Protein profile and alpha-lactalbumin concentration in the milk of standard and transgenic goats expressing recombinant human butyrylcholinesterase

The expression of recombinant proteins of pharmaceutical interest in the milk of transgenic farm animals can result in phenotypes exhibiting compromised lactation performance, as a result of the extraordinary demand placed on the mammary gland. In this study, we investigated differences in the protein composition of milk from control and transgenic goats expressing recombinant human butyrylcholinesterase. In Experiment 1, the milk was characterized by gel electrophoresis and liquid chromatography/mass spectrometry in order to identify protein bands that were uniquely visible in the transgenic milk and/or at differing band densities compared with controls. Differences in protein content were additionally evaluated by computer assisted band densitometry. Proteins identified in the transgenic milk only included serum proteins (i.e. complement component 3b, ceruloplasmin), a cytoskeleton protein (i.e. actin) and a stress-induced protein (94 kDA glucose-regulated protein). Proteins exhibiting evident differences in band density between the transgenic and control groups included immunoglobulins, serum albumin, β-lactoglobulin and α-lactalbumin. These results were found to be indicative of compromised epithelial tight junctions, premature mammary cell death, and protein synthesis stress resulting from transgene expression. In Experiment 2, the concentration of α-lactalbumin was determined using the IDRing^® assay and was found to be significantly reduced on day 1 of lactation in transgenic goats (4.33 ± 0.97 vs. 2.24 ± 0.25 mg/ml, P < 0.01), but was not different from non-transgenic controls by day 30 (0.99 ± 0.46 vs. 0.90 ± 0.11 mg/ml, P > 0.05). We concluded that a decreased/delayed expression of the α-lactalbumin gene may be the cause for the delayed start of milk production observed in this herd of transgenic goats.     

Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland

The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1-5 g/l) of recombinant human butyrylcholinesterase in the milk. Casein concentration, as evaluated by acid precipitation, was significantly reduced in the transgenic compared with the control goats throughout lactation (P < 0.01). Milk fatty acid composition for transgenic goats, as determined by gas chromatography, was found to have significantly fewer short chain fatty acids (P < 0.01) and more saturated fatty acids (P < 0.05) compared to controls, suggesting an overall metabolic stress and/or decreased expression of key enzymes (e.g. fatty acid synthase, stearoyl-CoA desaturase). The concentration of Na(+), K(+), assessed by atomic absorption spectrophotometry, and serum albumin, determined by bromocresol green dye and scanning densitometry, were similar in transgenic and control goats during the first several weeks of lactation. However, as lactation progressed, a significant increase in Na and serum albumin concentrations and a decrease in K(+) concentration were found in the milk of transgenic goats, while control animals remained unchanged (P < 0.01). These findings suggest that: (a) high expression of recombinant proteins may be associated with a slow-down in other synthetic activities at the mammary epithelium, as evidenced by a reduced casein expression and a decreased de-novo synthesis of fatty acids; (b) the development of permeable tight junctions may be the main mechanism involved in the premature cessation of milk secretion observed in these transgenic goats.     

Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk

The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range 1–5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications. As a result, this development constitutes another successful example in the application of transgenic animal technology.     

Mammary Specific Transgenic Over-expression of Insulin-like Growth Factor-I (IGF-I) Increases Pig Milk IGF-I and IGF Binding Proteins,with no Effect on Milk Composition or Yield

IGF-I regulates lactation by stimulating mammary mitogenesis, inhibiting apoptosis, and partially mediating the effects of growth hormone on lactogenesis. Herein, lactation performance during first and second parity was assessed in transgenic swine (TG) that over-expressed human IGF-I in milk under the control of the bovine α-lactalbumin promoter, regulatory regions and signal peptide coding sequence. Milk samples were collected throughout lactation (farrowing to d24) from TG sows and non-transgenic littermates (CON) and IGF-I, IGF-II, and IGFBP determined. Colostral (<24 h postpartum) IGF-I content was 26-fold greater (p < 0.001) in TG sows (949 ± 107 μg/L; range 228–1600 μg/L) than CON (36 ± 17.8 μg/L) and was 50- to 90-fold greater (p < 0.001) in mature milk (d2-24 postpartum). There was no effect of parity on milk IGF-I content. Milk IGF-II concentration was unaffected by IGF-I over-expression. Low molecular weight IGFBP (IGFBP-2 and -5) in the milk of TG sows were higher (p = 0.02) than CON in the early postpartum period, but did not differ in mature milk. Milk yield, determined by weigh-suckle-weigh, was similar in TG and CON as was litter weight gain. Milk nutrient composition was not significantly affected by IGF over-expression. Thus, mammary specific transgenic over-expression of IGF-I significantly increased milk IGF-I and IGFBP content, but did not impact lactation performance in swine.     

Changes in sow milk lipidome across lactation occur in fatty acyl residues of triacylglycerol and phosphatidylglycerol lipids,but not in plasma membrane phospholipids

Milk fats are vital to neonate survival and development, but vary highly by diet, maternal metabolic state and stage of lactation. To gain a better understanding of changes in lipid composition of sow milk across lactation, milk was collected from nine multiparous sows on days 0, 3, 7, and 14, relative to birth and lipids were profiled using multiple reaction monitoring (MRM) profiling. Percent fat was determined by creamatocrit, and found to be different (P < 0.05) between day 0 (12.36 ± 5.90%) and day 3 (16.22 ± 3.65%) but not between day 7 (13.13 ± 2.19%) and day 14 (12.13 ± 2.45%). Fat was extracted from milk (n = 6/day) using the Bligh-Dyer method and profiled using tandem mass spectrometry MRM to determine the abundance of lipids defined by class and fatty acyl residue composition. Lipid species relative concentration was calculated from internal standards, and data analysis was performed using Metaboanalyst 4.0. Concentration of phosphatidyl-choline, -serine, -ethanolamine, -inositol, cholesteryl ester and sphingomyelin did not vary across lactation days, nor did the distribution of associated fatty acyl residues. The total abundance of triacylglycerides (TGs) and phosphatidylglycerols (PGs) increased (P < 0.05) from colostrum (day 0) to transitional (days 3 and 7) and mature milk (day 14). As lactation days increased from day 0 to day 14, the number of carbons and unsaturation within fatty acyl residues decreased (P < 0.05) in both TGs and PGs. The proportion of TGs and PGs increased (P < 0.05) relative to other lipid classes. Changes in composition of milk triglycerides and phosphatidylglycerols likely reflect the metabolic activity of the mammary gland and developmental needs of neonates.     

Plasma prolactin,blood metabolites and milk production in bromocryptine-treated crossbred goats

To determine the effect of bromocryptine on plasma prolactin, metabolites and milk production, six healthy crossbred goats in their second or third lactation were selected from the institute’s goat herd. The goats were injected with bromocryptine @ 5 mg per day intramuscularly for a period of 5 days during 55–60 days of lactation (period I) and treatment was repeated after 14 days for an additional 5 days (period II). Blood samples were collected for 5 consecutive days before, during and after administration of bromocryptine during both periods of the study. Milk samples were collected on days coinciding with the days of blood sampling. During period I, bromocryptine administration decreased (P < 0.01) milk yields by 27.4%. After withdrawal of bromocryptine the yields increased (P < 0.01); however, during period II, bromocryptine treatment did not decrease milk yields. During period I bromocryptine treatment suppressed prolactin level (P < 0.01) which coincided with decrease in milk yield, but in period II such decreases in prolactin did not coincide with changes in milk yields. Fat content increased (P < 0.01) after bromocryptine administration in both periods. A decrease (P < 0.01) in protein and lactose content indicated a possible role of prolactin in the synthesis of these constituents by the mammary gland cell in goats. Blood glucose and NEFA were not affected by bromocryptine in period I but in period II an increase in blood glucose with a simultaneous decrease in NEFA was observed (P < 0.01).From this study it is concluded that prolactin has a role in the maintenance of milk secretion through protein synthesis by the mammary gland. However, it does not seem to have any direct role on milk fat synthesis.     

Variation in blood leucocytes,somatic cell count,yield and composition of milk of crossbred goats

Ten multiparous crossbred goats, five each of alpine × beetal (AB) and saanen × beetal (SB) were selected from the National Dairy Research Institute goat herd immediately after parturition. These were managed as per the practices followed in the institute’s goatherd. Blood and milk samples were collected at biweekly intervals from day 14 post-kidding for 22 weeks (154 days). Somatic cell count, electrical conductivity, fat, protein and lactose contents of milk were determined using standard methods. In the blood samples total leucocytes and differential leucocytes were also determined. Somatic cell counts were high immediately after parturition on day 14 of lactation and declined gradually with advanced lactation. There were individual variations (P < 0.01) in somatic cell counts between different lactation periods. Somatic cell count of milk was negatively correlated with neutrophils only (P < 0.05) and was neither correlated with milk yield, or with fat, protein, lactose content of milk. Electrical conductivity of milk was low up to four weeks of lactation and thereafter increased as the lactation advanced. Lactose content of milk declined gradually with the advancement of lactation. Fat content of milk was stable up to the eighth week and thereafter increased with advancement of lactation while the protein content of milk did not change significantly during lactation.     

Effect of Different Iodine Nutrition on Cerebellum Pcp-2 in Rat Offspring During Lactation

The thyroid functions of breastfed infants, as well as (indirectly) the development of their central nervous system, are dependent on the iodine status of the lactating mother. Purkinje cell protein-2 is a cell-specific marker of the cerebellum Purkinje cell and is a suitable indicator for observing the postnatal development of the cerebellum after birth. We measured the Purkinje cell protein-2 mRNA and protein levels in the rat cerebellum in the critical postnatal (14 days after birth) and maturation periods (28 days after birth) to determine the effect of different nutritional iodine levels on cerebellum growth in the offspring during lactation. We found that severe iodine deficiency resulted in thyroid dysfunction in lactating rats and their offspring on both 14 and 28 days, showing maternal total T₄ 16.7 ± 12.0 vs 36.4 ± 15.0, P < 0.05 (14 days) and 22.6 ± 18.7 vs 53.4 ± 9.4, P < 0.01 (28 days), and neonatal total T₄ 10.6 ± 2.3 vs 16.4 ± 4.7, P < 0.01(14 days) and 12.8 ± 2.9 vs 16.7 ± 3.4, P < 0.05 (28 days), respectively. The Purkinje cell protein-2 mRNA and its protein levels in offspring rats were significantly reduced that showed Purkinje cell protein-2 mRNA 1.12 ± 0.04 vs 2.25 ± 0.53, P < 0.05 (14 days) and 1.74 ± 0.94 vs 8.69 ± 2.71, P < 0.01 (28 days). However, mild iodine deficiency and excessive iodine maintained almost normal thyroid function in maternal and neonatal rats and normal Purkinje cell protein-2 mRNA and protein levels in offspring’s cerebellum. We conclude that severe iodine deficiency could significantly reduce Purkinje cell protein-2 mRNA and its protein levels, indicating that the cerebellum development was retarded, but mild iodine deficiency and excessive iodine could maintain them at an approximately normal level by the mother’s and offspring’s compensations, especially by the mother’s mammary glands.     

Large-scale production of functional human lysozyme in transgenic cloned goats

Human lysozyme (hLZ), an essential protein against many types of microorganisms, has been expressed in transgenic livestock to improve their health status and milk quality. However, the large-scale production of hLZ in transgenic livestock is currently unavailable. Here we describe the generation of transgenic goats, by somatic cell-mediated transgenic cloning, that express large amounts of recombinant human lysozyme (rhLZ) in milk. Specifically, two optimized lysozyme expression cassettes (β-casein/hLZ and β-lactoglobulin/hLZ) were designed and introduced into goat somatic cells by cell transfection. Using transgenic cell colonies, which were screened by 0.8 mg/mL G418, as a nuclear donor, we obtained 10 transgenic cloned goats containing one copy of hLZ hybrid gene. An ELISA assay indicated that the transgenic goats secreted up to 6.2 g/L of rhLZ in their milk during the natural lactation period, which is approximately 5–10 times higher than human milk. The average rhLZ expression levels in β-casein/hLZ and β-lactoglobulin/hLZ transgenic goats were 2.3 g/L and 3.6 g/L, respectively. Therefore, both rhLZ expression cassettes could induce high levels of expression of the rhLZ in goat mammary glands. In addition, the rhLZ purified from goat milk has similar physicochemical properties as the natural human lysozyme, including the molecular mass, N-terminal sequence, lytic activity, and thermal and pH stability. An antibacterial analysis revealed that rhLZ and hLZ were equally effective in two bacterial inhibition experiments using Staphylococcus aureus and Escherichia coli. Taken together, our experiments not only underlined that the large-scale production of biologically active rhLZ in animal mammary gland is realistic, but also demonstrated that rhLZ purified from goat milk will be potentially useful in biopharmaceuticals.     

Effects of Chromium and Carnitine Co-supplementation on Body Weight and Metabolic Profiles in Overweight and Obese Women with Polycystic Ovary Syndrome: a Randomized,Double-Blind,Placebo-Controlled Trial

The primary aim of our study was to determine the influence of taking chromium plus carnitine on insulin resistance, with a secondary objective of evaluating the influences on lipid profiles and weight loss in overweight subjects with polycystic ovary syndrome (PCOS). In a 12-week randomized, double-blind, placebo-controlled clinical trial, 54 overweight women were randomly assigned to receive either supplements (200 μg/day chromium picolinate plus 1000 mg/day carnitine) or placebo (27/each group). Chromium and carnitine co-supplementation decreased weight (− 3.6 ± 1.8 vs. − 1.0 ± 0.7 kg, P < 0.001), BMI (− 1.3 ± 0.7 vs. − 0.3 ± 0.3 kg/m², P < 0.001), fasting plasma glucose (FPG) (− 5.1 ± 6.0 vs. − 1.1 ± 4.9 mg/dL, P = 0.01), insulin (− 2.0 ± 1.4 vs. − 0.2 ± 1.2 μIU/mL, P < 0.001), insulin resistance (− 0.5 ± 0.4 vs. − 0.04 ± 0.3, P < 0.001), triglycerides (− 18.0 ± 25.2 vs. + 5.5 ± 14.4 mg/dL, P < 0.001), total (− 17.0 ± 20.3 vs. + 3.6 ± 12.0 mg/dL, P < 0.001), and LDL cholesterol (− 13.3 ± 19.2 vs. + 1.4 ± 13.3 mg/dL, P = 0.002), and elevated insulin sensitivity (+ 0.007 ± 0.005 vs. + 0.002 ± 0.005, P < 0.001). In addition, co-supplementation upregulated peroxisome proliferator-activated receptor gamma (P = 0.02) and low-density lipoprotein receptor expression (P = 0.02). Overall, chromium and carnitine co-supplementation for 12 weeks to overweight women with PCOS had beneficial effects on body weight, glycemic control, lipid profiles except HDL cholesterol levels, and gene expression of PPAR-γ and LDLR. Clinical trial registration number: http://www.irct.ir: IRCT20170513033941N38.

     

Effect of administration of live Saccharomyces cerevisiae on milk production,milk composition,blood metabolites,and faecal flora in early lactating dairy goats

The effects of live Saccharomyces cerevisiae (CNCM I-1077) supplementation to 72 Saanen dairy goats in early lactation were investigated. Animals were divided into treatment and control groups balanced for parity and milk production in the first 3 weeks of lactation. Treated animals received 0.2 g/day yeast supplement (Levucell SC20) corresponding to 4 × 10⁹ cfu/day S. cerevisiae, beginning week 3 of lactation, and lasting 15 weeks. Dry matter intake (DMI), body condition score (BCS), milk production and composition were recorded to day126 postpartum. Milk samples were analysed weekly for fat, protein, lactose, urea and SCC. Blood samples were analysed monthly for glucose, BHBA, NEFA, GOT and GGT. Intestinal microflora were assessed monthly in faeces, assaying total bacteria, enterobacteria, E. coli, coliform bacteria, clostridia, lactobacilli, molds and yeast. BCS was not affected by the treatment, but treated animals had greater DMI (2.71 kg/day versus 2.35 kg/day, S.E. ±0.12, P < 0.001) and produced more (P < 0.05) milk (2.38 kg/day versus 2.08 kg/day, S.E. ±0.14) of lower fat content (P < 0.05). Plasma metabolites and liver enzymes (GOT, GGT) were similar between groups, although BHBA levels tended to be lower (P = 0.06) in treated animals. Faecal E. coli were reduced (0.72 log₁₀ cfu/g versus 2.39 log₁₀ cfu/g, S.E. ±0.38, P < 0.05) and lactobacilli increased (P < 0.05) by the treatment. Yeast faecal levels were higher (P < 0.001) in treated animals during supplementation. In this study, live yeast supplementation to early lactating dairy goats significantly increased milk production, in relation to increased DMI, without decreasing BCS. Plasma metabolites (glucose, BHBA and NEFA) were consistent with the BCS findings (no differences between groups) and in addition there was a significant reduction of faecal E. coli content together with increase in lactobacilli in the treated animals, suggesting greater stability of the intestinal ecosystem. Results suggest that the inclusion of Saccharomyces cerevisae in the diet of early lactating dairy goats can be recommended under field conditions.     

Lactoferrin concentrations in goat milk throughout lactation

Lactoferrin is an iron-binding glycoprotein and is considered a major part of the non-specific disease resistance complex in the mammary gland. For cows, the influence of physiological factors on the lactoferrin concentration in milk has been reported. In addition, lactoferrin concentrations have been demonstrated to be proportional to somatic cell counts (SCC) in cows milk. In this study, we aimed to analyse the effects of lactational stage, lactation number and SCC in 19 goats throughout an entire lactational period. Lactoferrin concentrations in weekly composite milk samples were analysed with a competitive ELISA developed for caprine lactoferrin. Maximal lactoferrin concentrations were observed in the colostral samples (387 ± 69 μg/ml). In the following week, less than 20% of these concentrations were observed (62 ± 25 μg/ml) and thereafter until week 32 p.p., the weekly mean concentrations ranged between 10 and 28 μg/ml. Toward the end of lactation, approximately during the 33rd week, the concentrations began to increase and were reaching about 3.2-fold higher values in week 44 (107 ± 19 μg/ml). SCC were only available in monthly intervals and could thus not be directly related to the weekly lactoferrin recordings. When classifying the individual goats according to the median of their SCC values obtained during midlactation, the goats with SCC medians >430,000 had higher lactoferrin milk concentrations during this time than the ones with SCC below this threshold (P < 0.05). In addition, sampling week and parity significantly affected the lactoferrin concentration (P < 0.05 and P < 0.01, respectively). Comparing SCC and lactoferrin, both parameters are significantly affected by various physiological factors. Further studies are needed to clarify if the relationship between Lf and bacterial counts is closer compared to the relationship between SCC and bacterial counts.     

Effect of artificial vs. natural rearing on milk yield,kid growth and cost in Payoya autochthonous dairy goats

Seventy full lactations of Payoya dairy goats were used to study the effect of two different kid-rearing systems, natural or artificial, on milk yield, composition, hygiene-sanitary quality, kid growth and rearing cost. Two animal groups were established, one with goats under natural rearing (NS) and the other under artificial rearing (AR). In the NS group, the kids had free access to goat milk 18–20 h a day and were suckled up to 5 weeks of age and then the dams were milked twice daily. Dams in the AR were separated from their kids at 48 h post-partum; then, kids were reared artificially, and the dams were milked twice daily. The number of animals used in each type of rearing system was 35 (23 goats giving birth to twins and 12 goats giving birth to a single kid). Each week during suckling phase the volume of milk produced was measured, and individual samples were taken. From 5th week and until 210 days lactation, test-day yields recorded at intervals of 30 ± 3 days were obtained. The chemical composition of the milk, the bacteriology, and the somatic cell count was analyzed. The kids of both sexes were assigned to two groups, natural suckling (NS, n = 58) and ad libitum artificial rearing (AR, n = 58). Within each treatment, two groups of kids were formed depending on prolificacy: first group with kids from single birth (n = 12) and the second with twin kids (n = 46). Birth weight and weight every week upto the 4th week of life were recorded. During the 5 weeks of lactation the total milk yield per goat was higher for the NS group (140.2 L vs. 95.4 L; P < 0.001), although the total amount of marketable milk was greater for the AR group, with a difference of some 21 L (P < 0.05). Throughout the entire lactation the milk yield was higher in the group of natural rearing (total yield of 508 L vs. 400 L; P < 0.05). Although a significant effect of prolificacy was found during suckling phase (P < 0.001), during 30 weeks of lactation this factor did not affect milk yield (P > 0.05). For the milk composition and hygiene-sanitary quality there were no effects on the type of rearing system or the type of birth (P > 0.05). No significant effect was observed either for the feeding system or the sex or the prolificacy on the live weight of the kids at 28 days and the postnatal growth rate from birth to 28 days (P > 0.05). Natural rearing system had higher cost per kid comparing with artificial rearing system (€18.63/kid vs. €14.70/kid, respectively). However, when comparing total incomes during a full lactation, goats with natural rearing system had a higher income because of increment of total milk production (€29.95/kid).     

Superoxide signaling in perivascular adipose tissue promotes age‐related artery stiffness

We tested the hypothesis that superoxide signaling within aortic perivascular adipose tissue (PVAT) contributes to large elastic artery stiffening in old mice. Young (4–6 months), old (26–28 months), and old treated with 4‐Hydroxy‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (TEMPOL), a superoxide scavenger (1 mm in drinking water for 3 weeks), male C57BL6/N mice were studied. Compared with young, old had greater large artery stiffness assessed by aortic pulse wave velocity (aPWV, 436 ± 9 vs. 344 ± 5 cm s^‐1) and intrinsic mechanical testing (3821 ± 427 vs. 1925 ± 271 kPa) (both P < 0.05). TEMPOL treatment in old reversed both measures of arterial stiffness. Aortic PVAT superoxide production was greater in old (P < 0.05 vs. Y), which was normalized with TEMPOL. Compared with young, old controls had greater pro‐inflammatory proteins in PVAT‐conditioned media (P < 0.05). Young recipient mice transplanted with PVAT from old compared with young donors for 8 weeks had greater aPWV (409 ± 7 vs. 342 ± 8 cm s^‐1) and intrinsic mechanical properties (3197 ± 647 vs. 1889 ± 520 kPa) (both P < 0.05), which was abolished with TEMPOL supplementation in old donors. Tissue‐cultured aortic segments from old in the presence of PVAT had greater mechanical stiffening compared with old cultured in the absence of PVAT and old with PVAT and TEMPOL (both, P < 0.05). In addition, PVAT‐derived superoxide was associated with arterial wall hypertrophy and greater adventitial collagen I expression with aging that was attenuated by TEMPOL. Aging or TEMPOL treatment did not affect blood pressure. Our findings provide evidence for greater age‐related superoxide production and pro‐inflammatory proteins in PVAT, and directly link superoxide signaling in PVAT to large elastic artery stiffness.     

Production of recombinant albumin by a herd of cloned transgenic cattle

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1–2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1–2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.     

Generation of transgenic goats by pronuclear microinjection: a retrospective analysis of a commercial operation (1995–2012)

Production of transgenic founder goats involves introducing and stably integrating an engineered piece of DNA into the genome of the animal. At LFB USA, the ultimate use of these transgenic goats is for the production of recombinant human protein therapeutics in the milk of these dairy animals. The transgene or construct typically links a milk protein specific promoter sequence, the coding sequence for the gene of interest, and the necessary downstream regulatory sequences thereby directing expression of the recombinant protein in the milk during the lactation period. Over the time period indicated (1995–2012), pronuclear microinjection was used in a number of programs to insert transgenes into 18,120, 1- or 2- cell stage fertilized embryos. These embryos were transferred into 4180 synchronized recipient females with 1934 (47%) recipients becoming pregnant, 2594 offspring generated, and a 109 (4.2%) of those offspring determined to be transgenic. Even with new and improving genome editing tools now available, pronuclear microinjection is still the predominant and proven technology used in this commercial setting supporting regulatory filings and market authorizations when producing founder transgenic animals with large transgenes (> 10 kb) such as those necessary for directing monoclonal antibody production in milk.     

K<Subscript>ATP</Subscript> channel Kir6.2 E23K variant overrepresented in human heart failure is associated with impaired exercise stress response

ATP-sensitive K⁺ (K_ATP) channels maintain cardiac homeostasis under stress, as revealed by murine gene knockout models of the KCNJ11-encoded Kir6.2 pore. However, the translational significance of K_ATP channels in human cardiac physiology remains largely unknown. Here, the frequency of the minor K23 allele of the common functional Kir6.2 E23K polymorphism was found overrepresented in 115 subjects with congestive heart failure compared to 2,031 community-based controls (69 vs. 56%, P < 0.001). Moreover, the KK genotype, present in 18% of heart failure patients, was associated with abnormal cardiopulmonary exercise stress testing. In spite of similar baseline heart rates at rest among genotypic subgroups (EE: 72.2 ± 2.3, EK: 75.0 ± 1.8 and KK: 77.1 ± 3.0 bpm), subjects with the KK genotype had a significantly reduced heart rate increase at matched workload (EE: 32.8 ± 2.7%, EK: 28.8 ± 2.1%, KK: 21.7 ± 2.6%, P < 0.05), at 75% of maximum oxygen consumption (EE: 53.9 ± 3.9%, EK: 49.9 ± 3.1%, KK: 36.8 ± 5.3%, P < 0.05), and at peak VO₂ (EE: 82.8 ± 6.0%, EK: 80.5 ± 4.7%, KK: 59.7 ± 8.1%, P < 0.05). Molecular modeling of the tetrameric Kir6.2 pore structure revealed the E23 residue within the functionally relevant intracellular slide helix region. Substitution of the wild-type E residue with an oppositely charged, bulkier K residue would potentially result in a significant structural rearrangement and disrupted interactions with neighboring Kir6.2 subunits, providing a basis for altered high-fidelity K_ATP channel gating, particularly in the homozygous state. Blunted heart rate response during exercise is a risk factor for mortality in patients with heart failure, establishing the clinical relevance of Kir6.2 E23K as a biomarker for impaired stress performance and underscoring the essential role of K_ATP channels in human cardiac physiology.     

Production GH transgenic goat improving mammogenesis by somatic cell nuclear transfer

Growth hormone is a positive regulator of mammary gland development. Dairy animals that are administered growth hormone display enhanced lactation performance, a desirable agricultural trait. The objective of the current research was to generate an improved milk production phenotype in a large animal model using over-expressed GH in the mammary gland to promote mammogenesis. To this end, we constructed a mammary gland-specific expression vector, pcGH, and demonstrated effective GH expression in goat mammary epithelial cells in vitro by ELISA. Then, to produce transgenic offspring that were capable of stable GH expression in vivo, the linearized pcGH vector was electroporated into goat fetal fibroblasts. Cell colonies that were positive for GH were used as donors for nuclear transfer to enucleated oocytes. A total of 253 morulae or blastocytes developed from the reconstructed embryos were transferred to 56 recipients, resulting in 24 pregnancies at day 35. Finally, six transgenic goats were born. PCR detection confirmed the success of the cloning procedure. To observe the mammogenesis of dairy goats, the GH transgenic goats were mated with a completely healthy buck. In the later pregnancy period, the mammary gland of the GH transgenic goats were extensive than non-transgenic goats. These experiments indicated that the pcGH vector was incorporated into the transgenic goats and affected mammogenesis, which laid a solid foundation for elucidating the impact of GH on mammogenesis and lactation performance.     

Supplementing sow diets with palm oil during late gestation and lactation: effects on milk production,sow hormonal profiles and growth and development of her offspring

The supplementing of sow diets with lipids during pregnancy and lactation has been shown to reduce sow condition loss and improve piglet performance. The aim of this study was to determine the effects of supplemental palm oil (PO) on sow performance, plasma metabolites and hormones, milk profiles and pre-weaning piglet development. A commercial sow ration (C) or an experimental diet supplemented with 10% extra energy in the form of PO, were provided from day 90 of gestation until weaning (24 to 28 days postpartum) in two groups of eight multiparous sows. Gestation length of PO sows increased by 1 day (P<0.05). Maternal BW changes were similar throughout the trial, but loss of backfat during lactation was reduced in PO animals (C: −3.6±0.8 mm; PO: −0.1±0.8 mm; P<0.01). Milk fat was increased by PO supplementation (C day 3: 8.0±0.3% fat; PO day 3: 9.1±0.3% fat; C day 7: 7.8±0.5% fat; PO day 7: 9.9±0.5% fat; P<0.05) and hence milk energy yield of PO sows was also elevated (P<0.05). The proportion of saturated fatty acids was greater in colostrum from PO sows (C: 29.19±0.31 g/100 g of fat; PO: 30.77±0.36 g/100 g of fat; P<0.01). Blood samples taken on 105 days of gestation, within 24 h of farrowing, day 7 of lactation and at weaning (28±3 days post-farrowing) showed there were no differences in plasma concentrations of triacylglycerol, non-esterified fatty acids, insulin or IGF-1 throughout the trial. However, circulating plasma concentrations of both glucose and leptin were elevated during lactation in PO sows (P<0.05 and P<0.005, respectively) and thyroxine was greater at weaning in PO sows (P<0.05). Piglet weight and body composition were similar at birth, as were piglet growth rates throughout the pre-weaning period. A period of 7 days after birth, C piglets contained more body fat, as indicated by their lower fat-free mass per kg (C: 66.4±0.8 arbitrary units/kg; PO: 69.7±0.8 arbitrary unit/kg; P<0.01), but by day 14 of life this situation was reversed (C: 65.8±0.6 arbitrary units/kg; PO: 63.6±0.6 arbitrary units/kg; P<0.05). Following weaning, PO sows exhibited an increased ratio of male to female offspring at their subsequent farrowing (C: 1.0±0.3; PO: 2.2±0.2; P<0.05). We conclude that supplementation of sow diets with PO during late gestation and lactation appears to increase sow milk fat content and hence energy supply to piglets. Furthermore, elevated glucose concentrations in the sow during lactation may be suggestive of impaired glucose homoeostasis.