Treatment of subclinical mastitis in Damascus goats during lactation

This study was carried out to determine the efficacy of three treatment protocols for subclinical mastitis during lactation in Damascus goats. For this purpose intramammarian ampicillin dicloxacillin, intramuscular amoxycillin clavulonic acid and the combination of intramammarian ampicillin dicloxacillin and intramuscular amoxycillin clavulonic acid were used in goats with subclinical mastitis during lactation.The microbiological treatment rates in the intramammarian ampicillin dicloxacillin, intramuscular amoxycillin clavulonic acid and the combination of intramammarian ampicillin dicloxacillin and intramuscular amoxycillin clavulonic acid groups in the 7th and 21st days after the treatment were 62.5% and 92.5%, 62.5% and 70%, 87.5% and 87.5%, respectively. The intramammarian and intramuscular combination group was found to have a statistically higher treatment rate than other two groups on the 7th day. On the 21st day intramammarian and combination groups were found to have statistically better treatment rates than that of intramuscular group.It was concluded that the goat subclinical mastitis could be successfully treated during lactation. While the intramammarian ampicillin dicloxacillin treatment had the best treatment rates, the combination of intramuscular amoxycillin clavulonic acid was also successful. Intramuscular amoxycillin clavulonic acid as the sole treatment was not as effective as intramammarian therapy.     

Energy requirements and efficiency of Alpine goats in early lactation

Dairy goats may rely heavily on body fat and protein reserves in early lactation. Therefore, we aimed to determine the energy requirement and estimate the efficiency of utilization the nutrients of tissues mobilized in the first 8 weeks of lactation for milk production using the comparative slaughter technique. The average initial body mass of 51 multiparous goats was 57.19 ± 8.38 kg and a body condition score of 3.0 ± 0.5. Three goats were slaughtered at the beginning of the experiment to serve as baseline animals to estimate initial empty BW and initial body composition. We used a complete randomized design in which the factor was the day of lactation for slaughtering (the 7th, 14th, 21st, 28th, 35th, 42nd, 49th and 56th day), with six repeats, totalling 48 goats. No fasting before slaughtering. All animals received a single experimental diet. The efficiency of transferring energy from body reserves to milk was estimated using a multiple linear regression equation yielding a value of 0.76. The total energy stored in the empty body decreased over the eight lactation weeks, from 726.47 ± 26.19 to 316.18 ± 49.21 MJ, a 56.47% reduction, mainly because of a reduction in the energy from internal fat of 3.96 ± 1.98 MJ/day. In conclusion, the net energy required for maintenance is 60 ± 30 kJ/BW^0.75 per day, and the net energy required for lactation decreases 70 ± 30 kJ/day during the first eight lactation weeks.     

Hormonal regulation of phosphate homeostasis in goats during transition to rumination

Regulatory processes in phosphorus (P) homeostasis in small ruminants are quite different compared to monogastric animals. Adaptive responses of modulating hormones [parathyroid hormone (PTH) and calcitriol] to feeding variable amounts of P are lacking. Therefore, the aim of this study was to examine the influence of high dietary P intake (control diet: 4 g kg(-1) dry matter; high-P diet: 8 g kg(-1) dry matter) on the expression levels of PTH receptor (PTHR), vitamin D receptor (VDR) and Na+-dependent Pi transporters (NaPi II) in kidney and jejunum of goats starting rumination. After 3 months of feeding, plasma phosphate (Pi) and PTH concentrations were increased in the high-P diet group, whereas calcium and calcitriol were not changed. The intestinal Na+-dependent Pi transport capacity was not influenced by a high-P diet and the expression of jejunal VDR, PTHR and NaPi IIb was not modified. Interestingly, renal Na+-dependent Pi transport capacity was significantly reduced and concomitantly the expression of PTHR and NaPi IIa was decreased. In conclusion, the adaptive response of renal Pi reabsorption in goats, which were in transition from non-ruminant to ruminant stage was comparable to that of monogastric animals. In contrast, the modulation of the intestinal Pi absorption was like in adult ruminants.     

Hormonal control of casein synthesis in mammary explants from pregnant goats

The effects of insulin, cortisol, prolactin, 3,3',5-triiodo-L-thyronine (L-T3) and progesterone on the synthesis of total protein and casein in mammary explants from pregnant goats were studied. In the absence of hormones and in the presence of insulin plus cortisol the rate of incorporation of 14C-leucine into proteins that were precipitated with the anti-casein antibody decreased during culture. The addition of prolactin to hormonal combination of insulin and cortisol caused large stimulation of rates of casein synthesis. Maximum incorporation of leucine was attained between 3 and 5 days of culture in the presence of 0.5 microgram ml-1 of prolactin. Prolactin stimulated-casein and total protein synthesis were not consistently affected by the addition of L-T3 or progesterone. The inhibition of DNA synthesis by hydroxyurea or cytosine-arabinofuranoside had no effect on casein synthesis in mammary explants from pregnant goats.     

Fatty acids in the milk of goats after cessation of lactation

Regular hand milking of six goats was discontinued after 32-35 weeks of lactation. A few days after milking out ceased, the concentration of triglyceride in peripheral blood plasma increased. Over a period of weeks, the concentration of triglyceride in small samples of fluid taken from the teat canal fell gradually. Lipase activity of the milk fluid was temporarily reduced shortly after milking out ended, but, despite this, its concentration of free fatty acids increased. It is suggested that free fatty acids are released during clearance of milk triglyceride from residual fluid in the mammary gland after cessation of lactation.     

人乳铁蛋白(hLF)转基因山羊的遗传背景分析

以随机整合方式获得的转基因动物外源基因的拷贝数、整合位点及染色体核型等遗传背景并不清楚,可能会存在外源基因的沉默整合、无效整合、毒性整合以及其表达水平不可预测等问题。文中选取了6只原代(F0)及其相对应的子一代(F1)的人乳铁蛋白(hLF)转基因山羊作为研究对象,分别颈静脉采血、提取DNA,通过染色体核型分析、实时荧光定量PCR(qPCR)、ELISA和Westernblotting等检测技术,研究其外源基因的遗传背景与表达水平。结果显示,6只F0代转基因山羊的染色体没有明显的形态变异、数量改变等异常情况。相对拷贝数高低不同(2–16),且能够稳定地遗传给下一代,F0和F1代hLF基因拷贝数一致。F1代转基因山羊表达hLF水平最高可达1.12 g/L(L3-1,拷贝数8)。结果表明,整合的外源基因能够稳定地遗传下一代,也没有对转基因山羊个体的生长发育造成障碍,而且拷贝数高低与hLF表达水平无明显的相关性,这为转基因山羊及其他转基因动物的新品种培育奠定了基础,解析了遗传背景。     

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.     

Changes induced in rat plasma composition by lactation

Changes in rat plasma composition in post-partum period were studied from day 1 to post weaning recovery situation (day 40). Urea levels showed a significant increase at the peak of lactation, and glycerol levels showed a decrease towards late lactation. Changes in the plasma aminogram were more marked during the maximal intensity of lactation (days 10-20) and in postlactation. Low trypthophan levels were sustained throughout the lactation period. The combined amino acids showed significant increases on day 10 of lactation, and increases in non-essential and total amino acids in post-lactation (40 days). These results point towards increased amino-acid utilization during lactation.     

Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats

The production of valuable pharmaceutical proteins using transgenic animals as bioreactors has become one of the goals of biotechnology. However, the efficiency of producing transgenic animals by means of pronuclear microinjection is low. This may be attributed in part to the low integration rate of foreign DNA. Therefore, a large number of recipients are required to produce transgenic animals. We recently developed a transgenic procedure that combined the techniques of goat oocyte in vitro maturation (IVM), in vitro fertilization (IVF), microinjection, preimplantation selection of the transgenic embryos with nested PCR and transferring the transgenic embryos into the recipient goat uterus to produce transgenic goats. Thirty-seven transgenic embryos determined by nested PCR were transferred to thirty-two recipient goats. In the end, four live-born kids were produced. As predicted, all the live kids were transgenic as identified by PCR as well as Southern blot hybridization, The integration rate was 100% (4/4) which was completely in accordance with the results of embryo preimplantation detection. The results showed a significant decrease in the number of recipients required as only 8 recipients (32/4) were needed to obtain one live transgenic goat. We suggest that the transgenic system described herein may provide an improved way to efficiently produce transgenic goats on a large scale.     

Genetic and phenotypic parameters for lactation traits in a flock of Saanen goats in Mexico

Heritability, repeatability and genetic and phenotypic correlations of total milk production (TM), cumulated milk production to 120 days (M120) and lactation length (LL), were estimated from the analysis of 1413 records (566 does) obtained from a Saanen goat flock in Querétaro, Mexico, during 1991–1997. Variance components were estimated using a restricted maximum likelihood procedure and applying an animal model. The model included the fixed effects of year–season of kidding, litter size and age of doe, and random effects of animal, permanent environment and residual. For TM, M120 and LL estimates of heritability were 0.22 ± 0.07, 0.12 ± 0.05 and 0.04 ± 0.03, and of repeatability were 0.40, 0.27 and 0.11, respectively. Genetic correlations between TM and M120, TM and LL, and M120 and LL, were 0.98, 0.58 and 0.53, respectively. Phenotypic correlations ranged from 0.30 to 0.83. The correlated selection response for TM from indirect selection on M120 was 71.5% of that expected from direct selection. Efficient selection for TM is possible and M120 could be used as an early indicator of TM, due to the high indirect selection response calculated.     

Expression of active recombinant human lactoferrin in the milk of transgenic goats

This report details the establishment of a transgenic goat model in order to produce human lactoferrin (hLf) in the mammary gland for large-scale application and research. Two transgenic male goats were generated by microinjecting sequence encoding hLf cDNA to the pronuclear. In the two lines, derived from the two founders, eight lactating female goats could secrete recombinant human lactoferrin (rhLf) at concentrations of up to 0.765 mg/ml. The method of purifying the rhLf from the milk was achieved using ion-exchange chromatography and resulted in 97% purity. Biochemical and physicochemical characteristics of rhLf were similar to native lactoferrin (nhLf); this included N-terminal sequence, isoelectric point, molecular mass, glycosylation, iron-binding/releasing ability, thermal stability, and proteolysis. The rhLf showed broad spectrum antibacterial activity inhibiting the growth of several pathogenic bacterial strains. Also investigated, although to a lesser degree, was a practicable pasteurization method for the downstream processing of rhLf and, further, a method for the oral administration of rhLf. On the basis of these results, our studies show an optimistic and promising approach for the large-scale production and therapeutic application of rhLf expressed in 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.     

Hormonal evidence for induced ovulation in Monodelphis domestica.

Breeding activity in the South American grey opossum, Monodelphis domestica, is stimulated by the presence of males. This study presents the first analysis of changes in concentrations of plasma progesterone during pregnant and nonpregnant cycles. In Expt 1, females were paired with either intact or vasectomized males to stimulate a reproductive cycle, or were isolated from males. Within 4-8 days of pairing, females showed marked changes in the size of the urogenital opening (vulva), which were paralleled by similar changes in body weight (11.4 +/- 2%; mean +/- SEM; n = 13). There was a second increase and decrease in body weight (21.7 +/- 3.3%; n = 13) in pregnant and nonpregnant cycles during the luteal phase of the cycle. Changes in concentrations of plasma progesterone, determined from samples collected three times a week, were similar in pregnant and nonpregnant cycles. Plasma progesterone concentration, which was increased for 15-18 days, showed two distinct peaks. The first peak, 3-4.5 ng ml-1, occurred immediately after the decrease in vulval swelling, and the second peak (1-2 ng ml-1) occurred 8-12 days later. Concentrations of plasma progesterone were low before parturition occurred. Females isolated from males for 35 days showed no changes in body weight or vulval swelling and concentrations of plasma progesterone remained undetectable. In Expt 2, concentrations of luteinizing hormone (LH) in plasma were determined twice a day for the first few days after pairing. In three of five females an LH peak was detected coincident with the maximal swelling of the vulva.