Search

CASE HISTORY AND CLINICAL FINDINGS: A dairy cow, from a herd in the Waikato region of New Zealand, was reported with regenerative anaemia on 12 September 2014. Testing of blood from the animal using PCR assays forTheileria orientalisproduced a negative result for both Chitose and Ikeda types. LABORATORY FINDINGS: Using PCR and DNA sequencing, blood from the cow was positive forCandidatusMycoplasma haemobos. Further testing of another 12 animals from the case herd, 27 days after the affected cow was first reported, showed 11 animals were positive forCandidatusM. haemobos orMycoplasma wenyoniiin the PCR. None of these cattle were clinically anaemic or positive forT. orientalisIkeda type using PCR. A convenience sample of 47 blood samples from cattle throughout New Zealand, submitted to the Investigation and Diagnostic Centre (Ministry for Primary Industries) for surveillance testing forT. orientalisIkeda, was selected for further testing for bovine haemoplasmas. Of these samples, 6/47 (13%) and 13/47(28%) were positive forM. wenyoniiandCandidatusM. haemobos, respectively. There was no difference in the proportion of samples positive for the bovine haemaplasmas between cattle with anaemia that were negative forT. orientalis(6/20, 33%), or without anaemia orT. orientalis(10/18, 56%), or from cattle herds experiencing anaemia and infection withT. orientalisIkeda type (3/9, 33%). DIAGNOSIS: Bovine haemoplasmosis. CLINICAL RELEVANCE: The presence of bovine haemoplasmas in blood does not establish causality for anaemia in cattle. Diagnosis of anaemia associated with haemoplasmosis would require exclusion of other causes of regenerative anaemia and an association of the agent with anaemia in affected cattle herds. The data collected in this study did not provide evidence that bovine haemoplasmas were associated with a large number of outbreaks of anaemia in cattle in New Zealand. [ABSTRACT FROM AUTHOR]

A study was performed to investigate interlaboratory test agreement between a research and a commercial veterinary diagnostic laboratory on blood and urine samples, and to investigate test agreement between blood, urine, and kidney samples (research laboratory) for leptospirosis diagnosis. Samples were sourced from 399 sheep and 146 beef cattle from a local abattoir. Interlaboratory agreement for real-time quantitative polymerase chain reaction (qPCR) results on urine samples was almost perfect (kappa = 0.90), despite the use of different amplification targets (DNA gyrase subunit B gene vs. 16s ribosomal RNA gene), chemistries (SYTO9 vs. TaqMan probe), and pre-PCR processing. Interlaboratory agreement for microscopic agglutination test (MAT) positivity was almost perfect (kappa = 0.93) for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjobovis) but moderate (kappa = 0.53) for Leptospira interrogans serovar Pomona (Pomona). Among animals that had different titers recorded, higher Hardjobovis and lower Pomona titers were reported by the commercial laboratory than by the research laboratory (P < 0.005). These interlaboratory comparisons can assist researchers and diagnosticians in interpreting the sometimes discrepant test results. Within the research laboratory, the comparison of qPCR results on urine and kidney showed almost perfect agreement (kappa = 0.84), suggesting that the qPCR on these 2 specimens can be used interchangeably. The agreement between MAT positivity and urine and kidney qPCR results was fair (kappa = 0.32 and kappa = 0.33, respectively). However, the prevalence ratio of urine and kidney qPCR positivity in Hardjobovis-seropositive versus Hardjobovis-seronegative sheep indicated that Hardjobovis seropositivity found in sheep may be able to predict shedding or renal carriage., (© 2014 The Author(s).)

An increase in veterinary and farmer interest in bovine viral diarrhoea (BVD) in New Zealand over recent years led to requests for cost-effective identification of BVD virus (BVDV) infected herds and individuals. This study was undertaken to determine if the use of real-time reverse transcriptase polymerase chain reaction (RT-PCR) technology and dairy cow production data could identify persistently infected (PI) animals in milking herds. Milk samples were collected from the vats of dairy herds and tested for the presence of BVDV by RT-PCR till four herds were found containing PI animals. Individual serum samples were then collected from every cow in the herd and tested by both RT-PCR and antigen capture enzyme-linked immunosorbent assays (ACE) to identify the PI animals. Individual animal testing found 1/223, 1/130, 2/800 and 1/275 PI's respectively in the four herds. Based on these results a maximum pool size of 400 cows contributing to the bulk tank milk was selected. After removal of the PI from the herds, further bulk milk samples were shown to be BVDV negative by RT-PCR. All the PI animals identified by this method were found in the lowest producing 10-20% of herd. This approach of targeted testing of dairy herds using PCR technology, in conjunction with animal production information, markedly reduced the cost of diagnostic testing for BVDV in dairy herds in New Zealand. Questionnaire follow-up on 81 BVDV-positive herds (15% of those tested) indicated the stratification approach identified milking PIs successfully over 90% of the time and reduced the number of individual tests to 12% of the milking herd., (Copyright 2009 Elsevier B.V. All rights reserved.)

Sheep chromosome 2q (OAR2q), which is homologous with human chromosome 2q (HSA2q), and cattle chromosome 2 (BTA2), is known to contain several loci contributing to carcass traits. However, the chromosomal rearrangements differentiating these chromosomes among the three species have not yet been determined and thus precise correspondences between the locations of sheep and human genes are not known. Twenty-six genes from HSA2q (2q21.1-->2q36) have been assigned to OAR2q by genetic linkage mapping to refine this area of the sheep genome. Seventy-six genes were initially selected from HSA2q. Sixty-eight percent of the PCR primer sets designed for these genes amplified successfully in sheep, and 34% amplified polymorphic products. Part of the proximal arm of OAR2q was found to be inverted compared with HSA2q. The breakpoint has been localised near the growth differentiation factor 8 gene (GDF8), spanning 380 kb between the positions of the hypothetical protein (FLJ20160) (HSA2:191008944-191075046) and glutaminase (GLS) (HSA2:191453847-191538510) (Build36.1)., (Copyright 2007 S. Karger AG, Basel.)

The objective of this study was to determine whether differences in mRNA levels of key pituitary genes that regulate GH production, pituitary development, and growth were present and/or associated with divergent body composition phenotypes observed between sheep from genetically divergent lean and fat selection lines. Real-time PCR transcription profiles for pituitary specific transcription factor 1, prophet of pit1, GH, GH receptor, GH secretagogue receptor, GHRH receptor, leptin receptor, and somatostatin receptors 1 and 2 were determined in pituitary tissue. There was a difference in the amount of both GH (P < 0.001) and GH secretagogue receptor (P < 0.001) mRNA between the selection lines (5 females and 5 males per line; 20 wk of age); the lean line had greater abundance than the fat line, irrespective of which endogenous control gene was used. The results obtained for GHRH receptor were equivocal but suggestive; there were greater GHRH receptor mRNA levels (P < 0.001) in the lean line using beta-2-microglobulin as the endogenous control but not when hypoxanthine phosphoribosyltransferase and glyceraldehyde-3-phosphate dehydrogenase were used. No difference in pituitary specific transcription factor 1, prophet of pit1, GH receptor, leptin receptor, or somatostatin receptors 1 and 2 mRNA concentration was observed between the lines. The greater abundance of GH mRNA in the pituitary somatotropes from genetically lean animals appears to be associated with increased levels of GH secretagogue receptor mRNA and possibly GHRH receptor mRNA. This suggests that the difference in GH secretion between the lines may be due to differences in the afferent signals, such as ghrelin and/or GHRH, arising from the hypothalamus, or as a result of differential pituitary sensitivity to these hormones.

The potential of recombinant poxviruses as expression vectors has been extensively studied using Vaccinia virus but there has been only limited transfer of this technology to the Parapoxvirus genus. We detail here the construction of a recombinant Orf virus that expresses an antigenic peptide (EG95) of the causative agent of cystic hydatid disease, Echinococcus granulosus. Expression of this foreign antigen was regulated by a synthetic early/late poxvirus transcriptional promoter and levels of expression comparable to that achieved by a similar vaccinia virus recombinant were observed. The expression cassette was inserted into a unique orf virus gene (G1L) thereby confirming the non-essential nature of that gene and identifying a novel genomic insertion site. This recombinant will be a valuable tool with which to assess the potential of recombinant orf viruses to deliver vaccine antigens to sheep.

The aim of this study was to determine 1) the time of onset and cellular localization of gene expression for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase/Delta(5),Delta(4) isomerase (3beta-HSD), and the cytochrome P450 enzymes for cholesterol side-chain cleavage (P450(scc)), 17alpha-hydroxylase (P450(17alphaOH)), and aromatase (P450(arom)) during gonadal development; and 2) the amount of progesterone, androstenedione, testosterone, and 17beta-estradiol present in the fetal sheep gonad. Fetuses were collected on Days 24, 26, 28, 30, 32, 35, 40, 55, and 75 of gestation, and gene expression was determined by in situ hybridization. The steroid content of gonads collected on Days 30, 35, 55, and 75 of gestation was determined by RIA. Developing gonads collected from both male and female fetuses were steroidogenically active around the time of morphological sexual differentiation. In the female, the steroidogenic cells were initially located at the boundary of the cortex and medulla but become increasingly restricted to the mesonephric-derived cell streams. In the male, once tubules were identifiable, steroidogenesis was restricted to the interstitial regions. Interestingly, expression of both SF-1 and 3beta-HSD was observed prior to morphological sexual differentiation. In addition, expression of both of these genes was more widespread than the other genes in both males and females.

Animals heterozygous (I+) for the Inverdale prolificacy gene (FecX(I)) have an increased ovulation rate whereas those homozygous (II) for FecX(I) are infertile with "streak" ovaries and follicular development arrested at the primary (type 2 follicle) stage. The streak ovaries also contain small oocyte-free nodules with granulosa-like cells and often tumor-like structures. It has been hypothesized that these abnormal structures are of granulosa cell origin, and the aim of this study was to determine whether genes normally expressed in granulosa cells are also expressed in the nodules and tumor-like structures. The mRNAs encoding c-kit and its ligand stem cell factor (SCF), FSH receptor (FSH-R), follistatin, alpha-inhibin subunit, and the beta(A)- and beta(B)-activin/inhibin subunits were localized in ovaries of ewes with 0 (++), 1 (I+), or 2 (II) copies of the FecX(I) gene (n = 4-9 animals per genotype per gene) using in situ hybridization. Ontogeny of expression of all mRNAs examined was similar between ++ and I+ ewes. Expression of c-kit mRNA was observed in the oocyte of all follicular types present in ++, I+, and II ewes. Moreover, granulosa cells of type 2 (II) and type 2 and larger follicles (++, I+) expressed SCF mRNA. The mRNAs encoding FSH-R, follistatin, alpha-inhibin subunit, and beta(B)-activin/inhibin subunit were identified in type 3 and larger follicles of ++ and I+ ewes but not in follicles of II ewes that were only at the type 1, 1a, or 2 stages of development. However, the cells within the oocyte-free nodules of II ewes expressed all of these genes. The mRNAs encoding c-kit and beta(A)-activin/inhibin subunit were not observed in granulosa cells until antrum formation (type 5 follicles) or in the nodules of II ewes. Tumors from 4 ewes were obtained and classified as cystic, semisolid, or solid structures containing granulosa-like cells or as solid structures containing predominately fibroblast- and luteal-like cells. Often, two tumors were present on the same ovary. Tumors containing granulosa-like cells (n = 3-4 per gene) expressed the mRNAs encoding alpha-inhibin subunit, beta(A)-, and beta(B)-activin/inhibin subunits, follistatin, and the FSH-R but did not contain detectable amounts of mRNA for c-kit or SCF. Tumors composed predominately of fibroblast- and luteal-like cells expressed very low levels of SCF mRNA; of the other mRNAs examined, none were detected. Also, none of the genes examined were found to be expressed by the surface epithelium, theca externa, fibroblast, or vascular cells within the ovary of animals of any genotype. These findings are consistent with the hypothesis that the somatic cells in oocyte-free nodules and tumor-like tissue in II ewes originate from the granulosa cells of the small follicles.

The purpose of this paper is to review, using fetal sheep as the animal model, aspects of ovarian development related to follicular formation and to report on the identity of growth and paracrine factors which might be involved in this process. Before follicular formation there is a massive and sustained colonisation of the fetal ovary by mesonephric cells, which become a precursor source of follicular cells. From within the ovarian medulla, somatic 'cell-streams' branch into the cortex around nests of oogonia and oocytes. These 'cell-streams', which contain elongated cells with either flattened or cuboidal shaped nuclei, express steroidogenic factor-1 (SF-1), steroid acute regulatory protein (StAR), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450(scc), and P450(aromatase) mRNA and/or protein. Follicles form from the association of an oocyte with the 'cell-stream' with either a single layer of flattened cells (i.e. type 1 follicle) or with a mixture of flattened and cuboidal cells (i.e. type 1a follicle). These newly-formed follicles have between 3 and 57 somatic cells (i.e. granulosa cells) and contain oocytes which vary in diameter between 23 and 52 microm. Newly formed and early growing follicles have been identified with growth factors or growth factor receptors in either the oocytes or granulosa cells. Many of the growth factors are from the TGFbeta superfamily and are expressed in a cell- and stage-specific manner.

The aim of this study was to investigate stem cell factor and c-kit gene expression and protein localization in the mesonephros and ovary of sheep fetuses at different days of gestation, using RNA in situ hybridization and immunohistochemical procedures. At days 24 and 26 of gestation, stem cell factor mRNA and protein were present in cells throughout the developing gonad and mesonephros. From day 28 to day 40 of gestation, stem cell factor mRNA and protein became increasingly localized to the cortical region of the ovary, where most germ cells were present as actively proliferating oogonia. From day 40 to day 90 of gestation, stem cell factor mRNA and protein localization were confined mainly to the ovarian cortex. Moreover, within the cortical region, stem cell factor mRNA was low or absent where follicles were first forming and highest in the outer ovarian cortex, where germ cells were undergoing mitosis or the early stages of meiosis. In contrast, stem cell factor protein was present in newly forming follicles, as well as in mitotic and meiotic germ cells, which is consistent with the presence of both membrane-bound and soluble forms of this ligand. However, by day 90 of gestation, both stem cell factor mRNA and protein were observed in the granulosa cells of most (> 90%) primordial follicles. C-kit mRNA and protein were observed from day 24 of gestation in both germ cells and somatic cells but, with increasing gestational age, preferentially in germ cells (for example, pre-meiotic germ cells and both isolated oocytes and follicle-enclosed oocytes). C-kit protein, but not mRNA, was also observed in germ cells that were undergoing meiosis. The results indicate that the cells containing stem cell factor mRNA within the ovary up to day 90 of gestation originated from the gonadal blastema and from cells that migrated from the mesonephros before day 28 of gestation. Since stem cell factor mRNA was absent in both mesonephric cells migrating after day 28 of gestation and in regions where follicles were first forming, it is suggested that these later migrating mesonephric cells are the progenitors of the granulosa cells in the first forming follicles. In conclusion, during follicle formation, c-kit mRNA is localized to germ cells whereas c-kit, together with stem cell factor protein, is localized to both germ cells and somatic cells, consistent with the hypothesis that the presence of this receptor-ligand pair is essential to prevent apoptosis.

Early follicular growth refers to the development of an ovarian follicle from the primordial to early antral phase. In sheep and cows these phases of growth can be classified by the configuration of granulosal cells in the largest cross-section of the follicle as types 1 (primordial), 1a (transitory) 2 (primary), 3 and 4 (preantral) and 5 (early antral). Follicles classified as type 1 may be highly variable within each species with respect to number of granulosal cells and diameter of oocyte. Much of the variation in granulosal cell composition of type 1 follicles may occur at formation and this may account for the variability in granulosal cell composition throughout subsequent stages of growth. There appear to be important differences among species (for example sheep and cattle) in the number and function of granulosal cells relative to the diameter of the oocyte during the initiation of follicular growth. There is evidence that most, if not all, of the growth phases from types 1 to 5 are gonadotrophin-independent and that follicles develop in a hierarchical manner. In sheep, cows and pigs, numerous growth factor, growth factor receptor and gonadotrophin receptor mRNAs and peptides (for example c-kit, stem cell factor, GDF-9, beta B and beta A activin/inhibin subunit, alpha inhibin subunit, follistatin, FGF-2, EGF, EGF-R, TGF beta 1,2 and 3 FSH-R and LH-R) are expressed in a phase of growth (for example types 1-5)-specific and cell-specific manner. However, the roles of many of these factors remain to be determined.

The aims of the present study in ewes were 1) to test the hypothesis that apoptosis in granulosa cells is one of the processes involved in the structural demise of follicles and 2) to define the temporal relationships among the occurrence and degree of apoptosis in granulosa cells, aromatase activity, production of cyclic AMP (cAMP) by granulosa cells in response to FSH or LH, concentrations of estradiol 17 beta (E2) and progesterone in follicular fluid, and the characteristic morphometric changes associated with the process of follicular atresia. To address these aims, ewes were treated with either saline or steroid-free bovine follicular fluid (bFF) at 60 h after estrus, and ovarian follicles > or = 3 mm diameter were recovered at 0, 12, 18, or 24 h later. Apoptotic granulosa cells were identified by the presence of oligonucleosomes after 3'-end labeling of extracted DNA with [32P]alpha dideoxy ATP (ddATP). The degree of oligonucleosome formation, based on the intensity of radiolabeling, was given an apoptosis score (AP) of 0 (nondetectable), 1 (slight), 2 (moderate), or 3 (marked). Moreover, a labeling index (LI) was calculated from the amount of radiolabeled ddATP incorporated into low-molecular weight (< 4.2 kb) DNA fragments. On the basis of morphometric criteria, 73% (141 of 194) of the follicles classified as healthy had apoptotic granulosa cells compared to 86% (18 of 21) of the follicles classified as atretic. In the bFF-but not saline-treated ewes, the concentrations of plasma FSH had declined to basal values at 12 h after treatment. At the beginning of the treatment period, the degree of granulosa cell apoptosis was either undetectable (AP = 0, 47% of follicles) or slight (AP = 1, 44% of follicles) in the majority of follicles. After 12 h from the bFF but not the saline injection, there was a significant increase in the proportion of follicles (> or = 3 mm diameter) per ewe containing apoptotic granulosa cells (p < 0.001) and a significant decrease in the number of follicles per ewe with aromatase activity (p < 0.05) and with follicular fluid E2 > 20 ng/ml (p < 0.05). By 24 h after bFF treatment, apoptosis was evident in all follicles (> or = 3 mm diameter), fewer follicles contained FSH-responsive granulosa cells in terms of cAMP production (p < 0.05), and none were LH-responsive. A significant negative relationship was found between the degree of granulosa cell death as measured by L1 and follicular fluid E2 concentrations. In summary, the presence of apoptotic granulosa cells in an appreciable number of follicles considered to be healthy by morphometric criteria and before their commitment to preovulatory enlargement and ovulation suggests that apoptosis may be a physiological process in developing follicles and/or a very early event in atresia. Collectively, these data provide strong evidence that granulosa cells may die by apoptosis before there is an appreciable decrease in the capacity of the granulosa cell layer as a whole to respond to gonadotropins or to produce E2.

Two ovine stem cell factor (oSCF) cDNAs (822 bp and 738 bp) were generated from ovarian follicle mRNA by RT-PCR. Nucleotide sequencing revealed that the oSCF 822 bp cDNA encodes a precursor protein of 274 amino acids. An amino acid change 109E to 109Q was the only sequence difference from that previously described for this species. The smaller (738 bp) oSCF cDNA was shown by nucleotide sequencing to be an mRNA splice variant, equivalent to that found in other mammals, in which an exon (84 bp) encoding a potential proteolytic cleavage site is removed. Northern analysis revealed a single transcript of approximately 6.5 kb in follicles, corpora lutea and stroma of mid-luteal sheep ovaries. In situ hybridization was used to detect oSCF mRNA within ovaries of fetal sheep on days 90, 100, 120 and 135 of gestation (term = 147) and of adult sheep within the breeding season. In fetal and adult ovaries, oSCF mRNA was detected in the granulosa cells of follicles at all stages of follicle growth (primordial through to antral). The SCF gene was also expressed in granulosa cells of atretic follicles but appeared to be down-regulated in the cumulus cells surrounding the oocyte at more advanced stages of atresia. In fetal ovaries at day 90 of gestation (90DG), oSCF was expressed in the subepithelial mesenchymal cells of the ovarian cortex. By 100DG the gene expression in the subepithelial cells became restricted to a narrow region below the epithelium, and areas of expression were observed in groups of cells around isolated oocytes, primordial and primary follicles. oSCF gene expression also occurred in the surface epithelial cells of 90DG ovaries, the expression was absent from these cells by 135DG and in adult ovaries. Localization of oSCF mRNA was observed in the ovarian rete and endothelial cells of blood vessels of fetal ovaries. These results suggest that oSCF may have an important and continuous role in the development and/or maintenance of germ cells during follicle growth and atresia in sheep.

Reverse transcription-PCR has been used to obtain a cDNA sequence from the follicle-stimulating hormone (FSH) beta-subunit gene of the Australian brushtail possum (Trichosurus vulpecula). Comparisons of the possum FSHbeta-mRNA coding region nucleotide sequence with that of six eutherian mammal homologues reveals a mean percent identity of 77.3% and 76.8% at the nucleotide and predicted amino acid-sequence levels respectively. Furthermore, the predicted amino acid sequence of the possum FSHbeta mature protein shows evolutionary conservation of twelve cysteine residues and two potential N-linked glycosylation sites. The protein lacks the CAGY motif present in most reported glycoprotein beta-subunit sequences. The translation termination codon and consensus polyadenylation sequence overlap, a feature observed in other mammalian FSHbeta genes. Northern hybridization of total RNA from adult female possum pituitary revealed three hybridizing transcripts of approximately 2.8, 1.2 and 0.5 kb which may arise from utilizing alternative polyadenylation signals. In situ hybridization localized the FSHbeta transcripts to a sub-population of anterior pituitary cells interpreted as being gonadotropes. In summary the results indicate considerable evolutionary conservation of the structure of the FSH beta-subunit gene between the marsupial and eutherian mammalian lineages.

The c-kit protein is a transmembrane tyrosine kinase receptor that binds the growth factor stem cell factor. Mutant alleles of the genes coding for both the receptor (c-kit) and its ligand (stem cell factor) affect gametogenesis, development of melanoblasts and some aspects of haematopoiesis. The aim of this study was to examine expression of the c-kit gene during folliculogenesis in fetal sheep ovaries using in situ hybridization. A 422 bp cDNA encoding the extracellular domain of the c-kit protein was amplified from sheep ovarian RNA using reverse-transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. Riboprobes transcribed from the ovine cDNA encoding c-kit were used to detect the presence of mRNA encoding c-kit within the ovaries of fetal sheep on days 90, 100, 120 and 135 of gestation (term = 147 days). In day 90 and 100 fetal ovaries, mRNA encoding c-kit was not detected in association with oogonia during the period of meiosis (to prophase I) but was present in some of the isolated oocytes. In ovaries from day 90 to day 135, mRNA encoding c-kit was detected in the oocytes at every stage of follicular growth--primordial through to antral follicles. This pattern of localization is consistent with that demonstrated in mice.

A key question in elucidating the role of FSH in ovarian function is to determine when during follicular growth the FSH receptor first appears. The aim of this study was to examine the site and time of FSH receptor gene expression during early follicular growth. This study was carried out on ovaries of adult sheep during the luteal and prostaglandin-induced follicular phase of the oestrous cycle and also on ovaries of fetal sheep at 90, 100, 120 and 135 days of gestation (term = day 147). Using reverse transcription-PCR and a set of PCR primers spanning exons 8/9/10, two partial FSH receptor cDNAs (500 and 310 bp) were isolated from adult sheep ovary. It was shown by sequencing that exon 8 was deleted in the 310 bp cDNA, implying that this was part of an alternatively spliced FSH receptor transcript. Using RNA in situ hybridisation on ovaries of adult sheep, FSH receptor mRNA was observed in granulosa cells of early preantral follicles with one to two cell layers and it was seen that gene expression continued throughout folliculogenesis into advanced stages of atresia. Moreover, in the fetus, FSH receptor gene expression was detected in follicles with two or more layers of granulosa cells in ovaries taken at 100, 120 and 135 days of gestation. These results suggest that the FSH receptor gene is expressed after the granulosa cells of a follicle have begun to divide but not during the earliest stages of follicle growth, namely the transformation of a primordial follicle to a primary follicle.

In female sheep fetuses, the mesonephros and genital ridge can be identified at days 20 and 23 of gestation (term = 145 days), respectively. Moreover oogonia can be observed at the genital ridge from as early as day 23. Around day 55 of gestation, some germ cells enter meiosis coincident with the arrival of mesonephric-derived somatic cells (i.e. the rete ovarii). From day 75, 100, 120 and 135 of gestation, primordial (one layer of flattened granulosa cells), primary (one complete layer of cuboidal granulosa cells; early preantral), secondary (preantral) and tertiary (antral) follicles, respectively, develop within the innermost regions of the ovarian cortex. During the early neonatal period highly variable numbers of antral follicles may be present. After examination of Booroola fetuses from day 28 of gestation, it seems that the FecBB gene is associated with retarded development of the heart (day 28) mesonephros (days 30-40) and from day 30 to early neonatal life, the ovary. With respect to the ovary, fewer oogonia (days 30-40), primordial follicles (day 75-90) and growing follicles (day 120 to 6 weeks after birth) have been observed in females carrying the FecBB gene. By contrast, the FecBB gene is not associated with differences in plasma gonadotrophin or immunoreactive inhibin until early neonatal life. In Inverdale (I) fetuses heterozygous for the FecXI gene (I+), retarded germ cell development was observed at days 40 and 90 of gestation. In putative homozygous carriers (II) of the Inverdale gene, germ cell development appeared normal until day 100, but thereafter from day 120 normal secondary follicles were not observed, although many abnormal follicular-like structures were present. In both I+ and II fetuses no obvious differences in gonadotrophin concentrations have been noted. Collectively, the evidence suggests that the fecundity genes FecBB and FecXI, which affect ovulation rate in sexually mature females, are regulating organ differentiation or germ cell maturation or both processes during fetal life.

The 5' untranslated region (UTR) of beta A inhibin mRNA was compared in a variety of sheep tissues, using primer extension. Considerable variation in the length and number of 5' extended products were noted between tissues. Specific bands were noted in ovarian follicular RNA, which were also present in samples from corpora lutea, stroma, and placental cotyledon RNA. Other extended products were observed in RNA from corpora lutea, stroma, cotyledon, pituitary, bone marrow, frontal cortex, medial basal hypothalamus, adrenal, liver, and kidney, which were not present or weakly represented in follicular RNA. Additional tissue-specific bands were noted in testis and bone marrow RNA. No specific differences in the lengths of the 5' UTR of the beta A inhibin mRNA were observed in sheep homozygous for the Booroola fecundity gene FecB, in any tissue studied. The coding region of ovine beta A inhibin cDNA was sequenced and a genetic polymorphism confirmed within or close to the ovine beta A inhibin gene. We conclude that the beta A inhibin gene is expressed widely in the sheep. Furthermore there is variation in the length of the 5' UTR of beta A inhibin mRNA between male and female gonads and other tissues, implying that expression of this gene is differentially controlled. However, the FecB mutation does not affect mRNA splicing events or the initiation site used in ovarian transcription. The mechanism by which the FecB mutation influences the amounts of beta A inhibin mRNA, follicle-stimulating hormone (FSH) secretion and ovulation rate has still to be elucidated.

Apoptosis is a process of selective cell deletion implicated as a mechanism underlying the process of ovarian follicular atresia. The aims of this study were 1) to test the hypothesis that granulosa cell death during follicular (> or = 4 mm diameter) atresia in cows occurs by apoptosis and 2) to define relationships between the occurrence and degree of granulosa cell apoptosis, cAMP response to FSH or LH, extant aromatase activity, and other previously established biochemical and morphometric indices of granulosa cell function and follicular atresia in this species. Granulosa cells and follicular fluid from individual follicles 4-18 mm in diameter were collected from luteal-phase cow ovaries. Follicles were classified by morphometric criteria as "healthy" (n = 45) or atretic (n = 34). Apoptosis in granulosa cells from each follicle was inferred from detection of internucleosomal DNA cleavage by 3'-end radiolabeling; it was quantitated both subjectively from intensity of oligonucleosome labeling (apoptosis [AP] score = 0, 1, 2, or 3) and objectively by beta-counting of low-molecular weight gel fragments (labeling index; LI). Extant aromatase activity (ng estradiol produced/10(6) cells/3 h) and cAMP response (pmol/10(6) cells) to different doses of FSH or LH (1-10,000 ng/ml) was determined for granulosa cells from most healthy follicles (n = 39). Apoptosis was detected in granulosa cells from all atretic follicles as well as from 76% of healthy follicles, from 80% (16 of 20) of follicles with follicular fluid estradiol to progesterone ratios > 1, and from 71% (10 of 14) of follicles with extant aromatase activity (> 2 ng/10(6) cells/3 h). For healthy and atretic follicles, degree of DNA fragmentation was inversely related to the number of granulosa cells recovered (as percentage maximum by follicle size). In healthy follicles, FSH stimulated cAMP synthesis is a dose-dependent manner in granulosa cells from all follicles examined (> or = 4 mm), but only 36% of these had appreciable aromatase activity. The cAMP response to FSH (per cell) increased with follicle size from 4-7 mm in diameter and remained high in granulosa cells from follicles > or = 8 mm with aromatase activity; in cells without aromatase activity, cAMP response to FSH decreased with increasing follicle size > or 8 mm. The cAMP response to LH was generally low or undetectable in granulosa cells from 4-8-mm follicles; it then increased linearly with increasing follicle diameter > or = 8 mm, but to a greater degree in cells with aromatase activity than in cells without.(ABSTRACT TRUNCATED AT 400 WORDS)

The aim of this study was to investigate the sites of follistatin and alpha and beta A inhibin mRNA expression in the ovaries of female sheep fetuses at 90, 100, 120 and 135 days of gestation (term = day 147). At 90 and 100 days primordial follicles were formed, followed by the appearance of primary follicles at 100 days of gestation. At days 120 and 135, primordial, primary and preantral (i.e. secondary) follicles were present in the ovaries, but antral (i.e. tertiary) follicles were not observed at any of these gestational ages. Two Booroola genotypes were studied: homozygous carriers (BB) and non-carriers (++) of the fecundity gene (FecB). Irrespective of genotype no specific hybridization of the alpha and beta A inhibin riboprobes was detected in any ovarian cells at days 90, 100, 120 or 135 of gestation. In control mature ovaries, on the other hand, strong hybridization in the granulosa cells of antral follicles was observed. In contrast to alpha and beta A inhibin, follistatin antisense (but not sense) riboprobes hybridized specifically to the granulosa cells of preantral follicles with two or more layers of cells at days 120 and 135 of gestation. Moreover, hybridization was also evident in the cells of the ovarian rete at days 120 and 135, but not at 90 or 100 days. No follistatin mRNA expression was observed in the granulosa cells of primordial or primary follicles or in any other ovarian cell type at any of the gestational ages examined.(ABSTRACT TRUNCATED AT 250 WORDS)

The sites of follistatin and alpha and beta A inhibin gene expression were examined by in situ hybridization in sheep ovaries during the early and mid-luteal phases (days 3 and 10) of the oestrous cycle and a prostaglandin F2 alpha (PGF 2 alpha)-induced follicular phase. Follistatin mRNA was detected in the granulosa cells of preantral, antral and early atretic follicles at all stages of the oestrous cycle, and in the corpora lutea at the early and mid-luteal stages of the cycle. However, only low levels of expression of follistatin were observed in the presumptive preovulatory follicle at 56 h after treatment with PGF 2 alpha. Both alpha and beta A inhibin were shown to be expressed in ovaries at all stages of the oestrous cycle. In situ hybridization localized alpha subunit mRNA to the granulosa cells of most, but not all, healthy antral follicles, and to no other ovarian cell type. In contrast, expression of the beta A subunit was confined to a few medium-to-large healthy antral follicles. In antral follicles expressing beta A inhibin, mRNAs for alpha inhibin and follistatin were always detected, but the converse was not true. Unlike follistatin, no alpha and beta A inhibin expression was seen in preantral follicles, developing corpora lutea, or follicles undergoing atresia. These results show that, in the adult sheep ovary, follistatin gene expression is a constitutive event in all growing follicles from the early preantral stage, and also provide indirect evidence of the involvement of follistatin, but not inhibin or activin, in the early stages of ovarian follicle development in sheep.

We have isolated ovine follistatin cDNA from an ovarian follicle cDNA library and determined its sequence. The deduced amino acid sequence of the ovine follistatin precursor is highly homologous (greater than 97%) to the porcine, human and rat follistatins. Northern analysis was used to characterize follistatin gene expression in ovaries of adult ewes, collected from days 11 to 13 of the oestrous cycle. Two major (about 2.7 kb and 1.5 kb) and one minor (about 0.5 kb) transcripts were detected in polyadenylated RNA extracted from ovarian follicles and corpora lutea. The degree of expression of the transcripts varied in the two ovarian compartments, with the 2.7 kb species predominating in the follicles and the 1.5 kb species being more abundant in the corpora lutea. No transcripts were detected in stromal tissue containing preantral follicles of less than 1 mm.

Ovine cDNA probes for the alpha and beta A inhibin subunits and for follistatin were used to investigate the mRNA species for these hormones in ovaries obtained during the luteal phase of the oestrous cycle, from Booroola ewes which were homozygous carriers (BB) or non-carriers (++) of the FecB gene. BB ewes had significantly higher concentrations of peripheral FSH and LH immunoreactivity than ++ ewes, but the peripheral inhibin immunoreactivity and ovarian inhibin and progesterone secretion rates were not significantly different between genotypes. No gene-specific differences in the number or size of mRNA transcripts detected by Northern blotting were noted for any of these genes. A single alpha inhibin mRNA species at 1.5 kb was observed in the follicle RNA from ++ and BB ovaries. Low amounts of alpha inhibin hybridization were discerned occasionally in ++ and BB stroma and also in BB, but not in ++, corpora lutea. The beta A inhibin gene was expressed only in the follicles from both ++ and BB ovaries. At least three beta A inhibin transcripts were observed; one at 7.5 kb and at least two between 1.4 and 5.0 kb. The follistatin cDNA probe detected two major transcripts at 2.7 and 1.5 kb and a minor band at 0.5 kb in both follicle and corpora lutea RNA. Densitometry of the Northern blots revealed no significant gene-specific differences in the levels of alpha inhibin and follistatin gene mRNA transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

A total of 14 isolates of Aujeszky's disease virus were examined by restriction-endonuclease analysis using BamH1, Xho1 and Kpn1 restriction enzymes. BamH1 was the enzyme of choice as it produced only 15 major fragments. All isolates produced similar BamH1 patterns but minor variation in the mobility of fragments 5/5'/6, 10, 12 and 13 was evident. In a number of isolates, additional bands were present in fragment regions 4,5/5'/6 and 8. The technique of restriction-endonuclease analysis has proved to be an effective method for differentiating between Aujeszky's disease virus isolates, and has shown the BamH1 patterns generated in the NZ and Western Samoan isolates to resemble closely those described overseas as Group 1 patterns.

Streptococcus pneumoniae, a bacterium frequently associated with pneumonia in man, was isolated from the spleen, liver, lung and kidney of an Angora goat kid which had died suddenly. Signs of septicaemia were pronounced with widespread petechial haemorrhages on internal organs. Histologically, the spleen and liver were severely congested and necrotic changes were most marked in these organs. The goat kid had been reared as a household pet and the possibility that the goat kid had acquired the Str. pneumoniae from a human infection is discussed.