Genetic diversity among chicken breeds estimated through randomly amplified polymorphic DNA.

The randomly amplified polymorphic DNA (RAPD) markers were used to detect polymorphism among five breeds of chicken i.e. White Leghorn and Rhodes Island Red (selected for part period egg production and egg mass respectively), Red Cornish and White Plymouth Rock (selected for early body weights) and Kadaknath (native breed). Twelve of the fifty random primers screened yielded distinct polymorphic RAPD profiles. Of the total 96 fragments amplified, about 25% showed polymorphism. Using the RAPD data matrix, the within population and between population genetic similarity was estimated. The selected improved breeds showed higher within population genetic similarity in comparison to the native breed. The two meat type breeds showed a high level of genetic similarity between themselves. The White Leghorn breed showed a low genetic similarity with other breeds. The native breed showed highest similarity with Rhodes Island Red. The dendogram was constructed to show phylogenetic relationship among these breeds. As expected, the genetic distances were lowest within similar type breeds and were highest between dissimilar type breeds. The results indicated the effectiveness of RAPD in detecting polymorphism between chicken populations and their applicability in population studies and establishing genetic relationships among the chicken populations.

Measurement of within and between population genetic variability in quails.

1. To detect polymorphism in various quail lines, Randomly Amplified Polymorphic DNA (RAPD) markers were tested and were found to be effective. 2. Twenty decamer primers were selected at random and tested, and 6 of these generated distinct polymorphic patterns between the quail lines. 3. Out of a total of 60 bands amplified using 6 selected primers, 19 (31.7%) were found to be polymorphic. 4. Genetic similarity within the lines ranged from 0.726 in HBW to 0.836 in KLQ. 5. Genetic similarity estimates between the populations ranged from 0.709 to 0.808.

Characterization of androgen receptors in a well-differentiated endometrial adenocarcinoma cell line (Ishikawa).

Androgen receptors (AR) have been identified in the human endometrium, but their role in endometrial function and development towards endometrial receptivity remains poorly understood. In an effort to study the regulation and possible function in endometrial epithelium, we utilized the well-differentiated endometrial adenocarcinoma cell line, Ishikawa, as a model system. This cell line has proven to be stable, hormonally responsive, contains both estrogen and progesterone receptors, and has been shown to express endometrial proteins in a hormone responsive manner. In the present study, we demonstrate that Ishikawa cells also express AR, based on immunohistochemical staining, radioactive binding studies, RT-PCR and Northern blot analysis. The expression of AR is induced in Ishikawa cells by estrogens, similar to that reported for normal endometrium. Further, using an estrogen-responsive gene that has been characterized in this cell line, alkaline phosphatase, we show that androgens act as antiestrogens in diethylstilbestrol (DES) treated cells, inhibiting enzymatic activity in a dose-dependent manner. These data support a physiologic role for AR in the endometrium. Elevations in endometrial AR in certain clinical situations such as polycystic ovarian syndrome (PCOS) may amplify the effects of androgens on the endometrium leading to suspected defects in uterine receptivity, higher than expected infertility and high miscarriage rates observed in patients with this disorder.

Effect of the codon following the ATG start site on the expression of ovine growth hormone in Escherichia coli.

For expression of ovine growth hormone (OGH) in inclusion bodies without an affinity histidine tag at either end of the protein, three clones, differing only in the second codon following the ATG start site, were constructed. Their expression was studied by SDS-PAGE followed by immunoblotting. Clone Ala.OGH (clone 1), beginning with Met.Ala.Phe.Pro ellipsis, did not show any expression. Clone Phe.OGH (clone 3), beginning with Met.Phe.Pro ellipsis, gave very high levels of OGH expression following IPTG induction. However, in clone Gly.OGH (clone 2), in which the Ala codon was replaced with a Gly codon at the second position after the start site, a lower level of expression was obtained. Northern hybridization analysis showed that upon IPTG induction, OGH mRNA was transcribed from all three clones. These results therefore, imply that lack of expression in clone 1 and a lower level of expression in clone 2 are not due to a failure of transcription; however, they may be due to inefficient initiation of translation. The secondary structure analysis of mRNA predicts inaccessibility of different elements of the RBS in the case of Ala.OGH (clone 1). The present study highly underscores the importance of mRNA secondary structure at the start site in regulation of expression of a cloned gene in Escherichia coli, a prokaryotic expression system.

Cloning and sequencing of buffalo male-specific repetitive DNA: sexing of in-vitro developed buffalo embryos using multiplex and nested polymerase chain reaction.

Buffalo Y-chromosome specific repetitive DNA (BuRY.I) was cloned and sequenced in order to develop a sensitive method for sexing of buffalo preimplantation stage embryos using polymerase chain reaction (PCR). A highly sensitive and reliable sex determination assay using a primary (BRY.I), nested (BuRYN.I) and multiplex (BuRYN.I, ZFX/ZFY) PCR was developed. The BRY.I and BuRYN.I primers are targeted to amplify Y-specific sequences, while the ZFX/ZFY loci was amplified to serve as a positive control for both male and female samples. Accuracy of the sex determination assay was initially verified with genomic DNA obtained from blood of known gender. Further sensitivity and reproducibility of the assay was examined using DNA obtained from 1 or 2 blastomeres to demi embryos. Altogether, 80 IVF-derived embryos ranging from the 2 to 4 cell to the blastocyst stage were used for sex determination. Definite and clear signals following PCR amplification were obtained from all embryo samples. Accuracy of assays was determined by comparing results from a single cell with those of blastocyst stage embryos, thereby indicating that 1 or 2 blastomeres from a preimplantation buffalo embryo is sufficient for sex determination by PCR. No misidentification was observed within the embryo samples using nested (BuRY.I), primary (BRY.I) and multiplex (BuRYN.I; ZFX/ZFY) PCR, suggesting that this technique is a highly reliable method for sexing buffalo embryos.

High-level expression of ovine growth hormone in Escherichia coli: single-step purification and characterization.

The gene for ovine growth hormone (oGH) was expressed without signal sequences in Escherichia coli. A recombinant plasmid expression vector has been constructed which directs the synthesis of a fusion protein containing a stretch of six histidine residues (His6) at the amino-terminus under the control of a T5 promoter. Upon induction with isopropyl-beta-D-thiogalactopyranoside, the recombinant protein was synthesized and accumulated in the cytoplasm in the form of inclusion bodies, at levels of approximately 18% of the total cellular protein. The recombinant ovine growth hormone containing His tag was recovered and purified to >95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity Ni2+.NTA resin that has selectivity for proteins with neighboring histidine residues. Characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and amino terminal analysis demonstrated the authenticity of the fusion protein. The purified RoGH after refolding was found to be functionally active in terms of its receptor binding and antigenicity as analyzed by radio receptor assay and radio immuno assay. Yields of the purified expressed protein were found to be 32 microg/ml at a shake-flask level. Thus, results indicate that a combination of E. coli expression and affinity purification by Ni2+.NTA chromatography promises to be a rapid method to produce oGH for use in structure-function studies.

Differential cleavage and developmental rates and their correlation with cell numbers and sex ratios in buffalo embryos generated in vitro.

In vitro matured and fertilized buffalo oocytes were co-cultured with buffalo oviductal epithelial cells (BOEC) in CRlaa medium. Cleaved embryos were separated according to the time of completion of first cleavage (i.e., before 30 h and after 30 h post insemination) and cultured for 5 to 10 d and allowed to develop to the blastocyst stage. Zygotes cleaving before 30 h were termed fast-cleaving while those cleaving after 30 h were termed slow-cleaving. The results indicated that fast-cleaving embryos are more likely to develop into blastocysts (25%) than slow-cleaving embryos (7.8%). The quality and viability of fast-cleaving and fast-developing blastocysts was found to be better than that of slow-cleaving, slow-developing blastocysts as judged by cell numbers (67.7 +/- 3.7 vs 35.2 +/- 2.1). However, the mitotic index was not different between the 2 groups. The sex of fast-developing and slow-developing blastocysts was determined via the polymerase chain reaction (PCR) to correlate the rate of embryonic development with the sex ratio of the embryos. Embryos produced by Bull 293 and Bull M-82, irrespective of their being fast or slow-developing, gave rise to more females and males, respectively. From these results, we suggest that there may be a sire effect on sex ratio of in vitro produced buffalo embryos.

In vitro maturation of buffalo oocytes: role of insulin and its interaction with gonadotrophins.

A variety of signals is involved in the acquisition of competence to resume meiosis in buffalo oocytes. There is evidence that gonadotrophins are required for the resumption of meiosis and to achieve developmental competence. However, the source of the serum supplement also influences maturation of buffalo oocytes. Fetal calf serum was more effective at achieving maturational competence compared with oestrous serum. All the changes were found to be supported and strictly regulated by the somatic cells of the follicle. Further studies revealed that a variety of ovarian autocrine and paracrine factors may modulate the maturation of buffalo oocytes. The role of growth factors, particularly insulin, in in vitro maturation and granulosa cell differentiation is discussed.

Sex-specific identification of raw meat from cattle, buffalo, sheep and goat.

A simple and reliable method has been developed for accurate identification of male and female raw meats in cattle, buffalo, sheep and goat using the polymerase chain reaction (PCR) technique. The PCR assay was conducted on genomic DNA extracted from raw muscle tissue of male and female animals. The method has been found to be accurate, reliable and quick.

Biochemical studies on goat oocytes: Timing of nuclear progression, effect of protein inhibitor and pattern of polypeptide synthesis during in vitro maturation.

Temporal progression of nuclear events of goat oocytes matured in vitro was studied by adding a specific inhibitor to the culture medium at different time points, to investigate protein synthesis requirements and its pattern during in vitro maturation. Goat cumulus-oocyte complexes (COCs) were matured in vitro in TCM 199, fixed at different time intervals and stained with orcein to assess nuclear changes. The germinal vesicle (GV) stage was found to be present at 0 h, chromosomal condensation stage was observed at 8 h, metaphase I at 12 to 14 h, and metaphase II was begun after 16 h of maturation and was nearly completed at 24 h. Protein synthesis inhibitor, cycloheximide, blocked oocyte maturation at germinal vesicle breakdown(GVBD), if added to the maturation medium between 0 to 4 h, suggesting that protein synthesis is required for GVBD. The transition from metaphase I to metaphase II was also protein synthesis-dependent, as observed when cycloheximide was used between 8 to 10 h of culture. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was progressively restored, but many chromosomal abnormalities were noted. Changes in the protein synthesis pattern were studied by radiolabeling of oocytes with [(35)S]-methionine at 0, 7, 12 and 24 h of culture, corresponding with GV, GVBD, metaphase I and metaphase II stages. A polypeptide of 28.1 KDa appeared as a major band at the GV stage, and its size decreased greatly and disappeared after the GVBD stage. Three new polypeptides (35, 36.5 and 39 KDa) appeared at GVBD and were detectable at metaphase II. In conclusion, the synthesis of proteins is required for the maintenance and transition of goat oocytes from GV to metaphase II during in vitro maturation.

Ultrasonographic imaging to monitor early pregnancy and embryonic development in the buffalo (Bubalus bubalis).

Real time B-mode ultrasound was used to detect and monitor the early conceptus, its growth and its anatomical features in 26 buffalo between Days 18 and 62 of gestation. The buffalo were artificially inseminated, and the conceptuses were examined on alternate days beginning on Day 18. The embryonic vesicle and the embryo proper within the vesicle was first visible in 12 of the buffalo on mean Day (+/-SD) 19.00+/-2.1 and Day 19.0+/-1.69, respectively. The heartbeat of the embryo proper could be detected on Day 29.6+/-1.57. The heart rate of 203.8+/-9.0 beats per minute on the first day of detection decreased to 150 beats per minute on Day 62. The allantois, amnion, fore limbs, spinal cord and hind limbs were first identified on Day 30.0+/-1.14, Day 33.4+/-1.64, Day 34.6+/-1.34, Day 35.8+/-2.52 and Day 36.8+/-2.34, respectively. The optic area was first distinguished on Day 38.2+/-2.39. Split hooves, fetal movement, ribs and vertebra were identified on Day 46.0+/-2.64, Day 49.4+/-2.31 and Day 59.8+/-2.39, respectively. The mean length of the embryo proper was 4.2 mm on Day 19 which later increased to 53.6+/-2.1 mm on Day 62.

Polymerase chain reaction and its applications: special emphasis on its role in embryo sexing.

The polymerase chain reaction (PCR) has developed into one of the most promising methods for in vitro enzymatic amplification of DNA and has found widespread application in DNA cloning, sequencing and mutagenesis related studies. This innovative technique can selectively amplify a single target DNA molecule a billion-fold in a span of a few hours. Amplification of specific DNA sequences by PCR is useful in identification of sex, novel genes, pathogens and diseases. PCR has facilitated the establishment of evolutionary relationships among species and in revealing structural intricacies of single cells. In this article we review some of the major advances and applications of PCR, especially, its role in embryo sexing.