Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes

Follicular size, follicular atresia, and oocyte morphology were investigated for the possible relation of these characteristics to the developmental competence of bovine oocytes. Ovaries from a local slaughterhouse were dissected to obtain a heterogeneous population of follicles. Half of each follicle was fixed for histological analysis, and the oocytes were detached carefully and cultured individually. Before in vitro maturation, the oocytes were grouped into six different classes based on the morphology of the cumulus and the ooplasm: classes 1 and 2 represent oocytes with a homogeneous ooplasm plus a compact and complete cumulus, and classes 3–6 represent oocytes with a granulated ooplasm and an incomplete and/or expanded cumulus. Oocytes from class 3 (beginning of expansion in outer cumulus layers and slight granulations in the ooplasm) developed past the 16-cell stage significantly (P<0.05) more than oocytes with a compact and complete cumulus (classes 1 and 2) and oocytes from classes 4–6 (incomplete and/or expanded cumulus) after 5 days of in vitro culture. Oocytes from follicles measuring 3 mm or less did not develop past the 16-cell stage, whereas follicles of 3–5 mm and 5 mm or larger developed at similar rates (17% and 21% morulae, respectively). The state of the follicle did not affect whether an embryo reached at least the 16-cell stage, as comparable rates were obtained in all three groups of follicles: nonatretic (20%), intermediate (14%), and slightly atretic (16%). We concluded that oocytes acquire developmental competence late in the follicular phase, possibly when the first signs of atresia have appeared, and that oocytes with beginning signs of degeneration (class 3) will develop significantly more than all other classes. Class 3 oocytes originated from follicles that were generally atretic and therefore in later phases of follicular growth, suggesting that these oocytes, having been subjected longer to the follicular microenvironment, are more differentiated (possibly at the cytoplasmic level) than other classes of oocytes. © 1995 Wiley-Liss, Inc.     

A monoclonal anticarbohydrate antibody detecting superfast myosin in the masseter muscle

Adrenal medullary chromaffin cells secrete catecholamines through exocytosis of their intracellular chromaffin granules. Osmotic granule swelling has been implicated to play a role in the generation of membrane stress associated with the fusion of the granule membrane. However, controversy exists as to whether swelling occurs before or after the actual fusion event. Using morphometric methods we have determined the granule diameter distributions in rapidly frozen, freeze-substituted chromaffin cells. Our measurements show that intracellular chromaffin granules increase in size from an average of 234 nm to 274 nm or 277 nm in cells stimulated to secrete with nicotine or high external K⁺, respectively. Granule swelling occurs before the formation of membrane contact. Ammonium chloride, an agent which inhibits stimulated catecholamine secretion by approximately 50% by altering the intragranular pH, also inhibits granule swelling. In addition, ammonium chloridetreated secreting cells show more granule-plasma membrane contacts than untreated secreting cells. Sodium propionate induces granule swelling in the absence of secretagogue and has been shown to enhance nicotine- and high K⁺- induced catecholamine release. These results indicate that in adrenal chromaffin cells granule swelling is an essential step in exocytosis before fusion pore formation, and is related to the pH of the granule environment.     

Pro-enkephalin opioid peptides are abundant in porcine and bovine splenic nerves,but absent from nerves of rat,mouse, hamster,and guinea-pig spleen

The opioidergic innervation of the mammalian spleen and possible species differences were investigated. Light-microscopic immunohistochemistry revealed that splenic nerves of bovine and porcine spleen, but not of rat, mouse, hamster and guinea-pig spleen contained proenkephalin-derived opioidergic innervation. Immunoreactivity to both prodynorphin and pro-opiomelanocortin was absent from splenic nerves. In bovine and porcine spleen, fibers immunoreactive for met-enkephalin, met-enkephalin-Arg-Phe, met-enkephalin-Arg-Gly-Leu, leu-enkephalin and peptide F formed perivascular plexus, traveled in trabecular connective tissue, and extended into the capsule. Spatial relationships with immune cells were apparent in the white and red pulp, excluding lymphoid follicles. Colocalization of enkephalin immunoreactivity with immunoreactivities for tyrosin hydroxylase, dopamin--hydroxylase, and neuropeptide Y, but not for substance P or calcitonin gene-related peptide were found. Our results provide evidence that opioid expression in splenic innervation is strongly species-dependent and exclusively proenkephalin-derived. Colocalization with marker enzymes of noradrenergic neurons indicates a mainly postganglionic sympathetic origin of proenkephalinergic splenic innervation. Opioidergic perivascular nerves probably control the splenic blood flow. A close interrelationship of opioidergic fibers with immune cells provides the anatomical basis for direct effects of neurally released opioids on splenic immune functions.     

Immunohistochemical identification of calcitonin gene-related peptide and substance P in nerves of the bovine parathyroid gland

Summary Although peptide neurotransmitters have been shown to modulate hormone secretion in many glands, there are very few studies of neurotransmitters in the parathyroid gland. Bovine parathyroid glands were collected at a local abattoir, fixed with paraformaldehyde, sectioned using a cryostat, and stained by indirect immunohistochemistry for calcitonin gene-related peptide and substance P. We were able to positively identify both neuropeptides. Nerve fibres containing calcitonin gene-related peptide and substance P were identified in contact with the tunica media of arteries and arterioles and dispersed throughout the stroma of the gland. While many of the fibres encircled parenchymal lobules, no intimate contact with the peripheral chief cells was observed. All immunoreactive fibres were found to contain both neuropeptides. Since calcitonin gene-related peptide and substance P are vasodilators, they may increase blood flow within the gland. In addition, the neuropeptides may diffuse from perilobular nerve fibres into the parenchyma, thereby modulating secretion of parathyroid hormone.     

Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first-category COC predominantly possessed the penetrating CCPE, the fourth-category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus-oocyte contacts was assessed by means of incorporation of 3H-choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.     

Photoperiodic alteration of postpartum reproductive function in suckled cows

Three experiments were conducted using a total of 41 cows to determine if photoperiod modulates the establishment of postpartum estrous cycles and conception. Cows calving in the autumn and winter were exposed to either 18 hr light/day (18L:6D) or natural photoperiods. In Exp. 1, cows receiving 18L:6D had shorter (P<0.025) intervals from calving to estrus (61 +/- 3.8 days) than cows not receiving supplemental light (154 +/- 23.9 days). The same was true for primiparous cows in Exp. 2 (76 +/- 5.5 days vs 153 +/- 38.0 days; P<0.06) but not for the multiparous cows in Exp. 2 (56 +/- 5.2 days vs 40 +/- 7.4 days) or for all cows in Exp. 3 (60 +/- 10.1 days vs 70 +/- 13.5 days). Because conception rate was higher for cows exposed to 18L:6D for the multiparous cows in Exp. 2 and all cows in Exp. 3, interval to conception was significantly shorter for animals exposed to 18L:6D in all experiments. Generally, interval from calving to uterine involution was also reduced by exposure to 18L:6D. No effects of photoperiod were observed on body weight changes, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) or estradiol or on estradiol-induced release of gonadotropins. It was concluded that in certain situations day length can hasten estrus and conception in postpartum cows. The effect of photoperiod interacts with other conditions, one of which is parity. The endocrine basis for these effects are not known.     

Serum and milk progesterone in Syncro-Mate-B treated postpartum beef cows

Fifteen post-partum (61.5+/-6.1 days) cyclic beef cows (4.1+/-0.3 years old) were randomly assigned to receive the Syncro-Mate-B (SMB) treatment on day 0 (estrus), day 9 or day 18 of their cycle with implant removal nine days later. An additional five post-partum (41.7+/-5.6 days) beef cows (3.2+/-0.4 years old) not detected in estrus and without corpora lutea also received the SMB treatment. Blood samples (10ml) were obtained via venipuncture on the day of implantation and one, three, five, seven and nine days after implantation and ten days after the post-treatment estrus. Milk samples (25 ml) were obtained by hand stripping at the same periods. Nineteen of twenty cows were synchronized, i.e. estrus occurred within five days of implant removal. Progesterone concentrations in both serum and milk were similar and followed similar patterns for each group. The day-9 treated cows had greater serum and milk progesterone concentrations than the other groups of cows on days 1, 3, and 5 after implantation. In conclusion, cows treated with SMB had similar serum and milk progesterone concentrations, endogenous progesterone secretion was inhibited by SMB treatment and the inhibition was influenced by prior luteal function.     

Ecology of Hup+ Rhizobium strains of cow pea miscellany: native frequency and competence

Rhizobium strains nodulating summer legumes cow pea [Vigna unguiculata (L.)], green gram [V. radiata (L.) (Wilczek)], black gram [V. mungo (L.) (Hepper)] and cluster bean [Cyamopsis tetragonoloba (L.) (Taub)] and a winter legume chick pea [Cicer arietinum (L.)] were surveyed in the Northern Plains of India and screened for hydrogenase activity to determine distribution of Hup character in the native ecosystem. It was observed that 56% of the Rhizobium strains of summer legumes were Hup⁺ whereas that of the winter legume, chick pea, were all Hup^-. Ex planta acetylene reduction activity was observed in most of the Hup⁺ but not in the Hup^- strains of any of the host species. In summer legume, mixed inoculation of Hup⁺ and Hup^- strains, under sterilized as well as unsterilized soil conditions, showed that the host species were predominantly nodulated with Hup⁺ strain.     

Accuracy of pregnancy specific protein-B test for early pregnancy diagnosis in dairy cattle

The objective was to evaluate the accuracy of an ELISA for pregnancy specific protein B (PSP-B) for early pregnancy diagnosis in dairy cattle. Blood from lactating (>100 d postpartum) dairy cows (n = 738), was collected on Days 28, 30, and 35 (Day 0 = estrus), analyzed with an ELISA for PSP-B, and the cows designated as pregnant, probable, unlikely, or non-pregnant. Immediately after blood collection, transrectal ultrasonography (TRUS) was done for pregnancy diagnosis, and the results used as a criterion standard test for comparison with PSP-B. At Day 28, 46.3% were diagnosed by TRUS as pregnant. The PSP-B sensitivity was 93.9% on Day 28 and similar on Days 30 and 35. The PSP-B specificity, positive predictive value, negative predictive value, and accuracy were all >94% on Day 28 and similar on Days 30 and 35. However, the accuracy was significantly less compared to TRUS (P < 0.01). The percentage of all samples from cows that were probably pregnant or unlikely to be pregnant was 5.6%. At Days 28, 30, and 35, percentages of uncertain samples were 8.5, 4.8, and 3.3%, respectively (P < 0.01), and Kappa values were 0.92, 0.92, and 0.95. False negative and false positive results were attributed to low concentrations of PSP-B in pregnant animals and to persistence of pregnant concentrations of PSP-B in females with pregnancy loss, respectively. In conclusion, PSP-B ELISA was a sensitive, specific, and accurate test for pregnancy diagnosis (relative to TRUS) at Days 28, 30, and 35 after breeding.     

Changes in the gene expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in ovarian follicular cells of dairy cow at different stages of development

Adiponectin is one of the most important, recently discovered adipocytokines that acts at various levels to control male and female fertility through central effects on the hypothalamus-pituitary axis or through peripheral effects on the ovary, uterus, and embryo. We studied simultaneous changes in the gene expression pattern of adiponectin and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) in granulosa and theca cells, cumulus-oocyte complex, and in corpus luteum in healthy bovine (Bos tarus) follicles at different stages of development. The expression levels of adiponectin, AdipoR1, and AdipoR2 mRNA were lower (P < 0.05) in granulosa and cumulus cells in comparison with that in theca cells and oocyte. In contrast with the oocyte, AdipoR1 in granulosa, theca, and luteal cells was expressed (P < 0.05) more than AdipoR2. Adiponectin expression increased (P < 0.05) in granulosa cells and in cumulus-oocyte complex during follicular development from small to large follicles. Opposite results were observed in theca cells. Expression of adiponectin was highest in the late stages of corpus luteum (CL) regression, whereas lower expression was recorded in active CL (P < 0.05). AdipoR1 and AdipoR2 expression increased during the terminal follicular growth in granulosa and theca cells (P < 0.05) and during the luteal phase progress in CL. There was positive correlation between adiponectin mRNA level in granulosa cells from large follicles and follicular fluid estradiol concentration (r = 0.48, P < 0.05) and negative correlation between adiponectin mRNA abundance in theca cells and follicular fluid progesterone concentration (r = -0.44, P < 0.05). In conclusion, we found that the physiologic status of the ovary has significant effects on the natural expression patterns of adiponectin and its receptors in follicular and luteal cells of bovine ovary.