Single gene and gene interaction effects on fertilization and embryonic survival rates in cattle

Decrease in fertility and conception rates is a major cause of economic loss and cow culling in dairy herds. Conception rate is the product of fertilization rate and embryonic survival rate. Identification of genetic factors that cause the death of embryos is the first step in eliminating this problem from the population and thereby increasing reproductive efficiency. A candidate pathway approach was used to identify candidate genes affecting fertilization and embryo survival rates using an in vitro fertilization experimental system. A total of 7,413 in vitro fertilizations were performed using oocytes from 504 ovaries and semen samples from 10 different bulls. Fertilization rate was calculated as the number of cleaved embryos 48 h postfertilization out of the total number of oocytes exposed to sperm. Survival rate of embryos was calculated as the number of blastocysts on d 7 of development out of the number of total embryos cultured. All ovaries were genotyped for 8 genes in the POU1F1 signaling pathway. Single-gene analysis revealed significant associations of GHR, PRLR, STAT5A, and UTMP with survival rate and of POU1F1, GHR, STAT5A, and OPN with fertilization rate. To further characterize the contribution of the entire integrated POU1F1 pathway to fertilization and early embryonic survival, a model selection procedure was applied. Comparisons among the different models showed that interactions between adjacent genes in the pathway revealed a significant contribution to the variation in fertility traits compared with other models that analyzed only bull information or only genes without interactions. Moreover, some genes that were not significant in the single-gene analysis showed significant effects in the interaction analysis. Thus, we propose that single genes as well as an entire pathway can be used in selection programs to improve reproduction performance in dairy cattle.     

Dickkopf-1 secreted by decidual cells promotes trophoblast cell invasion during murine placentation

To investigate the physiological characteristics of the corpus luteum (CL) of pregnancy, we raised a mAb, human corpus luteum (HCL)-4, against human luteal cells obtained from CL of pregnancy. The affinity-purified antigen from human CL of pregnancy or placenta using HCL-4 was a 61 kDa protein. The partial amino acid sequence of the antigenic protein was identical to that of human monoamine oxidase A (MAOA, EC1.4.3.4). MAOA has been shown to catabolize catecholamines that were reported to regulate luteal function in CL and vasoconstriction in various organs. Immunohistochemistry using HCL-4 mAb showed that MAOA was intensely expressed on large luteal cells and moderately expressed on small luteal cells in the CL of pregnancy. In the CL of menstrual cycle, MAOA was weakly detected on large luteal cells but not detected at all on small luteal cells. Western blotting analysis confirmed the high expression of MAOA in CL of pregnancy. Northern blot analysis also showed the expression of MAOA mRNA in human CL, and showed that its expression was higher in CL of pregnancy than in CL of menstrual cycle. The increased expression of MAOA in the CL of pregnancy suggests the contribution of MAOA to the function of the CL of pregnancy.     

葡萄标准化生产与施肥

合理施肥是葡萄标准化生产中的一个重要环节。本文介绍了在葡萄无公害、绿色和有机栽培中的肥料使用规范,包括允许、限制和禁止使用的肥料种类以及基本的施肥原则,在葡萄正常生长发育的基础上保证葡萄产品质量安全,控制环境污染,实现土壤和水资源的可持续管理。     

Sperm proteasome and fertilization

The omnipresent ubiquitin-proteasome system (UPS) is an ATP-dependent enzymatic machinery that targets substrate proteins for degradation by the 26S proteasome by tagging them with an isopeptide chain composed of covalently linked molecules of ubiquitin, a small chaperone protein. The current knowledge of UPS involvement in the process of sperm penetration through vitelline coat (VC) during human and animal fertilization is reviewed in this study, with attention also being given to sperm capacitation and acrosome reaction/exocytosis. In ascidians, spermatozoa release ubiquitin-activating and conjugating enzymes, proteasomes, and unconjugated ubiquitin to first ubiquitinate and then degrade the sperm receptor on the VC; in echinoderms and mammals, the VC (zona pellucida/ZP in mammals) is ubiquitinated during oogenesis and the sperm receptor degraded during fertilization. Various proteasomal subunits and associated enzymes have been detected in spermatozoa and localized to sperm acrosome and other sperm structures. By using specific fluorometric substrates, proteasome-specific proteolytic and deubiquitinating activities can be measured in live, intact spermatozoa and in sperm protein extracts. The requirement of proteasomal proteolysis during fertilization has been documented by the application of various proteasome-specific inhibitors and antibodies. A similar effect was achieved by depletion of sperm-surface ATP. Degradation of VC/ZP-associated sperm receptor proteins by sperm-borne proteasomes has been demonstrated in ascidians and sea urchins. On the applied side, polyspermy has been ameliorated by modulating sperm-associated deubiquitinating enzymes. Diagnostic and therapeutic applications could emerge in human reproductive medicine. Altogether, the studies on sperm proteasome indicate that animal fertilization is controlled in part by a unique, gamete associated, extracellular UPS.     

Genotype by environment interaction for somatic cell score across bulk milk somatic cell count and days in milk

The objective of this paper was to investigate the importance of a genotype × environment interaction (G × E) for somatic cell score (SCS) across levels of bulk milk somatic cell count (BMSCC), number of days in milk (DIM), and their interaction. Variance components were estimated with a model including random regressions for each sire on herd test-day BMSCC, DIM, and the interaction of BMSCC and DIM. The analyzed data set contained 344,029 test-day records of 24,125 cows, sired by 182 bulls, in 461 herds comprising 13,563 herd test-days. In early lactation, considerable G × E effects were detected for SCS, indicated by 3-fold higher genetic variance for SCS at high BMSCC compared with SCS at low BMSCC, and a genetic correlation of 0.72 between SCS at low and at high BMSCC. Estimated G × E effects were smaller during late lactation. Genetic correlations between SCS at the same level of BMSCC, across DIM, were between 0.43 and 0.89. The lowest genetic correlation between SCS measures on any 2 possible combinations of BMSCC and DIM was 0.42. Correlated responses in SCS across BMSCC and DIM were, on some occasions, less than half the direct response to selection in the response environment. Responses to selection were reasonably high among environments in the second half of the lactation, whereas responses to selection between environments early and late in lactation tended to be low. Selection for reduced SCS yielded the highest direct response early in lactation at high BMSCC.     

Genotype by environment interaction for yield and somatic cell score with alternative environmental definitions

Differential genetic expression in high and low opportunity Sicilian Holstein-Friesian and Brown Swiss herd environments was investigated using endogenous and exogenous variables in a set of three definitions. Results of genetic by environmental interaction were compared using alternative environmental definitions: within herd-year standard deviation for mature equivalent milk yield (HYSD), detectable incidence of normal vs. abnormal (peakless) lactation and herds clustered by causal relationships from high and low frequency use of nutrition, milking, health and animal handling practices. Data for genetic analysis consisted of first-lactation standardized yields of milk, fat and protein, and weighted somatic cell score for 8897 daughters of 825 Holstein-Friesian sires and 1143 daughters of 220 Brown Swiss sires. Components of covariance, heritabilities, and genetic correlations were estimated using bivariate and multivariate sire models for average and contrasting environments for each definition. Sire variances for yields were consistently smaller in the low opportunity environments of both breeds. Except for differential incidence of abnormal lactation in Friesian herds, correlated yield response in less privileged environments was 0.41 to 0.81 as much as in high opportunity environments, a substantial loss. Genetic correlations between HYSD environments for yield traits of Friesian were 0.48 to 0.66 but exceeded 0.80 for other definitions. Less correlated response in somatic cell score was also predicted for environments with low use of yield-enhancing practices (0.66 for Friesian and 0.61 for Brown Swiss), which may have resulted from less health care and poorer milking management. Therefore, unfavorable management interactions likely foster unequal gains from selection in contrasting environments defined exogenously or by incidence of peakless lactation. Conversely, greater genetic as well as phenotypic response is expected from additional inputs of nutrition, health care and milking management.     

Early history of in vitro fertilization

Although in vitro fertilization (IVF) is used widely for a variety of purposes, it is often not appreciated how this technology was developed. A large number of experiments beginning in 1878 contributed to the first successful reports of IVF over 75 years later. The discovery of sperm capacitation in 1951 was central to the development of IVF technology, and it was rapidly followed by the first convincing reports of IVF in several species. The ability to fertilize oocytes in vitro has allowed major advances to be made into understanding the mechanisms involved in fertilization and early development, and IVF now supports reproductive biotechnology in animals and in humans. This article is a historical review of key experiments that helped to provide the basis for present day IVF procedures, placed into context with current practice.     

Centrosome changes during meiosis in horse oocytes and first embryonic cell cycle organization following parthenogenesis, fertilization and nuclear transfer

Various types of cell cycle organization occur in mammals. In this study, centrosome changes during meiosis in horse oocytes, and first cell cycle organization following fertilization, parthenogenesis and nuclear transfer, were monitored. Cumulus oocyte complexes harvested from horse ovaries obtained from slaughtered mares were cultured in vitro. Meiotic oocytes of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and II (MI and MII) stages were selected at various set times during in vitro maturation. Embryos at the first cell cycle stage were generated by subjecting MII stage oocytes to fertilization by intracytoplasmic sperm injection (ICSI), parthenogenetic treatment or nuclear transfer. Centrosome changes during meiosis and the first cell cycle organization were detected by indirect immunofluorescent staining, using a mouse anti-alpha-tubulin antibody for microtubules and a rabbit anti-gamma-tubulin antibody for centrosomes. These examinations showed that the centrosomes of the horse oocyte reorganize themselves from the beginning of GV stage to leave only PCM of gamma-tubulin surrounding both poles of the MI and MII stage spindles. These MII oocytes can organize the separation of metaphase chromosomes during the first embryonic cell cycle by parthenogenetic treatment. When the MII oocytes were subjected to ICSI or nuclear transfer, one or two red-stained centrosomes of gamma-tubulin were introduced by the fertilising spermatozoon or the donor cell which associated with the sperm chromatin in the fertilized embryos and with the donor cell chromatin and microtubules in the cloned embryos. This finding suggests that centrosomes are not an essential component in the formation of the metaphase spindle during meiotic maturation of horse oocytes, but they can be introduced from the spermatozoon or donor cell and are necessary for the organization of normal embryonic development.     

Dielectric response of cells and liposomes and its utilization for evaluation of cell membrane-protein interaction

The dielectric measurements of an Escherichia coli (E. coli) cell suspension and liposome suspensions were carried out in the frequency range between 0.1 and 100 MHz to detect the heat stress-mediated interaction between proteins and cell membranes. The dielectric relaxation dispersion was observed to be above 1 MHz. The dielectric parameter (amplitude of dispersion, deltaepsilon) based on the Cole-Cole equation was anomalously changed with increasing temperature. The value of deltaepsilon of liposomes was varied at various temperatures depending on the type of protein present. The change in deltaepsilon of liposomes correlated with the amount of protein translocated across the phospholipid membrane. The changes in the value of deltaepsilon of E. coli cells with temperature variation was similar to those of liposomes in the presence of proteins, suggesting that the variation in dielectric parameters reflected the interaction between the phospholipid membrane and proteins. It was found that the dielectric measurement could be utilized for the detection of the interaction between proteins and liposomes.     

Genotype by environment interaction and genetic correlations among parities for somatic cell count and milk yield

Lactation measures of somatic cell concentration and total SCC production were developed. Data were separated into three parity groups. Within parity, five data sets were created: four subsets by herd-year average SCC, and one with all records. Records on lactation SCC, total SCC production, and 305-d milk were analyzed by a sire model separately in each subset within parity. Variance components estimates were by REML. For SCC and total SCC production, heritability estimates averaged .12 and were lowest in the highest level of herd-year average SCC. Estimates of genetic correlation between SCC and total SCC production were over .95; between SCC and 305-d milk were around .25 in first and -.15 in later parities; between total SCC and 305-d milk were around .50 in first and .15 in later parities. Product-moment correlations between sire effects in different levels of herd-year average SCC were obtained. Ratios of product-moment correlations to their expected value were above .80 for all traits in all parities. High ratios indicated little genotype by environment interaction. A sire by herd interaction was fitted in the model and accounted for less than 2% of total phenotypic variance for SCC and total SCC production, and 4% for 305-d milk. Estimates of genetic correlation of first with later parities were .71 to .86 for all traits. Between second and third parity genetic correlation estimates were around unity for all traits. Records from all parities should be used for sire evaluation.     

Genotype x environment interaction for yield and somatic cell score with automatic and conventional milking systems

The objective of this study was to quantify genotype by environment interaction (G x E) between automatic milking systems (AMS) and conventional milking systems (CMS) for test-day milk, fat, and protein yield and for test-day somatic cell score (SCS) in The Netherlands. The G x E was studied in 2 ways: 1) between AMS farms and CMS farms in the same period and 2) within farms comparing the period before introduction of AMS with the period after introduction of AMS. For both sub-objectives, a separate data set was generated. Test-day records were used to be more flexible with respect to the introduction date of AMS. Multivariate, fixed regression, test-day sire models were used to estimate variance components. Genetic correlations between AMS farms and CMS farms in the same period were 0.93, >0.99, 0.98, and 0.79 for test-day milk yield, fat yield, protein yield, and SCS, respectively. Genetic correlations within farms between the period before and after introduction of AMS were lower for production traits and higher for SCS: 0.89, 0.91, 0.87, and >0.99, respectively, for test-day milk yield, fat yield, protein yield, and SCS. Heterogeneity of variance was observed between AMS and CMS in both data sets. Especially the residual variance increased with automatic milking. As a consequence, the heritability tended to be lower for automatic milking. It was concluded that effects of G x E are small between AMS and CMS. Therefore, AMS farms can select sires accurately based on national rankings.     

High expression of cell death-inducing DFFA-like effector a (CIDEA) promotes milk fat content in dairy cows with clinical ketosis

High blood concentrations of nonesterified fatty acids (NEFA) during ketosis represent a source of fatty acids for milk fat synthesis and explain the increase in milk fat content in ketotic cows. Cell death-inducing DFFA-like effector a (CIDEA) is a lipid droplet coat protein with important roles in the regulation of milk fat synthesis and secretion in mice. Whether ketosis alters the expression of CIDEA in mammary gland tissue and the extent to which it may contribute to regulation of milk fat synthesis and secretion are unknown. Mammary gland tissue and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) cows. Mammary epithelial cells isolated from cows were infected with CIDEA overexpression adenovirus for 48 h, treated with 0, 0.3, 0.6, or 1.2 mM NEFA for 24 h, or infected with CIDEA-silencing adenovirus for 48 h and treated with 1.2 mM NEFA for 24 h. Serum concentrations of NEFA and β-hydroxybutyrate were greater in cows with clinical ketosis, and milk production and dry matter intake were lower in cows with clinical ketosis. However, compared with healthy cows, the content of milk fat of cows with clinical ketosis was greater. Compared with healthy cows, abundance of mRNA and protein of CIDEA, fatty acid synthase (FASN), acetyl-coA carboxylase 1 (ACACA), butyrophilin (BTN1A1), and xanthine dehydrogenase (XDH) was greater in mammary tissue of cows with clinical ketosis. Overexpression of CIDEA in cultured mammary epithelial cells increased the abundance of FASN, ACACA, XDH, and BTN1A1, and increased triacylglycerol (TAG) content in mammary epithelial cells. Exogenous NEFA increased the abundance of CIDEA, FASN, ACACA, XDH, and BTN1A1, and increased TAG content in mammary epithelial cells. Importantly, knockdown of CIDEA reversed the upregulation of FASN, ACACA, XDH, and BTN1A1 abundance and TAG content induced by NEFA treatment. Overall, these data suggest that high levels of NEFA stimulate the expression of CIDEA and enhance de novo fatty acid synthesis and milk fat secretion. As such, these mechanisms explain in part the elevation of milk fat content in dairy cows with clinical ketosis.     

大棚高妻葡萄滴灌及施肥技术小结

从葡萄萌芽至葡萄成熟,降雨过多、降雨期过长成为制约南方葡萄产量和品质的主要因素。近年来,南方大棚葡萄栽培面积不断扩大,但5～7月的高温天气,大棚葡萄的灌溉和施肥成棘手的问题。为此,2003—2007年笔者对大棚栽培的高妻葡萄滴灌及施肥技术进行了探讨,技术小结如下。