This research communication describes the application of a fluorescent automatic cell counter Lactoscan SCC for simultaneous determination of somatic cell count and neutrophils in bovine milk. The obtained results were compared with results obtained by a flow cytometer and a light microscope. The Pearson correlations between the methods were calculated. A comparison between the main characteristics of the three kinds of analysis was made – the assay duration and the intra-assay precision. A relation between the SCC and neutrophil cells was observed in 55 milk samples. The obtained results confirm that the simultaneous determination of SCC and neutrophil analysis are necessary and support the early diagnosis of mastitis, the timely treatment of the animal and the avoidance of major economic losses.

Intraspecific and interspecific cloning via somatic cell nuclear transfer (iSCNT) is a biotechnique with great possibilities for wild mammals because it allows the maintenance of biodiversity by recovering species, nuclear reprogramming for the production of pluripotency-induced cells, and studies related to embryonic development. Nevertheless, many areas in cloning, especially those associated with wild mammals, are still in question because of the difficulty in obtaining cytoplasmic donor cells (or cytoplasts). Conversely, donor cell nuclei (or karyoplasts) are widely obtained from the skin of living or post-mortem individuals and often maintained in somatic cell banks. Moreover, the creation of karyoplast–cytoplast complexes by fusion followed by activation and embryo development is one of the most difficult steps that requires further clarification to avoid genetic failures. Although difficult, cloning different species, such as wild carnivores and ungulates, can be successful via iSCNT with embryo development and the birth of offspring. Thus, novel research in the area that contributes to the conservation of biodiversity and knowledge of the physiology of species continues. The present review presents the failures and successes that occurred with the application of the technique in wild mammals, with the goal of helping future work on cloning via iSCNT.

Buffalo milk production has become of significant importance on the world scale, however, there are few studies involving biotechnological tools specifically for buffalo. To verify the effects caused by subclinical mastitis on the components of milk and to study the innate immune system in the udder of dairy buffaloes with subclinical mastitis, we evaluated the levels of expression of the lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and toll-like receptors 2 (TLR-2) and 4 (TLR-4) genes in buffaloes with and without subclinical mastitis. Milk samples were collected for the determination of milk components: somatic cell score (SCS), fat, protein, lactose, total solids and solids-not-fat (SNF), as well as for RNA extraction of milk cells, complementary DNA synthesis, and expression profile quantification by quantitative real-time PCR. For gene expression, the ΔΔCt was estimated using contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both groups. Linear regression analysis was performed to determine the relationship between the genes studied and the milk components. Subclinical mastitis induced changes in the fat, lactose and SNF in milk of buffaloes, and the messenger RNA abundance was upregulated for TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes in milk cells of buffaloes with subclinical mastitis, whereas the LTF gene was not differentially expressed. Results of linear regression analysis showed that TLR-2 gene expression most explains the variation in SCS, and the change in a unit of ΔCt of the TNF-α gene would result in a higher increase in SCS. The study of these immune function genes that are active in the mammary gland is important to characterize the action mechanism of the innate immunity that occurs in subclinical mastitis in dairy buffaloes and may aid the development of strategies to preserve the health of the udder.

Somatic cell nuclear-transferred (SCNT) oocytes have a high potential for development in vitro, but a large proportion of embryos that are transferred to recipients is aborted before parturition. The precise mechanism for the high abortion rate is unknown, but abnormal placenta formation is frequently observed in SCNT-cloned pregnancies. The present study examined the effects of treating the recipients with cyclosporin A (CsA), an immunoprotectant, on the proportion of fetuses resulting from SCNT-cloned pregnancies. Cloned embryos developed from enucleated oocytes and receiving cumulus cells from F1 (C57BL/6 × DBA, H-2b/d) females were transferred to outbred ICR (in which the H-2 complex was not fixed) recipient females. Each recipient received an intraperitoneal injection of CsA or vehicle. Compared with vehicle, administration of CsA to recipients on day 4.5 of pregnancy significantly increased the proportion of fetuses observed on day 10.5. The proportion of fetuses at day 18.5 of pregnancy in recipients receiving CsA treatment was slightly higher than that in controls. This study is the first to report that CsA administration increases the proportion of fetuses resulting from SCNT-cloned pregnancies.

To facilitate nuclear reprogramming, somatic cells or somatic cell nuclear-transferred (SCNT) oocytes have been treated with the histone deacetylase inhibitor trichostatin A (TSA), or the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), to relax epigenetic marks of differentiated somatic cells. TSA-treated SCNT oocytes have increased developmental potential, but the optimal treatment period is unknown. Reduced methylation levels in somatic cells have no positive effect on SCNT oocytes, but the treatment of SCNT embryos with 5-aza-dC has not been investigated. We examined the effect of TSA treatment duration on the developmental potential of mouse SCNT oocytes and the effect of 5-aza-dC treatment on their in vitro and in vivo developmental potential. To determine the effects of TSA treatment duration, nuclear-transferred (NT) oocytes were cultured for 0 to 26 h with 100 nM TSA. SCNT oocytes treated with TSA for 8 to 12 h had the higher rate of development to blastocysts and full-term fetuses were obtained after treatment for 8 to 12 h. When oocytes were treated for 14 h and 26 h, blastocyst rates were significantly decreased and fetuses were not obtained. To examine the effect of 5-aza-dC, 2-cell stage SCNT embryos were cultured with 10 or 100 nM 5-aza-dC for 48 h to the morula stage and transferred. The potential of embryos treated with 5-aza-dC to develop into blastocysts was decreased and no fetuses were obtained after transfer. The findings demonstrated that long-term TSA treatment of SCNT mouse oocytes and treatment with 5-aza-dC inhibit the potential to develop into blastocysts and to fetuses after transfer.

Somatic cell count (SCC) in milk is considered to be a valuable indicator of cow mastitis. For assessment of SCC in milk, the bioluminescent assay based on determination of ATP from somatic cells ([ATPsom]) in milk was proposed earlier. However, this assay is still not widely used in practice owing to lower reliability compared with conventional methods such as direct microscopy and flow cytometry. We revised the bioluminescent SCC assay and developed a simple protocol based on determination of the total non-bacterial ATP concentration in milk. It was shown that the novel ATP-releasing agent Neonol-10 (oxy-ethylated iso-nonyl phenol) has superior performance providing 100% lysis of somatic cells while not disrupting bacterial cells of milk at a concentration of 1·5% w/w. There was high correlation (R2=0·99) between measured bioluminescence and SCC as measured by direct microscopy. The observed detection limit of the bioluminescent milk SCC assay was as low as 900 cell/ml, time of analysis was 2–3 min per sample. The proposed method has high potential for on-site mastitis diagnostics.

The level of gelatinases in surrounding body fluids of actively remodelling tissue is indicative of basement membrane and extracellular matrix degradation under various physiological and pathological circumstances. To elucidate the association of gelatinase with mammary tissue remodelling during gradual or acute involution, in the first trial, goats milked twice daily (lactation) and goats receiving decreased milking frequency (involution) served to provide a total of 12 milk samples and 11 mammary secretion samples, respectively. In the second trial, 6 cows served to provide samples of dry secretion in 3 consecutive weeks immediately following milk stasis. Gelatin zymography was applied for gelatinase phenotyping and quantification on milk, plasma and the degranulation medium/lysate of milk somatic cells. Results indicated that the most prevalent gelatinase subtype switched from gelatinase A in milk to gelatinase B in involution secretion. Mammary secretion of goats during involution contained marginally higher protein level, significantly lower casein ratio and greater specific capacity of gelatinase B compared with those of milk during lactation. Specific capacities of gelatinases A and B in plasma of goats were similar during lactation and involution, while gelatinase B capacity in degranulation medium/lysates based on unit number of goat somatic cell was significantly higher during involution than during lactation. Milk stasis of cows induced a significant increase in specific capacity of gelatinase B, but not gelatinase A, of dry secretion up to the third week. Results of both trials agree that regional selective accretion of gelatinase B in milk might have played a role in mammary tissue remodelling during involution induced by either decreasing milking frequency or milk stasis. It is suggested that infiltrated polymorphonuclear neutrophils are one of the potential contributors responsible for the accumulation of gelatinase B during involution.

The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80–100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.

A protocol was developed for collection and storage of somatic cell samples under adverse conditions with little infrastructure, for somatic-cell banks as a backup for endangered livestock breeds. The procedure, which is uniform across species, includes sample collection with ear taggers with an integrated tag/vial system, and recording of global positioning system (GPS) coordinates, digital images and breed information. In a pilot study, the procedures were tested on six local Vietnamese populations of pigs, sheep and goats. Initial investment was around €3000, while the total variable cost for sampling one breed with 25 females and 25 males was less than €1000. With support from local organisations, the sampling of six breeds with 300 animals was carried out in 2 months. The protocol and the complete workflow for setting up a somatic-cell bank, together with data collection, are described. The procedure has proved practicable and exceedingly cheap relative to the cryopreservation of semen or embryos.

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16 h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p<0.05), but significantly lower than in TR<>N chimeric blastocysts (p<0.001). Similarly, a higher proportion (p<0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.

In response to the rapid loss of animal genetic resources, a world wide emergency program is proposed to create national genebanks on the basis of somatic cells. Contrary to other procedures, like storing deep frozen semen or embryos, collection and storage of somatic cells can be done cheaply and rapidly for any species. Only in this way can an emergency store of animal genetic resources be created fast enough to forestall the ongoing rapid erosion of animal biodiversity. While animals can already be produced from somatic cells for 10 species, this number will continue to rise in the future. A layered strategy is proposed which is based on collection and storage within individual countries, thus leaving execution of the program, responsibility and ownership of these national cryobanks with those countries. After a pilot study, more country genebanks could be created as funds become available. With a limited effort, the creation of a network of national genebanks of last resort are a realistic option.

To enhance the probability of reprogramming somatic cell nuclei, fibroblast cells from an adult male rabbit and a 12-day-old fetus were fused with oocytes at the second metaphase. The chromosomes of recipient oocytes were then removed by treatment with demecolcine for 1 or 2 h after fusion. Demecolcine treatment of fused oocytes induced membrane protrusions that contained all the maternal chromosomes, thus making it possible to remove the chromosomes. The potential of nuclear-transferred oocytes to develop into blastocysts was high (48% and 59%) and the average cell number of the blastocysts was large (149 and 159) 96 h after in vitro culture. The proportions of nuclear-transferred oocytes enucleated 1 h after fusion and implanted after transfer to pseudopregnant recipients were relatively high (2.8% and 4.9%) compared with our previous reports (1.7%: Yin et al., 2000; 0.6% and 1.0%: Yin et al., 2002a) where donor cells were fused with previously enucleated oocytes. Of 34 adult somatic cell implantation sites, 6 had fetuses on day 12 or 14 of pregnancy, but none of the fetuses had a heart beat or developed to term. None of the nuclear-transferred oocytes whose chromosomes were removed 2 h after demecolcine treatment implanted after transfer to recipients. The possible reasons why the high-quality nuclear-transferred oocytes did not develop to term are discussed.

The somatic cell count of milk is routinely determined by the fluoro-opto-electronic method and sometimes by the direct epifluorescent filter technique (DEFT). This paper investigates the potential of solid phase cytometry (SPC), a novel technique combining aspects of both the fluoro-opto-electronic method and epifluorescence microscopy for somatic cell counting. In SPC, cells are retained on a membrane filter, fluorescently labelled and automatically detected on the entire membrane filter by means of a laser scanning instrument (ChemScan). Fluorescent spots can be visually inspected by an epifluorescence microscope with a computer-driven moving stage. The performance of SPC was compared with that of the fluoro-opto-electronic method using a Fossomatic 360 instrument for 68 milk samples with varying somatic cell counts (103–106/ml). The sample throughput and repeatability of SPC were inferior to those of the Fossomatic method and statistical analysis of the method comparison data using the approach of J. M. Bland & D. G. Altman (The Lancet 1986 February 8 pp 307–310) revealed a poor comparability between the two methods. Moreover, problems of milk filterability and the interference of fluorescent particles presently hamper the routine application of SPC. Nevertheless, this method represents the first example of the application of SPC to milk.