Relations Between Udder Infection and Somatic Cells in Camel (Camelus dromedarius) Milk

Quarter milk samples (n = 391) from 101 camels were examined to study the occurrence and causes of mastitis in traditionally managed camels in eastern Sudan and to evaluate the value of the California Mastitis Test (CMT), somatic cell count (SCC) and adenosine triphosphate (ATP) in the detection of subclinical mastitis in the camel. One hundred and seventy (43.5%) of the quarter milk samples yielded pathogenic bacteria. Streptococcus agalactiae, other Streptococcus spp., Staphylococcus aureus, coag–ulase–negative staphylococci, and Escherichia coli were isolated from milk. Thirty–two (8.2%) quarter milk samples yielded mixed cultures, and 189 (48.3%) yielded no growth. Mean values for CMT, SCC and ATP were higher for quarters infected with major pathogens. However, a significant number of quarter milk samples had elevated values in these tests but were from quarters from which no bacteria were isolated. The ability of the tests to predict a positive bacteriology increased slightly when 2 or 3 tests were combined. kw|Keywords|k]inflammation; k]diagnostic tests; k]Mastitis; k]CMT; k]ATP; k]bacteriology; k]Sudan     

Genetic evaluation of mastitis liability and recovery through longitudinal analysis of transition probabilities

Background

Many methods for the genetic analysis of mastitis use a cross-sectional approach, which omits information on, e.g., repeated mastitis cases during lactation, somatic cell count fluctuations, and recovery process. Acknowledging the dynamic behavior of mastitis during lactation and taking into account that there is more than one binary response variable to consider, can enhance the genetic evaluation of mastitis.

Methods

Genetic evaluation of mastitis was carried out by modeling the dynamic nature of somatic cell count (SCC) within the lactation. The SCC patterns were captured by modeling transition probabilities between assumed states of mastitis and non-mastitis. A widely dispersed SCC pattern generates high transition probabilities between states and vice versa. This method can model transitions to and from states of infection simultaneously, i.e. both the mastitis liability and the recovery process are considered. A multilevel discrete time survival model was applied to estimate breeding values on simulated data with different dataset sizes, mastitis frequencies, and genetic correlations.

Results

Correlations between estimated and simulated breeding values showed that the estimated accuracies for mastitis liability were similar to those from previously tested methods that used data of confirmed mastitis cases, while our results were based on SCC as an indicator of mastitis. In addition, unlike the other methods, our method also generates breeding values for the recovery process.

Conclusions

The developed method provides an effective tool for the genetic evaluation of mastitis when considering the whole disease course and will contribute to improving the genetic evaluation of udder health.     

Polymorphisms of the Toll-Like Receptor 2 of Goats (Capra hircus) may be Associated with Somatic Cell Count in Milk

Toll-like receptor 2 (TLR2) plays an important role in recognition by the innate immune system of Gram-positive bacteria. As Gram-positive bacteria cause mastitis, we examined variations in the region of the TLR2 gene that codes for the extracellular domain. Samples of forty goats from a single dairy herd were collected, half with low SCC (≤200,000 cells/mL), and half with higher SCC. Two synonymous single nucleotide polymorphisms (SNPs) were identified: 840G?>?A and 1083A?>?G. One nonsynonymous SNP 739G?>?A was identified. This coded for valine or isoleucine, which have similar physiochemical properties, and was not in a region coding for a known functional domain. Surprisingly, the least square mean SCC of the heterozygous goats (146,220) was significantly lower than the SCC of homozygous GG goats (537,700; p?=?0.004), although these two groups were similar in days in milk (p?=?0.984), and there was no significant difference by breed (p?=?0.941). Because factors other than mastitis can affect SCC and our sample sizes were limited, additional studies are needed to corroborate an association between TLR2 genotype and SCC or mastitis in goats.     

Relationships among somatic cell count, California mastitis test, impedance and bacteriological status of milk in goats and sheep in early lactation

The objectives of this trial were to evaluate and compare the test characteristics of a number of indirect tests of bacteriological status of the milk from goats and sheep and to assess the affect of varying levels of prevalence of infection on the performance of those tests.The somatic cell count, California mastitis test (CMT) score, electrical impedance and the bacteriological status of 220 and 262 milk samples from the glands of lactating dairy goats and sheep, respectively, were determined. The sensitivity and specificity of indirect tests in predicting the bacteriological status were compared by analyzing the areas under the receiver operating characteristic curve and calculating the likelihood ratio at various cut-off values.Bacteria were isolated from 17.7 and 4.6% of glands from goats and sheep, respectively. Somatic cell count was a better predictor of bacteriological status than either the CMT score or impedance in both goats and sheep. However, knowledge of the CMT score in both sheep and goats and the impedance in goats increases the likelihood of predicting the presence of a bacterial pathogen compared to no testing at all (P<0.05). Knowledge of the age and days postpartum of the animals at the time of testing did not increase the probability that infection status could be predicted. Variation in cut-off levels to predict infection status reported in the literature may be due to variation in the prevalence of infection in the populations studied. Use of likelihood ratio allows comparison of tests over a range of prevalence’s as likelihood ratios are independent of prevalence. The prevalence of infection within a herd should be considered when selecting a cut-off value for indirect testing for the presence of bacteria in milk of small ruminants.     

Prevalence of bovine subclinical mastitis and isolation of its major causes in Bishoftu Town,Ethiopia

Objective

A cross-sectional study was conducted from November 2015 to March 2016 to estimate the prevalence, to assess the risk factors and to isolate the major etiological agent of subclinical mastitis in Bishoftu town. The study was conducted on 262 cross breed lactating cows selected from 12 intensively managed dairy farms. California mastitis test (CMT) and bacteriological culture methods were used as diagnostic tools.

Result

From 262 cows examined, 105 (40.1%) and from 1048 quarters examined, 170 (16.1%) were positive for sub-clinical mastitis using CMT. All CMT positive samples were cultured for etiological agent identification. From 170 samples cultured, 153 were positive for known subclinical mastitis pathogens. The dominant bacteria isolated were Staphylococcus species from these Staphylococcus aureus (44.9%) was the major one followed by Streptococcus spp. (25.3%) and other gram negative enteric bacteria, Escherichia coli (8.8%). Age, body condition score, milk yield, and number of parity were considered as potential risk factors; among these, age and number of parity have statistically significance association with the occurrence of subclinical mastitis (P < 0.05) both in the CMT and the bacteriological tests.

     

Is there a special mechanism behind the changes in somatic cell and polymorphonuclear leukocyte counts,and composition of milk after a single prolonged milking interval in cows?

Background  

A single prolonged milking interval (PMI) e.g. after a technical stop in an automated milking system is of concern for the producer since it is associated with a short-lasting increase in milk somatic cell count (SCC), which is a major quality criterion used at the dairy plants. The content of polymorphonuclear leukocytes (PMN) and how the milk quality is influenced has not been much investigated. The SCC peak occurs without any obvious antigen challenge, possibly indicating a different leukocyte attraction mechanism after a PMI than we see during mastitis.     

Mastitis and related management factors in certified organic dairy herds in Sweden

Background  

Mastitis is one of the major threats to animal health, in organic farming as well as conventional. Preliminary studies of organic dairy herds have indicated better udder health in such herds, as compared to conventional herds. The aim of this paper was to further study mastitis and management related factors in certified organic dairy herds.     

Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10³ and 95.4×10³ CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10⁵ and 622.2×10⁵ cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.     

Structural equation models to estimate risk of infection and tolerance to bovine mastitis

Background

One method to improve durably animal welfare is to select, as reproducers, animals with the highest ability to resist or tolerate infection. To do so, it is necessary to distinguish direct and indirect mechanisms of resistance and tolerance because selection on these traits is believed to have different epidemiological and evolutionary consequences.

Methods

We propose structural equation models with latent variables (1) to quantify the latent risk of infection and to identify, among the many potential mediators of infection, the few ones that influence it significantly and (2) to estimate direct and indirect levels of tolerance of animals infected naturally with pathogens. We applied the method to two surveys of bovine mastitis in the Walloon region of Belgium, in which we recorded herd management practices, mastitis frequency, and results of bacteriological analyses of milk samples.

Results and discussion

Structural equation models suggested that, among more than 35 surveyed herd characteristics, only nine (age, addition of urea in the rations, treatment of subclinical mastitis, presence of dirty liner, cows with hyperkeratotic teats, machine stripping, pre- and post-milking teat disinfection, and housing of milking cows in cubicles) were directly and significantly related to a latent measure of bovine mastitis, and that treatment of subclinical mastitis was involved in the pathway between post-milking teat disinfection and latent mastitis. These models also allowed the separation of direct and indirect effects of bacterial infection on milk productivity. Results suggested that infected cows were tolerant but not resistant to mastitis pathogens.

Conclusions

We revealed the advantages of structural equation models, compared to classical models, for dissecting measurements of resistance and tolerance to infectious diseases, here bovine mastitis. Using our method, we identified nine major risk factors that were directly associated with an increased risk of mastitis and suggested that cows were tolerant but not resistant to mastitis. Selection should aim at improved resistance to infection by mastitis pathogens, although further investigations are needed due to the limitations of the data used in this study.     

Seasonal ovulatory activity exists in tropical Creole female goats and Black Belly ewes subjected to a temperate photoperiod

Background  

Seasonality of ovulatory activity is observed in European sheep and goat breeds, whereas tropical breeds show almost continuous ovulatory activity. It is not known if these tropical breeds are sensitive or not to temperate photoperiod. This study was therefore designed to determine whether tropical Creole goats and Black-Belly ewes are sensitive to temperate photoperiod. Two groups of adult females in each species, either progeny or directly born from imported embryos, were used and maintained in light-proof rooms under simulated temperate (8 to 16 h of light per day) or tropical (11 – 13 h) photoperiods. Ovulatory activity was determined by blood progesterone assays for more than two years. The experiment lasted 33 months in goats and 25 months in ewes.     

Higher somatic cells counted by the electronic counter method do not influence renneting properties of goat milk

Laws of different countries regarding SCC of goat milk are not in agreement with each other and sometimes they fix a threshold for the enhancement of dairy products. The aim of this study was to assess if renneting properties of goat milk are influenced by higher somatic cell count (SCC) measured by an electronic cell counter. Milk samples, taken throughout the lactation of 169 goats from three farms, were analyzed for chemical, physical, hygienic and renneting properties. Samples were divided into three levels on the basis of their SCC: L: low level, samples with SCC lower than 10⁶ cells/ml; M: medium, between 10⁶ and 2 × 10⁶ cells/ml; H: high, higher than 2 × 10⁶ cells/ml. Milk clotting time was between 12.07 and 13.31 min, curd firming time between 1.68 and 2.05 min and curd firmness between 41.66 and 48.97 mm. All the three renneting properties were not affected by the SCC level but they were highly correlated with other factors as protein content and pH. Furthermore, the microbial count showed a high positive correlation with SCC. These results showed that in goat milk, contrarily to other dairy species, higher SCC did not affect renneting properties and that counting of somatic cells by using electronic cell counters might be not suitable for the improvement of dairy products.     

Reference genome of wild goat (capra aegagrus) and sequencing of goat breeds provide insight into genic basis of goat domestication

Background

Domestic goats (Capra hircus) have been selected to play an essential role in agricultural production systems, since being domesticated from their wild progenitor, bezoar (Capra aegagrus). A detailed understanding of the genetic consequences imparted by the domestication process remains a key goal of evolutionary genomics.

Results

We constructed the reference genome of bezoar and sequenced representative breeds of domestic goats to search for genomic changes that likely have accompanied goat domestication and breed formation. Thirteen copy number variation genes associated with coat color were identified in domestic goats, among which ASIP gene duplication contributes to the generation of light coat-color phenotype in domestic goats. Analysis of rapidly evolving genes identified genic changes underlying behavior-related traits, immune response and production-related traits.

Conclusion

Based on the comparison studies of copy number variation genes and rapidly evolving genes between wild and domestic goat, our findings and methodology shed light on the genetic mechanism of animal domestication and will facilitate future goat breeding.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1606-1) contains supplementary material, which is available to authorized users.     

New mastitis phenotypes suitable for genomic selection in meat sheep and their genetic relationships with udder conformation and lamb live weights

Mastitis can prove expensive in sheep reared for meat production due to costs associated with treatment methods, poor lamb growth and premature culling of ewes. The most commonly used method to detect mastitis, in dairy systems, is somatic cell counts. However, in many meat-producing sheep flocks ewes are not routinely handled, thus regular milk sampling is not always possible. It is, therefore, worthwhile to investigate alternative phenotypes, such as those associated with udder conformation and methods of evaluating somatic cell counts in the milk, such as the California Mastitis Test. The main objectives of this study were therefore: (a) to estimate genetic parameters of traits relating to mastitis and udder conformation in a meat sheep breed; (b) estimate the level of association between somatic cell counts and the California Mastitis Test and (c) assess the relationships between mastitis and both udder conformation and lamb live weights. Data were collected from Texel ewes based on 29 flocks, throughout the UK, during 2015 and 2016. The ewes were scored twice each year, at mid- and late-lactation. Eight different conformation traits, relating to udder and teat characteristics, and milk samples were recorded. The data set comprised of data available for 2957 ewes. The pedigree file used contained sire and dam information for 31 775 individuals. The animal models used fitted relevant fixed and random effects. Heritability estimates for traits relating to mastitis (somatic cell score and the California Mastitis Test), ranged from 0.08 to 0.11 and 0.07 to 0.11, respectively. High genetic correlations were observed between somatic cell score and the California Mastitis Test (0.76 to 0.98), indicating the California Mastitis Test to be worthwhile for assessing infection levels, particularly at mid-lactation. The strongest correlations observed between the mastitis traits and the udder conformation traits were associated with udder depth (0.61 to 0.75) also at mid-lactation. Negative phenotypic correlations were estimated between mastitis and the weight of lamb reared by the ewe (−0.15 to −0.23), suggesting that lamb weights fell as infection levels rose. Genetic correlations were not significantly different from zero. Reducing mastitis will lead to improvements in flock productivity and the health and welfare of the animals. It will also improve the efficiency of production and the resilience to disease challenge. The economic benefits, therefore, of these results combined could be substantial not only in this breed but also in the overall meat sheep industry.     

Genetic Structure of Chinese Indigenous Goats and the Special Geographical Structure in the Southwest China as a Geographic Barrier Driving the Fragmentation of a Large Population

Background

China has numerous native domestic goat breeds, however, extensive studies are focused on the genetic diversity within the fewer breeds and limited regions, the population demograogic history and origin of Chinese goats are still unclear. The roles of geographical structure have not been analyzed in Chinese goat domestic process. In this study, the genetic relationships of Chinese indigenous goat populations were evaluated using 30 microsatellite markers.

Methodology/Principal Findings

Forty Chinese indigenous populations containing 2078 goats were sampled from different geographic regions of China. Moderate genetic diversity at the population level (H_S of 0.644) and high population diversity at the species level (H_T value of 0.737) were estimated. Significant moderate population differentiation was detected (F_ST value of 0.129). Significant excess homozygosity (F_IS of 0.105) and recent population bottlenecks were detected in thirty-six populations. Neighbour-joining tree, principal components analysis and Bayesian clusters all revealed that Chinese goat populations could be subdivided into at least four genetic clusters: Southwest China, South China, Northwest China and East China. It was observed that the genetic diversity of Northern China goats was highest among these clusters. The results here suggested that the goat populations in Southwest China might be the earliest domestic goats in China.

Conclusions/Significance

Our results suggested that the current genetic structure of Chinese goats were resulted from the special geographical structure, especially in the Western China, and the Western goat populations had been separated by the geographic structure (Hengduan Mountains and Qinling Mountains-Huaihe River Line) into two clusters: the Southwest and Northwest. It also indicated that the current genetic structure was caused by the geographical origin mainly, in close accordance with the human’s migration history throughout China. This study provides a fundamental genetic profile for the conservation of these populations and better to understand the domestication process and origin of Chinese goats.     

Identification and antibiogram of microbes associated with bovine mastitis

An investigation of Mastitis in cattle was carried out in Anand city and in nearby villages of Gujarat state using California Mastitis Test (CMT) kit. The prevalence of clinical and subclinical mastitis was found to be 5.5% and 15.75%, respectively. Staphylococcus aureus was identified through strain specific polymerase chain reaction; the remaining isolates identified on the basis of molecular analysis by 16S rDNA sequencing and phylogenetic analysis were Staphylococcus species, B. pumilus, Staphylococcus chromogenes, Bacillus species, and Pseudomonas species. In vitro antimicrobial susceptibility pattern of all the isolates was checked against 13 different antibiotics using the agar disc diffusion method. Highest bacterial resistance was observed with penicillin G and oxacillin antibiotics. It was also observed that the patterns of bacterial resistance have not changed in India over the years. The data supports the decrease in the incidence of mastitis but the rate of decrease is minimal. More effective control strategies are required.     

Application of optical coherence tomography enhances reproducibility of arthroscopic evaluation of equine joints

Background

Staphylococcus aureus is an important cause of clinical mastitis in dairy cows worldwide. The cure rate after antimicrobial treatment of clinical S. aureus mastitis is very variable due to both cow and bacterial factors. Studies have shown that bacterial genotype might affect short-term bacteriological and clinical cure, but the long-term outcome has been less studied. The objectives of this study were to investigate associations between bacterial genotype and long-term outcome of veterinary-treated clinical mastitis (VTCM) caused by S. aureus during a follow-up period of 120 days and to study genotype variation among Swedish S. aureus isolates. S. aureus isolates from cases of VTCM were genotyped by pulsed-field gel electrophoresis. Long-term outcome measurements used were somatic cell count (SCC), additional diagnoses of VTCM, milk yield and culling. Isolates were classified into clusters (>80% similarity) and pulsotypes (100% similarity). Clusters and pulsotypes were grouped according to occurrence. Multivariable mixed-effect linear regression models including cow and bacterial factors with possible influence on SCC or milk yield were used to calculate differences in SCC or milk yield between groups. Additional outcome measures were calculated using a test of proportions.

Results

The isolates (n?=?185) were divided into 18 clusters and 29 pulsotypes. Two pulsotypes were classified as common, and were found in 64% of the cases of VTCM. Remaining isolates were classified as less common or rare pulsotypes. The distribution was similar at cluster level. Outcome was calculated from follow-up data on 111 cows. Significantly lower SCC during the follow-up period was found in cows infected with common clusters compared to in cows infected with less common/rare clusters. The proportion of cows with SCC <200 000 cells/ml during the whole follow-up period was significantly higher in the group common clusters than in the group less common/rare clusters. Bacterial genotype did not influence the other outcome parameters.

Conclusions

In Sweden, two S. aureus pulsotypes, identified in about 64% of clinical S. aureus cases, were widespread. Cows infected with the common genotypes had significantly lower SCC during 120 days after treatment compared to cows infected with less common or rare genotypes.     

Mapping of the transcription start site (TSS) and identification of SNPs in the bovine neuropeptide Y (NPY) gene

Background

The domestic goat is one of the important livestock species of India. In the present study we assess genetic diversity of Indian goats using 17 microsatellite markers. Breeds were sampled from their natural habitat, covering different agroclimatic zones.

Results

The mean number of alleles per locus (NA) ranged from 8.1 in Barbari to 9.7 in Jakhrana goats. The mean expected heterozygosity (He) ranged from 0.739 in Barbari to 0.783 in Jakhrana goats. Deviations from Hardy-Weinberg Equilibrium (HWE) were statistically significant (P < 0.05) for 5 loci breed combinations. The D_A measure of genetic distance between pairs of breeds indicated that the lowest distance was between Marwari and Sirohi (0.135). The highest distance was between Pashmina and Black Bengal. An analysis of molecular variance indicated that 6.59% of variance exists among the Indian goat breeds. Both a phylogenetic tree and Principal Component Analysis showed the distribution of breeds in two major clusters with respect to their geographic distribution.

Conclusion

Our study concludes that Indian goat populations can be classified into distinct genetic groups or breeds based on the microsatellites as well as mtDNA information.     

Diagnosis of caseous lymphadenitis by ELISA in naturally infected goats from Venezuela

Due to the absence of previous reports, the goal of this work was to detect caseous lymphadenitis (CLA) in goat flocks from Venezuela using an indirect immunoenzymatic assay (ELISA). Eighteen farms were randomly selected in Falcon State, North-Western Venezuela. Blood samples were taken from 259 goats, 65 of them with abscesses. Experimental inoculations were made to healthy kids with 0.5 mL inocula containing 4.7 × 10⁵ of Corynebacterium pseudotuberculosis to observe the kinetics of antibody response. Immunoenzymatic assays were carried out using exotoxin of C. pseudotuberculosis as antigen. Antibody response in experimentally inoculated animals was detected 2 weeks after infection. Of 259 field goat sera, 55.98% were positive by ELISA. Of 65 goats with abscesses, 67.69% had CLA demonstrated by bacteriological methods; from these, 72.73% showed antibodies by ELISA. Of the remaining goats negative to CLA, 47.62% had antibodies by ELISA. Sensitivity was calculated in 72.73% and specificity in 67.74%. The immunoenzymatic assay applied in this research could be useful to detect CLA in naturally infected goat flocks from Venezuela.     

Mediation analysis to estimate direct and indirect milk losses associated with bacterial load in bovine subclinical mammary infections

Milk losses associated with mastitis can be attributed to either effects of pathogens per se (i.e. direct losses) or to effects of the immune response triggered by the presence of mammary pathogens (i.e. indirect losses). Test-day milk somatic cell counts (SCC) and number of bacterial colony forming units (CFU) found in milk samples are putative measures of the level of immune response and of the bacterial load, respectively. Mediation models, in which one independent variable affects a second variable which, in turn, affects a third one, are conceivable models to estimate direct and indirect losses. Here, we evaluated the feasibility of a mediation model in which test-day SCC and milk were regressed toward bacterial CFU measured at three selected sampling dates, 1 week apart. We applied this method on cows free of clinical signs and with records on up to 3 test-days before and after the date of the first bacteriological samples. Most bacteriological cultures were negative (52.38%), others contained either staphylococci (23.08%), streptococci (9.16%), mixed bacteria (8.79%) or were contaminated (6.59%). Only losses mediated by an increase in SCC were significantly different from null. In cows with three consecutive bacteriological positive results, we estimated a decreased milk yield of 0.28 kg per day for each unit increase in log₂-transformed CFU that elicited one unit increase in log₂-transformed SCC. In cows with one or two bacteriological positive results, indirect milk loss was not significantly different from null although test-day milk decreased by 0.74 kg per day for each unit increase of log₂-transformed SCC. These results highlight the importance of milk losses that are mediated by an increase in SCC during mammary infection and the feasibility of decomposing total milk loss into its direct and indirect components.     

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

Background

Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism.

Methodology/Principal Findings

Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats.

Conclusions/Significance

In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future.