Sexing greater flamingos by weight and linear measurements

The sex of 59 adult Greater Flamingos (Phoenicopterus ruber roseus L.) was determined by laparoscopic exam of the gonads. Concomitant body weight (kg) and linear measurements (mm) of the culmen (bill), tarsus, middle toe, and wing were taken and compared for males vs. females. Although an overlap between sexes existed in all measurements, males on average were larger than females. Student's t-test indicated significant sexual differences for all five measurements. Thus, weights and linear measurements—especially tarsus, middle toe, and wing length—appear to be a useful parameter in determining an individual's sex in lieu of laparoscopic, feather pulp, blood chromosome, or fecal steroid analysis.     

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo''s sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.     

牛冷冻试管胚胎性别鉴定的应用

利用双引物PCR技术对优质良种牛体外受精的冷冻胚胎进行性别鉴定。并对分割后的半胚分别进行PCR扩增和染色体分析,两种方法的判定结果相吻合,在本实验条件下,利用减少PCR体系中细胞的数量来确定最低的模板DNA的检出量,2－细胞不能检测,4－细胞检出率为62．5％,8－细胞为90％,16－细胞以上为100％。对23牧经过性别鉴定后的胚胎进行移植,获得5头纯种试管牛,产犊率为21．7％,与正常的胚胎移植 结果27．3％（3／11）无显著差异,预测性比为100％符合。     

Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.     

Sexing in vitro produced bovine embryos, at different stages of development, using rat H-Y antiserum

The male-specific H-Y antigen is present on mammalian cell membranes and has been identified by various methods, including antiserum cytotoxicity. The objective of the present study was to determine the sex of in vitro produced (IVP) bovine embryos, at varying stages of development, by culturing in the presence of rat monoclonal H-Y antibodies. Embryos derived from IVM/IVF were classified according to the interval after IVF (48, 96 or 120 h) as Category 1, 2 or 3 if they had 4 to 8, <32, and >32 cells, respectively. Embryos of each category were cultured for 24h in TCM-199 supplemented with bovine oviductal epithelial cells, fetal calf serum (FCS), and antibiotics (Control group), to which the following had been added: guinea pig serum (GPS; C' group); H-Y antiserum (HY group); or GPS and H-Y antiserum (C' + HY group). After culture, embryos were designated as "affected" when development was arrested or one or more blastomeres was degenerate; embryos lacking these changes were designated "unaffected." The sex of each embryo was subsequently determined by chromosome analysis. After 48h of IVF (Category 1), within each of the four treatments, the proportion of unaffected embryos was higher than the proportion of unaffected embryos (81% versus 19%, P < 0.05). Similarly, the Control, C' and HY groups of Categories 2 and 3 embryos had different proportions of unaffected versus affected embryos (75% versus 25%, P < 0.05). In all these groups, the male:female ratio did not significantly differ from 1:1. In contrast, in the C' + HY group of Categories 2 and 3 embryos, the ratio of unaffected versus affected embryos was 41% versus 59% (P < 0.05) and the male:female ratio differed (P < 0.05) from the expected 1:1 ratio (approximately 0.3:1 and 4.5:1 for unaffected versus affected, respectively). In conclusion, when bovine embryos were cultured in the presence of rat monoclonal H-Y antibodies and compliment, alterations occurred in embryos that were beyond the 8-cell stage; we inferred that the antibodies cross-reacted with H-Y antigens.     

PCR hot-start using duplex primers

A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirable products arising throughout PCR amplification thereby giving better results than a manual hot-start method.     

Vitrification of in vitro produced ovine embryos at various developmental stages using two methods

This study was conducted to evaluate the effects of developmental stage of in vitro produced (IVP) ovine embryos and the type of vitrification procedure used on embryo cryotolerance.The IVP embryos were vitrified at five different developmental stages: 4-, 8- and 16-cell, morula, and blastocyst. For each stage, half of the embryos were vitrified in either 30 μl 3.4 M glycerol + 4.6 M ethylene glycol in straw (method 1) or in <0.1 μl 2.7 M ethylene glycol + 2.1 M Me₂SO + 0.5 M sucrose placed on the inner surface of a straw (method 2) of vitrification solution, based on two different procedures. After warming embryo viability was determined by assessing the rates of re-expansion, survival, and blastocyst formation. The quality of surviving embryos was evaluated by their hatching rate and blastocyst cell numbers. In both vitrification methods, embryo survival progressively increased as the developmental stage progressed. In method 1 few of the early cleavage stage embryos (4-, 8- and 16-cell) could reach to the blastocyst stage following warming. There was no significant difference in blastocyst cell numbers (total, ICM, and trophectoderm cells) or hatching rate of blastocysts derived from vitrified embryos at different developmental stages. The number of dead cells in vitrified blastocysts in method 1 was higher than for non-vitrified blastocysts (P < 0.05). The number of apoptotic cells in vitrified blastocysts was higher than for non-vitrified counterparts (P < 0.05). In conclusion, both the developmental stage of IVP ovine embryos and the method of vitrification have a significant effect on the viability and developmental competence of sheep embryos.     

Sexing fish by palpation: a simple method for gonadal assessment of fluvial salmonids

This paper presents a non‐destructive sexing method that can be employed in fluvial salmonids and presumably some other taxa of fish. The sex of a fish is identified and assessed by indirectly palpating the gonad.     

A Comparison of Field and Molecular Techniques for Sexing Beavers

Abstract: The traditional method of sex identification in beavers (Castor canadensis) by external palpation can be inaccurate. We tested 2 genetic methods for determining sex in beavers, the zinc-finger DNA marker and the Y chromosome-specific sex determining region (SRY) marker. The SRY marker identified sex correctly in 57 of 67 (85%) beavers, whereas the zinc-finger technique was successful less often in only 48 of 67 (72%) animals. Sex was correctly assigned by palpation for 21 of 27 beavers (78%). Beaver studies in which accurate sex identification is critical may benefit by verifying the sex of individuals using one or both of these molecular markers.     

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.     

Ultrastructural characterization of fresh and cryopreserved in vivo produced ovine embryos

Controlled slow freezing and vitrification have been successfully used for ovine embryo cryopreservation. Selection of embryos for transfer is based on stereomicroscopical embryo scoring after thawing, but the subjectivity inherent to this selection step has been demonstrated by ultrastructural studies of controlled slow frozen, in vivo produced ovine morulae and blastocysts. These studies have shown that certain abnormalities remain undetected by stereomicroscopy only. In the present study, using ovine in vivo produced morulae and blastocysts, we have studied the ultrastructural alterations induced by open pulled straw vitrification (OPS) and controlled slow freezing, compared stereomicroscopical embryo scoring with light microscopy evaluation of embryo's semithin sections, and related the ultrastructural cellular damage with the embryo classification by stereomicroscopical embryo scoring of embryos’ and semithin section evaluation by light microscopy. The ultrastructural lesions found for OPS-vitrified and controlled slow frozen embryos were similar, independently of embryo stage. A significant higher number of grade 3 embryos was found at stereomicroscopical scoring after controlled slow freezing (P = 0.02), and a significant higher number of grade 3 blastocysts was found at semithin sectioning after OPS vitrification (P = 0.037). The extension of ultrastructural damage, especially of mitochondria and cytoskeleton, was related to the semithin classification but not to stereomicroscopical scoring at thawing. This suggests that semithin scoring is a useful tool for predicting ultrastructural lesions and new improvements in cryopreservation and thawing methods of ovine embryos are still warranted, including in the case of blastocysts cryopreserved by OPS vitrification.     

In vitro development of ovine embryos in CZB medium

One- to four-cell embryos were collected from multiparous crossbred ewes and were cultured in vitro for 120 hours in CZB medium. A 2x2 factorial treatment arrangement was used to examine the effects of glucose and ethylenediaminetetraacetic acid (EDTA) on in vitro embryo development. The embryos were examined every 12 hours, and all of the embryos were stained with a DNA-specific fluorochrome after the 120-hour evaluation to enable the counting of cell nuclei. Embryo development was analyzed for cleavage beyond 16 cells as well as for cleavage to at least the compact morula stage based upon both the 120-hour morphological evaluation and nuclear counts. Forty-eight percent of the embryos passed through the in vitro developmental block (i.e., cleaved beyond 16 cells), and 26% developed to 30 or more cells. Neither EDTA nor glucose affected in vitro embryo development based on the nuclear counts.     

Developmental,qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro

The objective of this study was to compare bovine and ovine oocytes in terms of (1) developmental rates following maturation, fertilization, and culture in vitro, (2) the quality of blastocysts produced in vitro, assessed in terms of their ability to undergo cryopreservation, and (3) the ultrastructural morphology of these blastocysts. In vitro blastocysts were produced following oocyte maturation/fertilization and culture of presumptive zygotes in synthetic oviduct fluid. In vivo blastocysts were used as a control from both species. In Experiment 1, the cleavage rate of bovine oocytes was significantly higher than that of ovine oocytes (78.3% vs. 58.0%, respectively, P < 0.001). The overall blastocyst yield was similar for both species (28.7% vs. 29.0%). However, when corrected for cleavage rate, significantly more ovine oocytes reached the blastocyst stage at all time-points (36.6% vs. 50.0% on day 8, for bovine and ovine, respectively, P < 0.001). Following vitrification, there was no difference in survival between in vivo produced bovine and ovine blastocysts (72 hr: 85.7% vs. 75.0%). However, IVP ovine blastocysts survived at significantly higher rates than IVP bovine blastocysts at all time points (72 hr: 47.4% vs. 18.1%, P < 0.001). At the ultrastructural level, compared with their in vivo counterparts, IVP blastocysts were characterized by a lack of desmosomal junctions, a reduction in the microvilli population, an increase in the average number of lipid droplets and increased debris in the perivitelline space and intercellular cavities. These differences were more marked in bovine IVP blastocysts, which also displayed electron-lucent mitochondria and large intercellular cavities. These observations may in part explain the species differences observed in terms of cryotolerance. In conclusion, the quality of ovine blastocysts was significantly higher than their bovine counterparts produced under identical in vitro conditions suggesting inherent species differences between these two groups affecting embryo quality.     

Determination of sex and scrapie resistance genotype in preimplantation ovine embryos

The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer to recipients. Sex and PrP genotypes were determined. Sex diagnosis was done by PCR amplification of ZFX/ZFY and SRY sequences after PEP-PCR while PrP genotype determination was performed after specific pre-amplification of specific target including codons 136, 154 and 171. Embryos were collected at Day 7 after oestrus. Blastocysts and expanded blastocysts were biopsied immediately after collection whereas compacted morulae were biopsied after 24 hr of in vitro culture. Eighty-nine biopsied embryos were frozen by vitrification. Fresh and vitrified whole embryos were kept as control. DNA of biopsies was extracted and pre-amplified. Sex diagnosis was efficient for 96.6% of biopsies and PrP genotyping was determined in 95.8% of codons. After embryo transfer, no significant difference was observed in lambing rate between biopsied, vitrified control and fresh embryos (54.5%, 60% and 66.6%, respectively). Embryo survival rate was not different between biopsied and whole vitrified embryos (P = 0.38). At birth, 96.7% of diagnosed sex and 95.4% of predetermined codons were correct. Lamb PrP profiles were in agreement with parental genotype. PEP-PCR coupled with sex diagnosis and nested PCR coupled with PrP genotype predetermination are very accurate techniques to genotype ovine embryo before transfer. These original results allow planning of selection of resistant genotype to scrapie and sex of offspring before transfer of cryopreserved embryo.     

PCR技术在性别鉴定及性别控制应用中的研究进展

PCR技术是一项发展迅猛的生物技术,因具有快速、灵敏、简便及特异性强等特点而被广泛应用于性别鉴定及其它许多相关研究领域。应用于性别鉴定的PCR方法从简单PCR法、双重或多重PCR法、巢式PCR法发展到改进的两温度梯度PCR法;而不同性别间除了呈现有或无关系（类似于Sry 基因）的基因序列外,也检测到了很多类似于锌指蛋白和牙釉蛋白的呈现不同性别特征的基因序列,这为性别鉴定引物的设计和PCR法进行性别鉴定提供了另一种全新的思路,即如果根据这种性别多态性DNA序列特点设计引物,采用两温度梯度PCR扩增技术进行PCR性别鉴定,则可望简化鉴定程序、降低检测时间、提高鉴定效率,使PCR性别控制技术更加成熟和实用化。随着研究的深入,PCR技术在性别鉴定及控制的应用中必将日益成熟,并推动此项技术在其它相关领域中的研究和应用取得更大的进展。     

运用PCR对小鼠植入前胚胎进行性别诊断

根据C57BL6小鼠Y染色体重复序列145C5的碱基顺序,设计并合成一对引物,运用PCR扩增昆明白小鼠入前胚胎卵裂球DNA,以确定其性别,共对108枚活检胚胎的相应卵裂球进行了性别诊断,获雄性胚46枚,雌性胚62枚,移植后分别获雄性仔鼠4只,准确率100％（4／4）,雌性仔鼠9只,准确率70％（9／13）,本研究结果表明小鼠Y染色体重复序列145C5的碱基顺序在C57BL6小鼠和昆明白小鼠中基本一致,为农牧业动物进行性别选择和运用PCR进行单基因病植入前遗传学诊断提供了方法学基础。     

应用双重PCR检测环境水体中的军团菌

建立双重PCR方法以检出环境水体中的军团菌。设计两对引物,分别扩增军团菌的16S rRNA和M ip基因,扩增片段长各为375bp和996bp。该方法检测军团菌的灵敏度为5.8×102cfu/m l,6株嗜肺标准军团菌均扩增出996bp和375bp两条带,4株非嗜肺军团菌扩增出375bp条带,4株非军团菌无条带;检测71份环境水样,5份出现两条条带,2份可见375bp条带,阳性率为7.0%。该方法快速、灵敏、特异,为水体中的嗜肺军团菌检测提供了有效方法。     

荧光定量PCR测定端粒长度

目的应用实时荧光定量聚合酶链式反应（Q—PCR）方法测定端粒长度。方法选取9种人类细胞株,提取基因组DNA,采用Q—PCR方法测定相对T／S比率,DNA印迹法测定末端限制性片段（TRF）长度,进行二者之间的相关性分析。结果定量PCR测定端粒长度相对T／S比率为0．68±0．57,DNA印迹法测量平均TRF值为8．57±2．34,两种方法测定结果的相关性分析R2=0．7807（P〈0．01）。结论采用荧光定量PCR方法测量端粒长度具有重复性好、省时、简便、可靠的特点,可高通量处理大量样品。     

In vitro developmental competence of ICSI-derived activated ovine embryos

The present study was conducted to determine the necessity for activation after intracytoplasmic sperm injection (ICSI) in sheep. The effect of chemical stimulation with either 5 μM ionomycin (I) for 5 min or ionomycin + 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h on the efficiency of ICSI, was compared in six experimental groups: (1) ICSI, (2) ICSI + I, (3) ICSI + I + 6DMAP, (4) Sham, (5) Sham + I, and (6) parthenogenetics (Sham and parthenogenetic groups were used as controls). In the present study, ovine oocytes needed additional chemical stimulation, after conventional ICSI, to activate (female pronucleous formation) and to form zygotes with male and female pronuclei (2PN). The percentage of cleaved embryos obtained and developed to blastocyst stage was higher (P < 0.001) for ICSI-derived zygotes, followed by activation (I and I + 6DMAP; 18.2 and 22.5%, respectively) than ICSI and Sham injection without activation (3.0 and 0.0%, respectively). There was, however, no significant difference between activation protocols I or I + 6DMAP. Furthermore, there was no significant difference among chemically activated, ICSI-derived zygotes in term of hatchability rate; however, the percentage was significantly higher in parthenogenetic and IVF groups than ICSI and Sham injection. In conclusion, neither sperm alone nor mechanical activation was sufficient for ovine oocyte activation and pronuclei formation. Therefore, in our study conditions for in vitro embryo development, chemical activation of oocytes must be considered an essential part of the ICSI procedure in sheep.