Udder Health Status of First Parity Dairy Cows in Early-Lactation Based on Somatic Cell Count Categories

The main aim of this study was to investigate the prevalence of intramammary infection (IMI) in early-lactation of primiparous cows using milk recording cow composite somatic cell count (CSCC) categories (combining the first 2 milk recording results after calving). Another aim was to evaluate the milk urea (MU) content as a potential supplementary indicator to SCC or CSCC for the identification of IMI in primiparous cows after calving. This retrospective observational study was conducted on records of test-day of primiparous cows over a period of 6 years (January 2016 to December 2021. The SCC data for 158 Holstein Friesian primiparous cows, with their first milk recording 5 to 35 days after calving and their second milk recording 28 to 56 days in milk (DIM), were identified. Each primiparous cow was assigned a CSCC category (low-low, low-high, high-low or high-high) based on the CSCC at the first 2 milking recordings using the following cut-offs: ≤150,000 cells/ml (low), >150,000 cells/ml (high). The association between CSCC categories and MV content was analyzed using correlation models. At the first milk recording, a proportion of 63.29% was in the low SCC category, and the rest (36.71%) was in the high SCC category. At the second milk recording, a proportion of primiparous cows in CSCC categories was 59.49%, 3.80%, 27.85% and 8.86% in low-low, low-high, high-low and high-high, respectively. At the second milk recording, a proportion of 12.66% of primiparous cows was in the high CSCC category and a proportion of 87.34% of primiparous cows was in the low CSCC category, indicating a poor and a good udder health, respectively. The association of SCC with MU content in low and in high SCC categories at the first milk recording was positive and moderate (+0.49) and negative and strong (-0.97), respectively. The association of CSCC categories with MU contents at the second milk recording was inconclusive. We concluded that CSCC categories may be a useful tool for identifying success and problems regarding the udder health of primiparous cows in early lactation.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned,ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.

Transcriptome‑wide mapping of milk somatic cells upon subclinical mastitis infection in dairy cattle

Background Subclinical intramammary infection(IMI)represents a significant problem in maintaining dairy cows’health.Disease severity and extent depend on the interaction between the causative agent,environment,and host.To investigate the molecular mechanisms behind the host immune response,we used RNA-Seq for the milk somatic cells(SC)transcriptome profiling in healthy cows(n=9),and cows naturally affected by subclinical IMI from Proto-theca spp.(n=11)and Streptococcus agalactiae(S.agalactiae;n=11).Data Integration Analysis for Biomarker discov-ery using Latent Components(DIABLO)was used to integrate transcriptomic data and host phenotypic traits related to milk composition,SC composition,and udder health to identify hub variables for subclinical IMI detection.Results A total of 1,682 and 2,427 differentially expressed genes(DEGs)were identified when comparing Prototheca spp.and S.agalactiae to healthy animals,respectively.Pathogen-specific pathway analyses evidenced that Proto-theca’s infection upregulated antigen processing and lymphocyte proliferation pathways while S.agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle,and carbohydrate and lipid metabolism.The integrative analysis of commonly shared DEGs between the two pathogens(n=681)referred to the core-mastitis response genes,and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells(r2=0.72),followed by the udder health(r2=0.64)and milk quality parameters(r2=0.64).Variables with r≥0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cyto-hubba plug-in.The genes in common between DIABLO and cytohubba(n=10)were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals(sensitivity>0.89,specificity>0.81,accuracy>0.87,and precision>0.69).Among these genes,CIITA could play a key role in regulating the animals’response to subclinical IMI.Conclusions Despite some differences in the enriched pathways,the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response.The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.

Relationship of Somatic Cell Count with Milk Yield and Composition in Chinese Holstein Population

The objective of this study was to analyze the relationship of somatic cell count （SCC） with milk yield, fat and protein percentage, fat and protein yield using analysis of variance and correlation analysis in Chinese Holstein population. The 10 524 test-day records of 568 Chinese Holstein Cattle were obtained from 2 commercial herds in Xi＇an region of China during February 2002 to March 2009. Milk yield, fat percentage, fat and protein yield initially increased and then dropped down with parity, whereas protein percentage decreased and SCC increased. Analysis of variance showed highly significant effects of different subclasses SCC on milk yield and composition （P〈 0.01）. Compared with milk yield with SCC ≤ 200 000 cells mL-1, milk yield losses with SCC of 200 000-500 000 cells mL-1, 501000-1 000 000 cells mL-1, ≥ 1 000 000 cells mL-1 were 0.387, 0.961 and 2.351 kg, respectively. The highly significant negative correlation coefficient between somatic cell score （SCS） and milk and protein yield, milk yield and fat and protein percentage, protein percentage and fat yield were -0.084, -0.037, -0.061, -0.168, and -0.088, respectively （P〈 0.01）. The highly significant positive correlation coefficients between SCS and fat yield and fat and protein percentage, milk yield and fat and protein yield, fat percentage and protein percentage and fat yield, protein yield and protein percentage and fat yield were 0.041, 0.177, 0.105, 0.771, 0.865, 0.122, 0.568, 0.318, and 0.695, respectively （P〈 0.01）. There was no significant relationship between fat percentage and protein yield （P 〉 0.05）. The results of the present study first time provide the relevant base-line data for assessing milk production at Xi＇an region of China.

PiggyBac Transposon Mediated Efficient eGFP Expression in Porcine Somatic Cells and Cloned Embryos

PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein （eGFP） and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer （SCNT）, the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.

Genetic parameters for somatic cell score and production traits in the fi rst three lactations of Chinese Holstein cows

The objectives of this study were to estimate genetic parameters of lactation average somatic cell scores （LSCS） and examine genetic associations between LSCS and production traits in the first three lactations of Chinese Holstein cows using single-parity multi-trait animal model and multi-trait repeatability animal model. There were totally 273605 lactation records of Chinese Holstein cows with first calving from 2001 to 2012. Heritability estimates for LSCS ranged from 0.144 to 0.187. Genetic correlations between LSCS and 305 days milk, protein percentage and fat percentage were -0.079, -0.082 and -0.135, respectively. Phenotypic correlation between LSCS and 305 days milk yield was negative （-0.103 to -0.190）. Genetic correlation between 305 days milk and fat percentage or protein percentage was highly negative. Genetic correlation between milk fat percentage and milk protein percentage was highly favorable. Heritabilities of production traits decreased with increase of parity, whereas heritability of LSCS increased with increase of parity.

Mapping of Microsatellite SW943 to Porcine Chromosome 12p11-(2/3p13) Using Primed in situ Synthesis and Somatic Cell Hybrid Panel

The porcine microsatellite SW943 was regionally localized on 12p11-(2/3p13) by the two methods: the Primed in situ (PRINS) labelling on the pachytene bivalents of pigs using the Dig-11-dUTP as the report molecule and pig X rodent Somatic Cell Hybrid Panel (SCHP) which contains 27 cell lines through PCR amplification. Advantages and disadvantages of the two methods for physical mapping of microsatellites were also discussed.

Somatic Cell Count in Relation to Udder and Morphometry in Holstein Friesian Dairy Cows

In the present study authors present some aspects regarding udder health as well as the udder teat morphometry.The udder health was defined on the basis of somatic cell count(SCC)in milk.The morphometry parameters included:distance from rear teat-tip to the floor,distance from fore teat-tip to the floor,udder depth,udder length,udder width,udder volume,fore teat length,rear teat length,fore teat diameter,and rear teat diameter.The present investigation involved 92 Holstein Friesian cows,from an experimental herd.Considering particular structural elements of a cow udder,three location groups were distinguished:1st,2nd,and 3rd(L_(1),L_(2) and L_(3) respectively).From the study result,primiparous cows(L_(1) group)were characterized by a relatively low SCC in milk,as compared to multiparous cows(L_(2) group or L_(3) group).The cows in the 3rd lactation in relation to the first lactation cows,head SCC increased over 3 times,which indicates the increase of the chance of the cows to be susceptible to intramammary infections.All data showed a gradual increase in depth length,width and volume of the udder as the number of parity increases.The differences observed in udder morphometry in different parities were found significant(p<0.05)to highly significant(p<0.01).The values of the coefficients of correlation for the analyzed udder and teat conformation traits and SCC in milk,were statistically varied and ranged from-0.32 to+0.28.Accordingly,the udder and teat morphometry characteristics such as distance from rear are fore teat-tip to the floor,udder depth,volume,teat diameter,can be said to have some degree of association with the udder health in Holstein Friesian cows evaluated in this study.Hence their inclusion in breeding program as indicator traits may help reduce the incidence of intramammary infections.

Impact of Bacterial and Somatic Cells Content on Quality Fresh Milk in Small-Scale Dairy Farms in Kosovo

The basic goal of this research was to determine the impact of the presence of bacterial (CFU) and somatic cells count content (SCC) in quality of fresh milk in some small cattle farms in Kosovo. The survey was based on existing standards for milk quality in Kosovo placed under administrative guidance MA-no. 20/2006. The study was based on fresh milk analysis of 150 farms performed during the period September-December 2012, which was obtained in 9 different localities (collection points) of the Kosovo. Our study reveals that CFU and SCC in fresh milk were significantly affected by a number of factors, as: sampling period (repetition), locality, breed, and time of sampling (evening or/and morning). According to the results for CFU and SCC, there were big differences between the farms (milk collection points) included in the study (P < 0.0403) and (P < 0.0293). The results show that small size breed like Busha and its crosses tend to be less exposed to SCC/mL in milk (72.840) and (293.592), compared to Black Holstein (613.462), Simmental (521.519) and Brown Swiss (418.44). Milk produced in evening tended to be of better quality (259.854 CFU/mL) compared to the one from morning milking (576.689 CFU/mL). Fresh milk quality analyzed in the third repetition was better for about 33.3% compared with the repletion first. For CFU and SCC, the analyses show that about 74.7% and 64.7% of milk produced belongs to extra quality, while lower quality of milk of category three is 12.0% and 23.3%, respectively. Considering that about 85% of milk produced in Kosovo comes from small-scale dairy farms, the current study sets out that small-scale milk production system cannot be neglected by interest parties in dairy sector and needs permanent following up and improvement.

Effect of Bacterial Content and Somatic Cell Count on Sheep Milk Quality in Kosovo

The main objective of this study was to analyze the effect of bacterial content (CFU) and somatic cell count (SCC) in milk quality in sheep (Ovis aries) farms according to the existing standards for fresh milk quality in Kosovo. A total of 2256 milk test day records from 379 Bardhoka (BAR), Sharri sheep (SHA), Kosovo sheep (KOS) and Balusha (BAL) ewes in eight herds across the country were collected and analyzed through a period April-October 2010. The general linear model and Duncan’s test were used to analyze the effect of different variables on presence of CFU and SCC in fresh milk. The effect of all variables was considered as a fixed. The overall results show that farm (P 0422), respectively, had a significant effect on presence of SCC. For CFU farm (P 0422) and breed (P 1211.17 to 6425.76 CFU/mL and 846.07 to 2043.15 SCC/mL milk, respectively. Although the rate of fresh milk contamination with SCC tends to be relatively low compare with CFU, by all means both variables should not be underestimated bearing in mind that a high rate of them on fresh milk are negatively correlated with farmer’s profit, consumer food safety and overall animal health.

China Succeeded in Somatic Cell Cloning

Chinese scientists have succeeded in cloning a colony of cattle from fully differentiated somatic cells. The news was announced jointly by the Chinese Academy of Sciences (CAS), National Natural Science Foundation of China (NSFC) and the government of Shandong Province at a press conference held on March 7, 2002.……

Single‑cell sequencing reveals the reproductive variations between primiparous and multiparous Hu ewes

Background In the modern sheep production systems,the reproductive performance of ewes determines the economic profitability of farming.Revealing the genetic mechanisms underlying differences in the litter size is important for the selection and breeding of highly prolific ewes.Hu sheep,a high-quality Chinese sheep breed,is known for its high fecundity and is often used as a model to study prolificacy traits.In the current study,animals were divided into two groups according to their delivery rates in three consecutive lambing seasons(namely,the high and low reproductive groups with≥3 lambs and one lamb per season,n=3,respectively).The ewes were slaughtered within 12 h of estrus,and unilateral ovarian tissues were collected and analyzed by 10×Genomics single-cell RNA sequencing.Results A total of 5 types of somatic cells were identified and corresponding expression profiles were mapped in the ovaries of each group.Noticeably,the differences in the ovary somatic cell expression profiles between the high and low reproductive groups were mainly clustered in the granulosa cells.Furthermore,four granulosa cell subtypes were identified.GeneSwitches analysis revealed that the abundance of JPH1 expression and the reduction of LOC101112291 expression could lead to different evolutionary directions of the granulosa cells.Additionally,the expression levels of FTH1 and FTL in mural granulosa cells of the highly reproductive group were significantly higher.These genes inhibit necroptosis and ferroptosis of mural granulosa cells,which helps prevent follicular atresia.Conclusions This study provides insights into the molecular mechanisms underlying the high fecundity of Hu sheep.The differences in gene expression profiles,particularly in the granulosa cells,suggest that these cells play a critical role in female prolificacy.The findings also highlight the importance of genes such as JPH1,LOC101112291,FTH1,and FTL in regulating granulosa cell function and follicular development.

Single-cell RNA-seq analysis of testicular somatic cell development in pigs

Spermatogenesis is the process by which diploid male germ cells propagate and differentiate into haploid flagellated spermatozoa.This highly complicated process is dependent on testicular somatic cells maturation.While the role of these somatic cells in spermatogenesis is relatively well established,knowledge about their development and maturation,particularly at the molecular level,is limited.In this study,we profiled the testicular single-cell transcriptomes of Guanzhong black pigs at the age of 7,30,60,90,and 150 days.Five types of Sertoli cells,five types of Leydig cells,and four types of peritubular myoid cells were identified.Histological analysis revealed the changes in proliferation levels and marker gene expressions,and the prion-like protein gene(PRND)was identified as a novel marker for Sertoli cells.Additionally,integrated analyses of porcine and human datasets revealed similarities between human and pig testicular somatic cells.Overall,the data obtained in this study contribute to the understanding of testicular development in pigs as a model species.

Generation of cloned calves from different types of somatic cells

Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nu-clear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated. (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respec-tively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively. (ii) Development rates to the blastocyst stage for SCNT embryos from diffetent individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P <0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively. (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively, P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9 % and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. For in vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%A, 29.4%B, 37.9%A and 41.5%C, respectively (PABC<0.05); for in vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respec-tively.

Exploring the developmental potential of adult mammalian somatic cells

In the traditional views on developmental biology, the process of a mammal from a zygote to. an adult individual follows continuous changes of space and time environments and is the result of different expressions of target genes. It has long been known that this process is irreversible and the terminal differentiated adult cells, such as cardiac myocytes and neurons, will not divide and differentiate. But recent reports on the two hottest fields - cloning medicine and stem cell biology doubted these concepts. This may lead to a further understanding of the potentiality of mammal development and may provide great chances for commercial and clinical practice.

Shared Gene Regulation during Human Somatic Cell Reprogramming

Human induced pluripotent stem （iPS） cells have the ability to differentiate into all somatic cells and to maintain unlimited self- renewal. Therefore, they have great potential in both basic research and clinical therapy for many diseases. To identify potentially universal mechanisms of human somatic cell reprogramming, we studied gene expression changes in three types of cells undergoing reprogramming. The set of 570 genes commonly regulated during induction of iPS cells includes known embryonic stem （ES） cell markers and pluripotency related genes. We also identified novel genes and biological categories which may be related to somatic cell reprogramming. For example, some of the down-regulated genes are predicted targets of the pluripotency microRNA cluster miR302/367, and the proteins from these putative target genes interact with the stem cell pluripotency factor POU5F1 according to our network analysis. Our results identified candidate gene sets to guide research on the mechanisms operating during somatic cell reprogramming.

Effect of Rare Earth Elements on the Induction Frequency of the Somatic Cell Embryo in The Fruit of Chinese Wolfoerry

A part of lanthanides could raise the induction fraquency(IF) of the somatic cell embryo(SCE) in the fruit of Chinese wolfbeny. The effect of thorium and yttrium used for this purpose is not obvious and even plays a role of inhibition when they are used with a concentration of more than 4 ppm.When the combinations of different rare earth elements (RE) are used, the diversity of the effects amongthem is large. Some of them help to raise the iF of the SCE while the others inhibit the generation of SCE.The mischmetal results in the best effect, giving a relative IF of 295.4%.

Generation of ApoE deficient dogs via combination of embryo injection of CRISPR/Cas9 with somatic cell nuclear transfer

Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors （WHO）. Athero- sclerosis is a chronic inflammatory disease characterized by accumulation of lipids in the arterial wall （Gofman and Lindgren, 1950）. Tremendous clinical and experimental efforts have been made to reveal the pathogenesis of the disease. Nevertheless, the mechanism of atherosclerosis is still unclear. A suitable animal model to study metabolic disorders and subsequent atherosclerosis is a necessity. The traditional method by feeding high fat diet to establish animal models of atherosclerosis disease is time- consuming and laborious, and in many circumstances, the pheno- types are not consistent among the individual models.

Reprogramming somatic cells to cells with neuronal characteristics by defined medium both in vitro and in vivo

Background:Currently,direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds.Delivery of transcriptional factors may affect genome stability,while small-molecule compounds may require more evaluations when applied in vivo.Thus,a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation.Results:Here,we report that a defined medium(5C)consisting of basic fibroblast growth factor(bFGF),N2 supplement,leukemia inhibitory factor,vitamin C(Vc),andβ-mercaptoethanol(βMe)induces the direct conversion of somatic cells to cells with neuronal characteristics.Application of 5C medium converted mouse embryonic fibroblasts(MEFs)into TuJ+neuronal-like cells,which were capable of survival after being transplanted into the mouse brain.The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury,possibly by inducing similar conversions,when infused into the mouse brain in vivo.Crucially,5C medium could also induce neuronal characteristics in several human cell types.Conclusions:In summary,this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.

Oocyte-associated transcription factors in reprogramming after somatic cell nuclear transfer:a review

Oocytes are unique cells with the inherent capability to reprogram nuclei.The reprogramming of the somatic nucleus from its original cellular state to a totipotent state is essential for term development after somatic cell nuclear transfer.The nuclear-associated factors contained within oocytes are critical for normal fertilization by sperm or for somatic cell nuclear reprogramming.The chromatin of somatic nuclei can be reprogrammed by factors in the egg cytoplasm whose natural function is to reprogram sperm chromatin.The oocyte first obtains its reprogramming capability in the early fetal follicle,and then its capacity is enriched in the late growth phase and reaches its highest capability for reprogramming as fully-grown germinal vesicle oocytes.The cytoplasmic milieu most likely contains all of the specific transcription and/or reprogramming factors necessary for cellular reprogramming.Certain transcription factors in the cytoplast may be critical as has been demonstrated for induced pluripotent stem cells.The maternal pronucleus exerts a predominant,transcriptiondependent effect on embryo cytofragmentation,with a lesser effect imposed by the ooplasm and the paternal pronucleus.With deep analysis of transcriptomics in oocytes and early developmental stage embryos more maternal transcription factors inducing cellular reprogramming will be identified.