Evaluation of bovine cervical mucus during estrous cycle by nuclear magnetic resonance

Bovine cervical mucus exhibited cyclical change of nuclear magnetic resonance properties in relation to stage of estrous cycle. The spectral lineshapes, as measured by asymmetry indices calculated for widths at 20 and 50% peak height, showed an abrupt change between early and midestrus. A second transition was at 6 to 15 days postestrus. The estrus-related change is postulated as reflecting primarily increase of water content of cervical mucus, whereas the postestrus transition appeared to be the consequence of decreased mucous hydration in concert with molecular changes of structure of mucous polymers.     

Influence of environmental heat on peripheral plasma progesterone and cortisol during the bovine estrous cycle.

Plasm progesterone and cortisol were measured in jugular blood by competitive protein-binding assay. Six cycling, estrus-synchronized Guernsey heifers were followed for four consecutive estrous cycles under two controlled temperature (18.2 C, 55% relative humidity; and 33.5 C, 55% relative humidity) conditions. Prolonged exposure to heat of Guernsey heifers increased plasma progesterone on days 2 to 19 of the first cycle and only on days 2 to 8 of the second cycle. However, heat stress depressed plasma cortisol in both cycles and reduced the correlation coefficient between these steroids relative to specific stages of the estrous cycle.     

Dihydrotestosterone influenced numbers of healthy follicles and follicular amounts of LH receptor mRNA during the follicular phase of the estrous cycle in gilts

The present experiments were conducted to determine androgenic effects on numbers, health, and amounts of gonadotropin receptor mRNA in late developing follicles of gilts. Gilts (n=5 per group) received daily injections of one of the following treatments on days 13-16 or days 13-18 of the estrous cycle: corn oil, 5alpha-dihydrotestosterone (DHT, 10 mg), flutamide (1.5 g, an androgen receptor inhibitor), DHT (10 mg) plus flutamide (1.5 g), testosterone (10 mg), and testosterone (10 mg) plus flutamide (1.5 g). Ovarian follicles > or =5 mm in diameter were evaluated on day 17 or 19, 24 h after receiving the last treatment dose. Follicles were classified as healthy (H), moderately atretic (MA), or very atretic (VA). Treatment with DHT increased (P<0.05) the numbers of H follicles relative to control gilts on days 17 and 19. DHT administration from days 13 to 16 diminished (P<0.05) the amounts of LH receptor (LHR) mRNA in H follicles from day 17 (relative amounts: 1.45+/-0.33 and 2.72+/-0.33 for DHT- and vehicle-treated gilts respectively). The effects of DHT on numbers of H follicles and LHR mRNA were not observed in gilts receiving DHT plus flutamide. Androgens did not influence numbers of MA, VA, and total follicles, or follicular estradiol-17beta concentrations and amounts of FSHR mRNA. Treating gilts with DHT during follicular recruitment and selection did not induce changes in the numbers of total follicles > or =5 mm, but rather increased the numbers of healthy follicles in this follicular population in association with decreased amounts of LHR mRNA.     

Influence of stage of cycle, corpus luteum location, follicle size, and number of large follicles on estradiol-17 beta concentrations in bovine follicles

Concentrations of estradiol-17 beta in follicular fluid were correlated to follicular size, stage of estrous cycle, location of corpus luteum, and presence of large follicles. Paired ovaries were obtained from 481 nonpregnant cows at slaughter and follicles were classified as ipsilateral or contralateral to the corpus luteum. Follicular fluid estradiol-17 beta concentrations from 2494 small, 1485 medium, and 396 large follicles were quantified by radioimmunoassay. Stage of estrous cycle was estimated by visual examination of the corpus luteum. Follicles in stage 1 of the estrous cycle (d 1 to 4) had the highest estradiol-17 beta concentration and the smallest mean follicular diameter. Location of follicles relative to the corpus luteum had no influence on estradiol-17 beta concentrations. As follicular size increased, concentration of estradiol-17 beta also increased. The presence of a single large follicle did not affect the concentration of estradiol-17 beta in medium or small follicles. In contrast, if multiple large follicles occurred in the same cow, concentrations of estradiol-17 beta were significantly lower in medium but not small follicles.     

Regulation of apoptosis in the atresia of dominant bovine follicles of the first follicular wave following ovulation

During atresia of bovine follicles, granulosa cells are lost through the controlled form of cell death, apoptosis. The purpose of this study was to characterize the regulation of apoptotic death of granulosa cells in dominant bovine follicles during the first wave of follicular development. Dominant follicles were collected from Holstein heifers on days 4, 6 or 8 of the first follicular wave (n = 5/day). Regulation of apoptosis in granulosa cells was examined by annexin V and propidium iodide staining; measurement of relative levels of mRNA encoding Bcl-2, Bcl-xL and Bax; and activity of caspase-3, -8 and -9. Steady-state levels of mRNA encoding four oxidative stress-response proteins were determined. Compared with day 4, the incidence of apoptotic and nonviable granulosa cells tended to increase on day 6, and numbers of nonviable cells were higher on day 8. The ratios of relative levels of mRNA encoding Bcl-2 to Bax and Bcl-xL to Bax were higher on day 6 than days 4 and 8. Activity of caspases-3 and -9 in granulosa cells did not change among the 3 days, while caspase-8 activity decreased on day 8 compared with days 4 and 6. Amounts of GSHPx, MnSOD and Cu/ZnSOD mRNA in granulosa cells were higher on day 8 than day 6. In theca interna, amounts of Cu/ZnSOD mRNA decreased between days 4 and 6. From the decreased production of estradiol and increased numbers of apoptotic and nonviable granulosa cells, we conclude that atresia of the dominant follicle is initiated between days 4 and 6 of the first follicular wave. However, apoptosis of granulosa cells does not appear to be initiated by changes in expression of oxidative stress-response proteins.     

Expression and localization of apelin and its receptor APJ in the bovine corpus luteum during the estrous cycle and prostaglandin F2alpha-induced luteolysis

Angiogenesis, changes in blood flow, and extracellular matrix remodeling are the processes associated with the development and demise of the bovine corpus luteum (CL) during the estrous cycle. APJ (putative receptor protein related to angiotensin type 1 receptor) is a G-protein-coupled receptor, and its ligand, apelin, has been identified as a novel regulator of blood pressure and as an angiogenic factor. We hypothesized that the apelin-APJ system is involved in luteal function. This study investigated whether apelin-APJ exists in bovine CL and determined their expression profiles and localization during luteal phase and prostaglandin F(2)(alpha) (PGF(2)(alpha))-induced luteolysis. During the luteal phase, apelin mRNA expression increased from early to late CL and decreased in regressed CL. APJ mRNA expression increased from early to mid-CL and remained elevated in late and regressed CL. Apelin and APJ proteins were immunohistochemically detected only in the smooth muscle cells of intraluteal arterioles during the luteal phase. PGF(2)(alpha) stimulated apelin and APJ mRNA expression at 0.5-2 and 2 h respectively, and then the mRNA expression of apelin-APJ was inhibited from 4 h during PGF(2)(alpha)-induced luteolysis. Additionally, apelin mRNA and protein were stimulated at 1 h after PGF(2)(alpha) injection only in the periphery of mid- but not early CL. The present study indicated that the apelin-APJ was localized in the smooth muscle cells of intraluteal arterioles, and responded to PGF(2)(alpha) at the periphery of mid-CL in the cow. Thus, the apelin-APJ system may be involved in the maturation of CL and the luteolytic cascade as a regulator of intraluteal arterioles in cow.     

The effect of day of the estrous cycle, location of ovulatory structure, and progesterone on in vitro bovine endometrial secretions.

Endometrial tissue was collected by biopsy from mature Holstein cows either on d 0 (estrus) or on d 9, 14, or 18 of the estrous cycle to determine the effects of day of the cycle, uterine horn, and in vitro progesterone on endometrial protein secretion. Tissue was incubated for 24 h in supplemented media containing 14C amino acids and either 0, 4.7, or 47 ng of progesterone/ml. Media were analyzed for total protein, radiolabeled protein, and profile of protein released during incubation. Endometrial tissue at d 0 released more protein than did tissue collected on all other days. Radiolabeled proteins were greater on d 0 and 18 than on d 9 and 14. Endometrium from the uterine horn contralateral to the corpus luteum released more radiolabeled protein than endometrium from the uterine horn ipsilateral to the corpus luteum. Seventeen protein bands were identified by electrophoresis. Proximity of the uterine horn to the site of ovulation affected the distribution of specific bands 21,400, 55,000, 74,600, and 88,100 molecular weight. The proportion of released proteins represented by proteins of molecular weights 12,700, 19,100, 21,400, 32,000, and 66,500 was affected either by day of the estrous cycle, proximity of uterine horn to the site of ovulation, or progesterone concentration. The results show that day of the estrous cycle and uterine horn not only alter overall endometrial protein secretion and synthetic activity but also have specific effects on distribution of individual proteins.     

Composition and morphology of the follicular basal lamina during atresia of bovine antral follicles

The fate of the follicular basal lamina during atresia was investigated using bovine follicles, in which different follicle phenotypes have been observed. These phenotypes include: healthy follicles with rounded basal granulosa cells with an aligned basal lamina or follicles with columnar basal granulosa cells with a basal lamina of many loops (loopy), and atretic follicles in which either the antral granulosa cells (antral atresia) or the basal cells (basal atresia) die first. Loopy lamina and basal atresia occur only in small antral follicles < 5 mm in diameter. Follicles were collected from cattle of unknown reproductive history and processed for immunohistochemistry and electron microscopy, and from animals in which follicle growth had been monitored by daily measurements of follicle diameter by ultrasonography. Electron microscopic observations of dominant follicles during the growth phase, plateau and regression showed that the basal lamina was still visible and intact upon atresia. These follicles had a conventional aligned basal lamina, which they retained, except for some degree of folding, as they progressed into antral atresia. In small follicles (2-5 mm in diameter), the basal cell shape (rounded or columnar) and appearance of the basal lamina (aligned or of many loops) did not appear to be related to the type of atresia. On atresia the follicular basal laminae retained immunoreactive laminin alpha1 and beta2, type IV collagen alpha1 and nidogen. Laminin alpha2, which may come from the theca, was present in the follicular basal lamina of only 22% of healthy follicles, but was expressed very commonly in 71% of the atretic follicles. Laminin alpha2 expression was found in both phenotypes of healthy follicles, antral and basal atretic follicles, and follicles with aligned or loopy basal laminae. It is concluded that the basal lamina is not degraded upon atresia, but does undergo a variety of other changes.     

Visceral tissue growth and proliferation during the bovine lactation cycle

Twenty one multiparous, nonpregnant, lactating dairy cows were used to assess the impact of stage of lactation on visceral tissue mass and small intestinal cell proliferation. Cows were slaughtered at each of 4 stages of lactation: 14, 90, 120, and 240 d of lactation. With stage of lactation, DMI increased through d 90 and thereafter remained similar through d 240 (quadratic). Carcass weight and empty body weight (EBW) declined with stage of lactation through d 120 and increased thereafter (quadratic). As a percentage of EBW, rumen, small intestine, and liver weights increased with increasing stage of lactation (quadratic), increasing from 14 to 120 d and declining through 240 d. Stage of lactation did not have a measurable affect on reticulum, omasum, abomasum, or large intestine weights as a percentage of EBW. Visceral adipose mass as a percentage of EBW declined with stage of lactation to a minimum at 120 d and increased by 240 d (quadratic). Concentrations of RNA and DNA of digestive tract organs were largely unaffected by stage of lactation with the exception of the liver DNA concentration through d 120 (quadratic). The proliferative growth fraction (Ki67) was unaffected by stage of lactation. However, bromo-deoxyuridine labeling of jejunal crypts exhibited a cubic response with stage of lactation and tritiated thymidine incorporation by duodenal epithelium increased with stage of lactation through d 120, declining thereafter (quadratic). Mass of visceral tissues increase to meet the energetic demands of lactation and that increased absorption capacity of the intestines is achieved by hyperplastic growth of the intestinal epithelium.     

Prolactin in blood plasma and milk of buffalo during estrous cycle and early pregnancy

Concentration of prolactin was measured by radioimmunoassay for 11 primiparous and 16 multiparous buffalo. Prolactin concentration of blood plasma on day of insemination was about 200 ng/ml for primiparous and about 315 ng/ml for multiparous animals and fluctuated between 130 and 200 ng/ml for primiparous and between 250 and 345 ng/ml for multiparous pregnant animals. For the nonpregnant group prolactin fluctuated between 145 and 240 ng/ml for primiparous and between 210 and 310 ng/ml for multiparous animals with minor elevations 1 to 2 days before estrus. Concentration of prolactin of milk was not significantly different from that of plasma and was positively correlated .68 for nonpregnant and .93 for pregnant animals.     

Changes in milk characteristics and fatty acid profile during the estrous cycle in dairy cows

The relationship of the estrous cycle to milk composition and milk physical properties was assessed on Holstein (n = 10,696), Brown Swiss (n = 20,501), Simmental (n = 17,837), and Alpine Grey (n = 8,595) cows reared in northeastern Italy. The first insemination after calving for each cow was chosen to be the day of estrus and insemination. Test days surrounding the insemination date (from 10 d before to 10 d after the day of the estrus) were selected and categorized in phases relative to estrus as diestrus high-progesterone, proestrus, estrus, metestrus, and diestrus increasing-progesterone phases. Milk components and physical properties were predicted on the basis of Fourier-transform infrared spectra of milk samples and were analyzed using a linear mixed model, which included the random effects of herd, the fixed classification effects of year-month, parity number, breed, estrous cycle phase, day nested within the estrous cycle phase, conception, partial regressions on linear and quadratic effects of days in milk nested within parity number, as well as the interactions between conception outcome with estrous cycle phase and breed with estrous cycle phase. Milk composition, particularly fat, protein, and lactose, showed clear differences among the estrous cycle phases. Fat increased by 0.14% from diestrus high-progesterone to estrous phase, whereas protein concomitantly decreased by 0.03%. Lactose appeared to remain relatively constant over diestrus high-progesterone, rising 1 d before the day of estrus followed by a gradual reduction over the subsequent phases. Specific fatty acids were also affected across the estrous cycle phases: C14:0 and C16:0 decreased (?0.34 and ?0.48%) from proestrus to estrus with a concomitant increase in C18:0 and C18:1 cis-9 (0.40 and 0.73%). More general categories of fatty acids showed a similar behavior; that is, unsaturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, trans fatty acids, and long-chain fatty acids increased, whereas the saturated fatty acids, medium-chain fatty acids, and short-chain fatty acids decreased during the estrous phase. Finally, urea, somatic cell score, freezing point, pH, and homogenization index were also affected indicating variation associated with the hormonal and behavioral changes of cows in standing estrus. Hence, the variation in milk profiles of cows showing estrus should potentially be taken into account for precision dairy farming management.     

Cation concentrations in fluid from the oviduct ampulla and isthmus of cows during the estrous cycle.

To detect variations in oviduct fluid cation concentrations, Ca++, Mg++, K+, and Na+ were determined for daily samples of blood serum and bovine oviduct fluid collected from indwelling isthmic and ampullary catheters. Isthmic oviduct fluid Ca++ concentration was significantly greater than that in ampullary fluid, particularly around estrus and ovulation. Maximum Ca++ concentrations found in isthmic oviduct fluid at estrus (2.57 +/- .22 mM) and at ovulation (2.50 +/- .29 mM) were similar to those of medium used for in vitro capacitation of bovine sperm. Concentrations of Mg++ in oviduct fluid differed significantly by estrous cycle stage, but not by oviduct region, and were consistently lower than those detected in serum. No relationships were found for K+ or Na+ with respect to region or stage, but K+ was generally higher in oviduct fluid than in serum. The concentration of K+ averaged over stage and region (4.46 +/- .13 mM) and the K+:Na+ ratio (.032 +/- .002) were similar to those reported in bovine in vitro capacitating and fertilizing media. Concentrations of Ca++ and Na+ from peritoneal fluid from nonstaged cows were similar to those of oviduct fluid or serum. The Mg++ concentration was greater, and K+ concentration was less, in peritoneal than in oviduct fluid.     

Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture

During ovarian folliculogenesis, ascorbic acid may be involved in collagen biosynthesis, steroidogenesis and apoptosis. The aims of this study were to determine the effects of ascorbic acid on bovine follicle development in vitro. Preantral follicles were cultured for 12 days in serum-free medium containing ascorbic acid (50 microg ml(-1)). Half of the medium was replaced every 2 days, and conditioned medium was analysed for oestradiol and matrix metalloproteinase 2 (MMP-2) and MMP-9 secretion. On day 12, cell death was assessed by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In the absence of serum, there was significant (P < 0.05) follicle growth and oestradiol secretion over the 12 day culture period. Ascorbic acid had no effect on these parameters. The addition of serum from day 0 stimulated follicle growth (P < 0.05), but compromised follicle integrity. By day 12 of culture, a higher proportion of follicles remained intact in the presence of ascorbic acid in serum-free conditions (P < 0.05), and significantly (P < 0.01) less granulosa and theca cell death was observed in these follicles than in control follicles. Moreover, ascorbic acid significantly (P < 0.05) increased production of MMP-9, an enzyme involved in basement membrane remodelling. In conclusion, this culture system was capable of supporting follicle differentiation over the 12 day culture period. Furthermore, ascorbic acid maintains bovine follicle health and basement membrane remodelling in vitro.     

Coculture of in vitro fertilized bovine embryos with oviductal epithelial cells originating from different stages of the estrous cycle.

Bovine embryos derived from in vitro fertilization procedures were cocultured in vitro with oviductal cells obtained from heifers between d 4 and 6 or d 14 and 16 of the estrous cycle. In addition, proteins secreted by oviductal cells isolated between d 4 and 6 or d 14 and 16 of the cycle were monitored. Embryos (2- to 4-cell) were incubated in Tissue Culture Medium-199 with 10% fetal bovine serum with or without oviductal cells at 39 degrees C for 10 d following in vitro insemination. There were more morulae, blastocysts, and hatched blastocysts following coculture with oviductal cells than with culture in medium alone. However, no differences were noted in embryo development following coculture with oviductal cells obtained between d 4 and 6 or d 14 and 16 of the estrous cycle. Also, no differences were detected in the amount of [35S]methionine-labeled proteins secreted by oviductal cells isolated from different days of the estrous cycle. These results indicate that oviductal epithelial cells isolated from early and late luteal phases of the estrous cycle will effectively support early embryonic development following prolonged in vitro culture.     

Prevalence and ultrastructural characteristics of bovine mammary corpora amylacea during the lactation cycle

Morphologic and histochemical study of spherical, lamellated inclusion bodies in bovine mammary parenchyma established presence of corpora amylacea. The majority (90%) were in alveolar lumina, followed by stroma (7%) and epithelium (3%). Occurrence of corpora amylacea was unrelated to lactation age, intramammary infection status, milk somatic cell concentration, or milk production. Quantitative analysis demonstrated a progressive increase of prevalence of these structures from the early to later stages of lactation, followed by a return during the late dry period to concentrations at early lactation. Ultrastructural study revealed deeply basophilic, dense, lamellated deposits (70%) and less dense, fibrillar deposits (30%). Dense bodies stained partially or not at all for amyloid but stained for presence of calcium salts, whereas fibrillar bodies were amyloid positive and calcium negative. Corpora amylacea were most prevalent in fully differentiated parenchymal areas (47.1 bodies/unit tissue area of 2.4 X 10(6) micron 2), less numerous in intermediately differentiated areas (23.4), and sparse in nonsecretory areas (7.1). Morphologic relationships between deposits and parenchyma suggest that corpora amylacea suppress milk secretion and flow during late lactation in isolated areas by engorging luminal spaces and clogging small ducts, leading to milk stasis and involution.