A genetic study of bovine lymphocyte antigens (BoLA) and their frequency in several breeds

Using sera which defined the BoLA specificities at the two International BoLA workshops (Edinburgh, 1978 and Wageningen, 1980) and the European Regional workshop (Paris, 1979),142 informative matings from 15 bulls have been studied. On the basis of this data, 11 of the 15 internationally agreed specificities and one of the regionally defined specificities behave as if controlled by alleles at a single autosomal locus. Data has not as yet been obtained for the other four internationally agreed specificities which are also believed to be at this locus. — The frequencies of 13 of the internationally agreed specificities and one of the regionally defined specificities have been studied for both sexes in one breed and for a single sex in another five breeds. The other two internationally agreed specificities are very recent and the populations have not been tested for them. The frequencies between sexes within a breed and within sexes between breeds differ significantly.     

Absence of BoLA class I antigens on bovine spermatozoa

Forty AI bulls were tested for BoLA class I antigens by means of eight specific polyclonal reagents. By means of immobilization and sperm penetration tests these antigens were not detected on sperm cells. Isoimmunization studies with the use of sperm as antigenic stimuli and insemination of frozen spermatozoa diluted in specific reagents did not prove the presence of BoLA class I antigens on bovine spermatozoa. The cytotoxic tests used in this investigation were not reliable.     

Absence of BoLA class I antigens on bovine spermatozoa

Summary. Forty AI bulls were tested for BoLA class I antigens by means of eight specific polyclonal reagents. By means of immobilization and sperm penetration tests these antigens were not detected on sperm cells. Isoimmunization studies with the use of sperm as antigenic stimuli and insemination of frozen spermatozoa diluted in specific reagents did not prove the presence of BoLA class I antigens on bovine spermatozoa. The cytotoxic tests used in this investigation were not reliable.     

Cryopreservation of in vitro produced bovine embryos: effects of lipid segregation and post-thaw laser assisted hatching

The objective was to determine if lipid segregation, with or without post-thaw laser assisted hatching (LAH) of in vitro produced (IVP) bovine embryos, would enhance in vitro survivability and development 24 h post-thaw. On Day 6 of culture (Day 0 = IVF), in vitro produced bovine embryos were divided into three developmental stages: 32-cell (n = 78), compact morula (CM n = 223), and blastocyst (n =56). Embryos within each stage were allocated to the following treatments prior to cryopreservation in 1.5M ethylene glycol: no treatment (Control), 7.5 μg/mL Cytochalasin B for 20 min (CB), or CB with centrifugation (16,000 × g) for 20 min (CBCF). All CB treatments were extended to include embryo freezing. Immediately post-thaw, one-half of the CBCF and Control groups were subjected to zona pellucida drilling (LAH), using the XY Clone® system, creating groups CBCFLAH and LAH, respectively. All thawed embryos were cultured for 24 h and evaluated. No treatment differences were observed for either post-thaw survival or 24 h development. Within the CM stage, CBCFLAH and LAH exhibited a greater number of both total and live cells than Control (total: 69.4, 69.3, 53.0, live: 56.4, 54.7, 39.3 respectively; P < 0.05). In conclusion, LAH post-thaw alone or in combination with CBCF improved embryo viability following cryopreservation.     

Effects of parental ploidy level and genetic divergence on chromosome elimination and chloroplast segregation in somatic hybrids within Brassicaceae

Summary Chromosome and organelle segregation after the somatic hybridization of related species with different degrees of genetic divergence were studied by comparing the interspecific somatic hybrids Brassica oleracea (CC) (+) B. campestris (AA), B. napus (AACC) (+) B. oleracea (CC) B. napus (AACC) (+) B. nigra (BB) and B. napus (AACC) (+) B. juncea (AABB) with the intergeneric somatic hybrids B. napus (AACC) (+) Raphanus sativus (RR) and B. napus (AACC) (+) Eruca sativa (EE). Within each combination, some hybrids were found whose DNA content was equal to the sum of parental chromosomes, others had a relatively higher DNA content and in most of the cases, some had a relatively lower content. However, the frequency distribution in these three classes differed significantly between the combinations. A positive correlation between the frequency of hybrids with eliminated chromosomes and the genetic distance between the species in each combination was found. Furthermore, by combining species with different ploidy levels we found a significantly higher degree of chromosome elimination compared to combinations of species with the same ploidy level. In the B. napus (+) B. Nigra, B. napus (+) R. sativus and B. napus (+) E. sativa combinations chromosomes from the B, R and E genomes appeared to be preferentially sorted out, as indicated by the fact that some of the nuclear markers from these genomes were missing in 7–46% of the plants, whereas no plants were lacking B. napus nuclear markers. Fertile hybrids were found in all but the B. napus (+) R. sativus fusion combination; the latter hybrids were male sterile, but female fertile. Hybrids between the A and C genomes were more fertile than hybrids obtained between the distantly related AC and B, R or E genomes, respectively. Analysis of the chloroplast RFLP pattern revealed that chloroplasts in the B. oleracea (+) B. campestris hybrids segregated randomly. A slightly biased segregation, favouring B. napus chloroplasts, was found in the B. napus (+) B. oleracea combination, whereas B. napus chloroplasts were strongly selected for in the B. napus (+) B. juncea, B. napus (+) B. nigra, B. napus (+) R. sativus and B. napus (+) E. sativa somatic hybrids.     

Sources of bovine lymphocyte antigen (BoLA) typing reagents*

Sera from about 1000 cows were tested for cytotoxicity against a panel of up to 100 lymphocyte samples. Cytotoxic antibodies presumably resulting from trans-placental immunization of the cow by her calf were found in about 45% of these sera. The antibody titers of sera from parous cows rarely exceed 4², some persisted for over one year, but decreased notably at calving. Thirty-five immune sera were also produced by alloimmunization with lymphocytes. They usually reached peak titers of up to 4⁴ at 2 or 3 weeks after the initial immunization. Subsequent immunizations produced sera with very high titers but they were much more polyspecific. High-titered antibodies were also produced by skin graft recipients. Useful cytotoxic antibodies were found in 19 of 111 colostrum whey samples. Studies on 13 dam-calf pairs showed that the newborn calf may acquire cytotoxic antibodies from its mother's colostrum, but the only cytotoxic antibodies detectable in this calf s serum are those not directed against its own lymphocyte antigens. It is concluded that efficient lymphocyte typing requires antibodies from a variety of sources.     

The segregation of inner and outer cells in porcine embryos follows a different pattern compared to the segregation in mouse embryos

The mammalian blastocyst consists of an inner cell mass (ICM) enclosed by the trophectoderm. The origin of these two cell populations lies in the segregation of inner and outer cells in the early morula. In the present study, the segregation of inner and outer cells has been studied in porcine embryos and is compared with segregation in mouse embryos. For this, nuclei of inner and outer cells were differentially labelled with two fluorochromes after partial complement-mediated lysis of the outer cells. In porcine and mouse embryos compaction and the first appearance of inner cells occur at different stages of development. In porcine embryos compaction was observed as early as the 4-cell stage, while in mouse embryos compaction occurred in the 8-cell stage. The first inner cells segregated in porcine embryos which were in the transition from four to eight cells and inner cells were added during two subsequent cell cycles. In mouse embryos inner cells segregated predominantly during the fourth cleavage division. From the results obtained we conclude that the segregation of inner and outer cells follows a different pattern in mouse and in porcine embryos.     

Nonrandom segregation of parental alleles in reovirus reassortants.

To test for nonrandom segregations among their 10 genomic RNA segments, we examined a set of 83 reassortants derived from mammalian reovirus type 1 Lang and type 3 Dearing. After confirming the genotypes of the reassortants, we performed statistical analyses on the distributions of parental alleles for each of the 10 gene segments, as well as for the 45 possible pairings of the 10 segments. The analyses revealed nonrandom associations of parental alleles in the L1-L2, L1-M1, L1-S1, and L3-S1 segment pairs, at levels indicating high statistical significance (P < 0.005). Such associations may reflect specific interactions between viral components (protein-protein, protein-RNA, or RNA-RNA) and may influence both the evolution of reoviruses in nature and their genetic analysis in the laboratory. The data may also support an hypothesis that reovirus reassortants commonly contain mutations that improve their fitness for independent replication.     

Association between BoLA and subclinical bovine leukemia virus infection in a herd of Holstein-Friesian cows

The role of the bovine major histocompatibility system (BoLA) in subclinical bovine leukemia virus (BLV) infection was investigated in a herd of Holstein-Friesian cows (n=240). The BoLA W8.1 allele was negatively associated with the presence of antibodies to the major BLV envelope glycoprotein, BLV-gp51 (corrected P<0.001, relative risk =0.31). These results suggest that a BoLA-linked gene(s) may influence the early spread of BLV infection. Since B cells are the primary target of BLV infection, we then determined the relationship between BoLA-A locus phenotypes and B-cell numbers in peripheral blood of seropositive and seronegative cows. There were no significant differences between BoLA-A alleles for any hematological parameter in seronegative cows. Seropositive cows with the W12.1 allele had significantly greater absolute numbers of lymphocytes per microliter and B cells per microliter than did seropositive cows with other BoLA-A phenotypes (P<0.01, respectively). The average effect associated with the W12.1 allele in BLV-infected cows was an increase of 2010 B cells per microliter of whole blood relative to BLV-infected cows with other BoLA-A phenotypes. These results demonstrate that susceptibility to the polyclonal expansion of BLV-infected B lymphocytes is associated with the W12.1 allele in Holstein-Friesian cattle. Compared with results of a previous study in a herd of Shorthorn cattle, it appears that resistance and susceptibility to subclinical progression of BLV infection are associated with different BoLA-A locus alleles in different cattle breeds.Abbreviations used in this paper AGID agar gel immunodiffusion - BLV bovine leukemia virus - BoLA bovine lymphocyte antigen - EBL enzootic bovine leukosis - HLA human leukocyte antigen - MHC major histocompatibility complex - PL persistent lymphocytosis     

Expression of a male-specific factor on various stages of preimplantation bovine embryos

Four-cell to blastocyst stage bovine embryos were collected from superovulated donors and cultured for 90 min in Ham's F-10 medium (HF-10) containing 10% (V/V) absorbed anti-histocompatibility (H)-Y antiserum. Embryos were then washed 3 times and placed in HF-10 supplemented with 10% (V/V) fluorescein isothiocynate (FITC)-conjugated goat anti-mouse gamma globulin. After an additional wash, embryos were placed in fresh drops of HF-10, individually evaluated at 200 X magnification, and classified as either fluorescent (H-Y-positive) or nonfluorescent (H-Y-negative). Embryos were then placed in drops of HF-10 containing 14% vinblastin and cultured for 4-6 h. Embryos were coded and individually karotyped, and the sex chromosomes were identified. H-Y antigen was detected as early as the eight-cell stage, but not at the four-cell stage. Seventy-nine percent of fluorescent embryos and 89% of nonfluorescent embryos were XY and XX, respectively. Another experiment was carried out in which H-Y antigen was detected on intact inner cell masses (ICM) isolated by immunosurgery from expanded blastocysts that also had been assayed for H-Y antigen. Eighty-eight and 92%, respectively, of ICM classified as fluorescent or nonfluorescent had been scored the same as intact blastocysts. It is concluded from these data that H-Y antigen can be detected on eight-cell to blastocyst stage bovine embryos. There appears to be a localization of detectable antigen in the area of the ICM at the expanded blastocyst stage. Detection of H-Y antigen is an effective, noninvasive method for identification of the sex of preimplantation bovine embryos.     

Differential staining and segregation of parental chromosomes in mouse-rabbit hybridomas.

Hoechst 33258 fluorescent staining can be coupled with G-banding to identify the chromosomal contribution of each parent in mouse-rabbit hybridomas. A fast and essentially complete segregation of rabbit chromosomes is obtained in these cells. The rabbit X chromosome is preferentially maintained in media imposing HGPRT activity for cell growth. Mouse-rabbit hybridomas, some of which secrete rabbit immunoglobulin chains, should be a convenient material for the identification of chromosomes governing rabbit Ig chain synthesis.     

Predicting parental genotypes and gene segregation for tetrasomic inheritance

Recent genome mapping projects in tetraploid plant species require a method for analysing the segregation patterns of molecular marker loci in these species. The present study presents a theoretical model and a statistical analysis for predicting the genotypes of a pair of tetraploid parents at a codominant (for example, RFLPs, microsatellites) or dominant (for example, AFLPs, RAPDs) molecular marker locus based on their and their progeny’s phenotypes scored at that locus (gel-band patterns). The theory allows for null alleles and for any degree of double-reduction to be modelled. A simulation study was performed to investigate the properties of the theoretical model. This showed that in many circumstances both the parental genotypes can be correctly identified with a probability of nearly 1, even when the molecular data were complicated by null alleles or double-reduction. Configurations where the parental genotype cannot be identified are discussed. The power to detect double-reduction varies considerably, depending on the proportion of identical alleles carried and shared by the parents, and the number of null alleles. Incorrect deductions of the occurrence of double-reduction were rare. The method was applied to data on a microsatellite locus segregating in the parents and 74 offspring of a tetraploid potato cross. Twentyfour parental configurations were consistent with the parental gel pattern, but only one of these was compatible with all the phenotypic data on the offspring. The feasibility for extending the present model to predict segregation of several linked loci, and particularly the linkage phase, is briefly discussed. Received: 7 June 1999 / Accepted: 28 September 1999     

Quick-splitting of bovine embryos

Described is a simplified method of bovine embryo bisection amenable to on-farm embryo transfer. Using a microblade operated by a hand-held micromanipulator, Day 7 bovine embryos were bisected while in the zona pellucida. With a vertical motion, the embryo was pinned between the blade and the bottom of a plastic petri dish and bisected. Demi-embryos were transferred nonsurgically (without zonae pellucidae) into synchronized recipients. Pregnancy rates were normal with 5 13 (38%) and 9 20 (45%) of recipients confirmed pregnant 70 to 80 d after receiving either twin or single half embryos, respectively. This compared to 12 28 (43%) of recipients becoming pregnant from transfer of whole embryos. These data confirm that bovine demi-embryos do not need zonae pellucidae on Day 7 and that simplified field methods of bisection give normal pregnancy results.