana de la Montaña (AM) and Holsteins, may underlie such differences. We compared the plasma metabolomes of Holstein, AV, AM, and crossbred cattle recipients selected for meta-analysis within an embryo transfer (ET) program. Blood samples were collected on day 0 (oestrus) and day 7 (prior to ET) (N = 234 samples × 2 days). Nuclear magnetic resonance quantified N = 36 metabolites in plasma, and more metabolic differences between breeds were found on day 0 (N = 19 regulated metabolites) than on day 7 (N = 5). AV and AM largely differed from Holstein cattle (N = 55 and 35 enriched metabolic pathways, respectively); however, AV and AM differed in N = 6 enriched pathways. Metabolic activity was higher in AV than in Holstein cattle, as explained in part by the mh phenotype. The metabolomic characterization of breeds facilitates biomarker research and helps to define the healthy ranges of metabolite concentrations.

Female segregation patterns of the putative Y-chromosome-specific microsatellite markers INRA124 and INRA126 do not support their use for cattle population studies.

Here we tested the segregation and paternal compatibility of markers INRA124 and INRA126 on female DNA in 10 different cattle families, in order to clarify the usefulness of these microsatellites for the study of male-mediated population processes in cattle. Their performance was compared with that of four microsatellites located in the PAR-BTAY (UMN0108, UMN0803, UMN0929 and UMN0905) and another one male-specific microsatellite (INRA189). INRA124 and INRA126 amplified the same sized fragment in both sexes. Same size alleles were sequenced and the high homology found allowed us to rule out non-specific female amplification. INRA124 showed full parental compatibility, whilst the locus INRA126 showed 55% parental incompatibility. Based on these observations, it is recommended that markers INRA124 and INRA126 should not be used in studies to characterize male-mediated genetic events in cattle.

Analysis of mitochondrial DNA diversity in Burkina Faso populations confirms the maternal genetic homogeneity of the West African goat.

To date, no comprehensive study has been performed on mitochondrial genetic diversity of the West African goat. Here, we analysed a 481-bp fragment of the HVI region of 111 goats representing four native West African populations, namely the three main Burkina Faso breeds, zoo-farm kept Dwarf goats and endangered Spanish goat breeds used as the outgroup. Analyses gave 83 different haplotypes with 102 variable sites. Most haplotypes (65) were unique. Only three haplotypes were shared between populations. Haplotypes were assigned to cluster A except for H45 (belonging to the Spanish Bermeya goat) which was assigned to cluster C. amova analysis showed that divergence between groups (Phi(CT)) was not statistically significant regardless of whether the partition in two hierarchical levels that was fitted included Spanish samples or not. The West African goat scenario shown here is consistent with that previously reported for the species: haplogroup A is predominant and has a very high haplotype diversity regardless of the geographic area or sampled breed. The large phenotypic differences observable between the West African Dwarf and Sahelian long-legged goat populations are not detectable with mitochondrial markers. Moreover, a previously suggested introgression of Sahelian goat southwards because of desertification could not be assessed using mtDNA information.

Microsatellite analysis characterizes Burkina Faso as a genetic contact zone between Sahelian and Djallonke sheep.

A total of 123 sheep belonging to the Djallonke, Mossi, and Burkina-Sahel breeds, along with 41 Spanish Xalda sheep were genotyped for 27 microsatellites. The pair Djallonke-Mossi had the highest between breeds molecular coancestry. Admixture analysis informed on the parental role of the Burkina-Sahel and Djallonke breeds. The Mossi breed was a hybrid population nearer to the Djallonke breed. Only half of the Mossi individuals were correctly assigned to their breed. The Burkina-Sahel and Djallonke breeds can be considered ancestrally different genetic entities. Differentiation between the Djallonke and Mossi breeds may be due to introgression of Sahelian sheep.

Improving the estimation of realized effective population sizes in farm animals.

Computation of inbreeding rate (DeltaF) must consider that inbreeding is delayed with one generation with respect to the idealized population when addressed using individual inbreeding coefficients. The expression relating inbreeding in generation t with inbreeding rate F(t) = 1 - (1-DeltaF)(t) should be more correctly written in real animal populations as F(t) = 1 - (1-DeltaF)(t-1), as changes in allele frequencies occur in the equivalent co-ancestries in the previous generation. This simple approach is tested on simulated and real pedigrees thus demonstrating that: (i) the adjusted individual increase in inbreeding becomes stable in populations under random mating while the unadjusted parameter does not; (ii) regression of the unadjusted parameter over generations in pedigrees under random mating is highly significant while after correction it is not significant; and (iii) the variance of the adjusted parameter is reduced with the generations.

Genealogical analyses in open populations: the case of three Arab-derived Spanish horse breeds.

This research assesses the genetic composition of three Arab-derived Spanish horse breeds as an example to highlight the major shortcomings related to genealogical analyses in open populations and to propose approaches useful to deal with this task. The studbooks of three Spanish Arab (SA)-derived horse breeds, Spanish Anglo-Arab (dAA), Hispano-Arab (dHA) and Spanish Sport Horse (dSSH) and those of their parental breeds SA, Spanish Purebred (SPB) and Thoroughbred (TB), totalling 211 754 individuals, were available. The genealogies of the dAA, dHA and dSSH were analysed not only using the corresponding studbook (breed exclusive dataset) but also including the genealogies of the founders from parental breeds (completed dataset). Coancestry analyses revealed that the present SA-derived populations share more genes with the Arab than with the other parental breeds. Effective population size was computed by accounting for migration rates to obtain an equivalent closed-population effective size ((eq)N(e)) of 39.2 for the dAA, 56.3 for dHA and 114.1 for dSSH. The essayed methodologies were useful for characterising populations involving migration. The consequences of the management of the analysed breeds are discussed. The results emphasize the need to include the complete genealogies of the individuals to attain reliable genealogical parameters.