Mounting behavior as affected by stage of estrous cycle in Holstein heifers

Nine Holstein heifers whose estrous cycles were synchronized were observed during three subsequent consecutive cycles. Observations of heifers during each periestrus period began when the first heifer reached day 15 of an estrous cycle and ended 36 h after onset of estrus for the ninth heifer. There were 118 1-h observations at 6-h intervals during the three periestrus periods. Estrous cycles were divided into postestrual, luteal, preestrual, and estrual stages by blood progesterone profiles and estrus. Heifers of these four stages accounted for 27.5, 48.3, 15.4, and 8.8% of heifers available to mount during the 118 observations, and heifers of each stage made 9.0, 5.2, 18.9, and 66.9% of attempted mounts. When number of heifers in estrus simultaneously increased from one to five, total attempted mounts and stands per hour increased from 10.1 to 63.5 and from 4.5 to 48.5. Stands per heifer per hour increased significantly as number of heifers in estrus increased from one to two and tended to increase as number in estrus increased further. Presence of one preestrual heifer in a group containing one estrual heifer increased total stands per hour from 2.3 to 5.0. Efficiency of detection of estrus may depend on proportion of animals within estrous cycle stages that have highest mounting activity.     

A macrophage and theca cell-enriched stromal cell population influences growth and survival of immature murine follicles in vitro

Innovations in in vitro ovarian follicle culture have revolutionized the field of fertility preservation, but the successful culturing of isolated primary and small secondary follicles remains difficult. Herein, we describe a revised 3D culture system that uses a feeder layer of ovarian stromal cells to support early follicle development. This culture system allows significantly improved primary and early secondary follicle growth and survival. The stromal cells, consisting mostly of thecal cells and ovarian macrophages, recapitulate the in vivo conditions of these small follicles and increase the production of androgens and cytokines missing from stromal cell-free culture conditions. These results demonstrate that small follicles have a stage-specific reliance on the ovarian environment, and that growth and survival can be improved in vitro through a milieu created by pre-pubertal ovarian stromal cell co-culture.     

Effect of steroid hormones on tissue remodelling and progesterone receptors in the uterus of seasonally anoestrous brushtail possums (Trichosurus vulpecula)

This study describes progesterone receptor (PR) location within uterine cells and associated morphological changes to the uterine luminal and glandular epithelium in seasonally anoestrous brushtail possums treated with oestradiol and/or progesterone. Twenty-four adult female possums (n = 6/group) were treated with oestradiol, progesterone, oestradiol followed by progesterone or with the oil vehicle alone for 6-day periods. Uterine tissue was recovered, weighed and processed for light and transmission electron microscopy and for immunohistochemistry for PRs. Stereological techniques were used to quantify epithelial cell and constituent volumes for both luminal and glandular tissues. Plasma concentrations of oestradiol and progesterone were determined by radioimmunoassay. Mean uterine wet weights were significantly heavier (P < 0.001) following oestradiol/progesterone treatment and maximum gland dilation, cellular and stromal growth, maximum cell height, and cell and constituent volumes were recorded after this treatment regimen. Cell nuclei and debris were commonly observed in gland lumina, and nuclear PRs were found predominantly in stromal cells following oestradiol-only treatment. Sequential treatment with oestradiol and then progesterone caused a decline in the number of positively stained cells. Epithelial cells contained extensive secretory organelles and degenerating cells were common within the glands. Oestradiol treatment induced cell and cell constituent growth and promoted PR formation in anoestrous possum uterine tissue. Subsequent exposure to progesterone stimulated uterine tissues to reach maximum wet weights and led to the cellular maturation necessary to remodel the uterus in preparation for pregnancy.     

Effect of concentrations of murine stromal and hematopoietic cells on the progenitors expansion in their three-dimensional coculture in nonwoven fabrics

The effects of concentrations of murine stromal and hematopoietic cells on their three-dimensional coculture in nonwoven fabrics were investigated for the construction of an ex vivo expansion system for hematopoietic progenitors without adding expensive cytokines. The increase in initial stromal cell concentration from 0.4 x 10(5) to 2.7 x 10(5) cells/ml increased progenitor concentration, while the increase in initial hematopoietic cell concentration from 0.6 x 10(5) to 6.1 x 10(5) cells/ml decreased the fold increase in hematopoietic cell concentration.     

Thyroid hormones in milk and blood of lactating donkeys as affected by stage of lactation and dietary supplementation with trace elements

The traditional utilization of donkeys (Equus asinus) as dairy animals has recently attracted substantial scientific interest with regard to human nutrition. Donkey milk is well tolerated by infants with cows' milk allergy, useful in the treatment of human immune-related diseases, in the prevention of atherosclerosis, and in-vitro studies showed an anti-proliferative effect. Active 3-3'-5-triiodothyronine (T3) in colostrum and milk could play different physiological roles, systemic and paracrine, for both the mother and the suckling offspring. The aim was to evaluate whether thyroid hormones (TH) concentrations in milk and blood of lactating donkeys change with the advancing lactation and whether they can be affected by dietary supplementation with several trace elements, some of them directly involved with TH synthesis (I), metabolism (Se) and action (Zn). Sixteen lactating jennies were divided into two groups (CTL and TE). Mixed feed for TE was added with Zn, Fe, Cu, Mn, I, Se and Co. Every 2 weeks milk and blood samples were collected at 11·00. Total concentrations of T3 in milk (T3M) and T3 and T4 in plasma (T3P and T4P) were assayed using ELISA kits, validated for the donkey species. T3M was not correlated with TH concentrations in blood, did not change with the stage of lactation, and was significantly higher in TE (4·09 ± 0·07 ng/ml, mean ± SE) than in CTL group (3·89 ± 0·08 ng/ml). T4P (81·8 ± 5·2 ng/ml) and T3P (15·2 ± 1 ng/ml) significantly changed with time, but were not significantly affected by dietary treatment. T3P/T4P ratio was significantly lower in TE group. This study indicates that in donkey milk the concentration of T3, a human-like bioactive compound, can be affected by trace elements intake.     

Characterization of cellular motions through direct observation of individual cells at early stage in anchorage-dependent culture

In the monolayer culture of murine fibroblasts, the characterization of cellular motions was performed by morphological observation using a tool consisting of an optical assembly connected to a computer-aided image analysis system. This tool enabled the estimation of the projected area of the cells with sufficient accuracy, being able to follow the serial behavior of the cells on the culture surface. Trypsinization was chosen as an external factor for altering the state of the cells, and the rate of cell spreading (r(S)) and time of first cell division (t1) were evaluated by subjecting the cells to trypsin treatment for t(T)=3 and 15 min. During culture of the cells treated for t(T)=3 min, the r(S) values of individual cells exhibited a broad variation, ranging from 41 to 321 microm2/h, and the average r(S) was 165+/-78 microm2/h , which was 1.5 times larger than that of the cells treated for t(T)=15 min. On the contrary, the average t1 of the cells treated for t(T)=3 min was 9.5 h which was 60% reduced as compared with that of the cells treated for t (T)=15 min. The prolonged time of trypsin treatment was considered to induce the decrease in r(S) and delay of the first cell division due to the requirement for the recovery from cell surface damage caused by trypsinization. The logarithmic plots of r(S) and t1 were found to have an inverse relation regardless of the state of the individual cells.     

Effect of temperature on cell population balance in Dexter's culture of murine bone marrow hematopoietic cells with stromal cells

The effect of temperature in the range from 25 to 37 degrees C on the population balance of stromal and multiple-lineage hematopoietic cells from murine bone marrow at various stages of differentiation in Dexter's culture was investigated. The length of time required for stromal cells to reach confluence after inoculation of both cell types harvested from murine bone marrow was shorter at higher temperatures. On the other hand, the hematopoietic cell concentration initially decreased more rapidly at higher temperatures until the confluence of stromal cells, which should then support the proliferation of hematopoietic cells. The concentration of hematopoietic cells began to increase after 2 weeks incubation at 33 and 37 degrees C and after 4 weeks at 29 degrees C. However, the growth of hematopoietic cells was not stable at 37 degrees C, and neither stromal nor hematopoietic cells showed significant growth at 25 degrees C. The specific growth rate of hematopoietic cells at 29 degrees C after 4 weeks was comparable or higher than that at 33 degrees C, while the final concentration of hematopoietic cells was maximal at 33 degrees C. Using a cultivation method in which hematopoietic cells were recharged onto a confluent layer of stromal cells prepared at 33 degrees C, the maintenance and growth of hematopoietic cells were better at 29 degrees C than at higher temperatures. The content of progenitor cells among the hematopoietic cells increased prior to the increase in the hematopoietic cell concentration, and the progenitor cell was greater content at the lower temperature. These results suggest that cultivation at 29 degrees C might be superior to 33 degrees C for the maintenance and growth of hematopoietic cells with a prepared confluent layer of stromal cells.     

Murine SPAM1 is secreted by the estrous uterus and oviduct in a form that can bind to sperm during capacitation: acquisition enhances hyaluronic acid-binding ability and cumulus dispersal efficiency

Sperm uptake of epididymal sperm adhesion molecule 1 (SPAM1) in vitro has recently been shown to be a marker of sperm maturation, since acquisition of this surface hyaluronidase increases cumulus dispersal efficiency. Here, we demonstrate that this glycosyl phosphatidylinositol-linked sperm antigen, previously shown to be expressed during estrous in the female reproductive tract, is secreted in the uterine and oviductal fluids (ULF and OF respectively) in a 67 kDa form, which can bind to sperm. We show that it can be acquired by caudal sperm from Spam1 null, Spam1-deficient mutant, and wild-type (WT) mice in vitro during incubation in ULF or OF at 37 degrees C, as detected by immunocytochemistry and flow cytometry. SPAM1 binding after ULF incubation was localized predominantly to the acrosome and the mid-piece of the flagella of Spam1 null sperm in a pattern identical to that of WT sperm. After ULF incubation, WT sperm demonstrated a significantly (P<0.001) enhanced hyaluronic acid-binding ability, and the involvement of SPAM1 in this activity was shown by a significant (P<0.001) decrease in binding when sperm were exposed to SPAM1 antiserum-inhibited ULF. Importantly, when Spam1 null sperm were exposed to ULF with SPAM1 accessible (in the presence of pre-immune serum) or inaccessible (in the presence of SPAM1 antiserum) for uptake, there was a significant difference in cumulus dispersal efficiency. Taken together, these results suggest that in the sperm surface remodeling that occurs prior to and during capacitation, the fertilizing competence of sperm is increased via acquisition of SPAM1, and likely other hyaluronidases, from the female tract.     

Postpartum ovarian follicular dynamics and estrous activity in lactating dairy cows

The objectives of this study were to characterize early postpartum follicular dynamics in dairy cows in relation to their estrual activity and subsequent reproductive performance using 50 (26 primiparous and 24 multiparous) lactating Holstein cows. Ovaries and uterine horns of postpartum lactating cows were examined by ultrasonography 3 times weekly and continued until first services occurred after a 45-d voluntary waiting period. No differences were detected in fertility between primiparous and multiparous cows. In 40 of 50 cows, first postpartum ovulation was observed within 4 follicular waves, and the follicular wave patterns and ovarian cycles in most cows returned to normal as in cattle having normal estrous cycles after the second postpartum ovulation. Cows with the longest intervals from calving to first ovulation produced the most milk and also had prolonged intervals to first estrous activity. Differences in follicular dynamics before first ovulation altered intervals to first estrus, first service, and uterine involution, but these differences did not affect pregnancy rate, number of services, and days open. First postpartum insemination after 3 follicular waves tended to have greater pregnancy rates than those after 2 follicular waves. First inseminations at first estrus could produce greater pregnancy rates than those at subsequent periods of estrus.     

Betaine reduces cellular melanin content via suppression of microphthalmia-associated transcription factor in B16-F1 murine melanocytes

Food Science and Biotechnology - Long-term topical skin care by traditional anti-melanogenic agents can raise several safety concerns. An understanding of the molecular mechanisms of active...     

Anogenital distance is associated with postpartum estrous activity,intensity of estrous expression,ovulation, and progesterone concentrations in lactating Holstein cows

The objectives of this retrospective observational study were to determine the associations of anogenital distance (AGD) with (a) postpartum estrous activity, (b) diameter of the preovulatory follicle, (c) intensity of estrous expression, (d) postestrus ovulation, (e) corpus luteum (CL) size, and (f) concentrations of progesterone at estrus and on d 7 after estrus. Lactating Holstein cows (n = 178; 55 primiparous, 123 multiparous) were enrolled into the study during the first postpartum week. All cows were continuously monitored by a pedometer-based automated activity monitoring (AAM) system for estrus. Postpartum estrous activity was assessed using the AAM estrus alerts, in which cows with at least one true estrus alert (i.e., a relative increase in steps from each cow's baseline detected by the AAM and the presence of at least one follicle >15 mm, a CL <20 mm, or no CL detected by ultrasound) by the first 50 d in milk (DIM) were considered to have commenced estrous activity. At the estrus alert >60 DIM, ovulation was determined by ultrasound at 24 h, 48 h, and 7 d after estrus, and blood samples were collected at estrus alert and on d 7 after estrus for progesterone analysis. The AGD was measured from the center of the anus to the base of the clitoris and classified as either short- or long-AGD using 2 cut-points of 148 mm (predictive of the probability of pregnancy to first insemination; short-AGD, n = 115; long-AGD, n = 63) and 142 mm (the median AGD; short-AGD, n = 90; long-AGD, n = 88). Regardless of the cut-point used, early postpartum estrous activity by 50 DIM (67 vs. 54%), duration of estrus (11.6 vs. 9.7 h), and preovulatory follicle diameter (20 vs. 19 mm) were greater in short-AGD than in long-AGD cows. Increased peak of activity at estrus in short-AGD cows (354 vs. 258% mean relative increase) was affected by an interaction between AGD and parity in which multiparous long-AGD cows had lesser relative increase in activity than primiparous cows (217 vs. 386%, respectively). Mean progesterone concentration at estrus was lesser in short-AGD (0.47 vs. 0.61 ng/mL) than in long-AGD cows. The ovulatory response at 24 h did not differ, but at 48 h (91 vs. 78%) and on d 7 after estrus (97 vs. 84%) it was greater in short-AGD cows. Although CL diameter on d 7 after estrus did not differ, short-AGD cows had greater progesterone concentration 7 d after estrus than long-AGD cows (4.1 vs. 3.2 ng/mL, respectively). In conclusion, greater proportions of short-AGD cows commenced estrous activity by 50 DIM, had larger preovulatory follicles, exhibited greater duration of estrus, had reduced progesterone concentration at estrus, had greater ovulation rates and progesterone concentration 7 d after estrus compared with long-AGD cows, with no difference in CL size between AGD groups. Because all the differences in physiological characteristics of short-AGD cows reported herein favor improved reproductive outcomes, we infer that these are factors contributing to improved fertility reported in short-AGD cows compared with long-AGD cows.     

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.     

The relationship between estrous behavioral score and time of ovulation in dairy cattle

One of the major contributors to poor fertility of a dairy herd is ineffective detection of estrus. Recently, it has become evident that cow factors contribute largely to low detection rates. Until now, standing behavior has been the symptom used to determine the right moment for insemination. However, standing behavior is not observed in over 50% of the cows in estrus in a number of herds, and no data are available to relate the moment of ovulation to other estrous symptoms. Therefore, in the present study, cows were observed for a number of visual signs of estrus, twice per day for 30 min, and the moment of ovulation was estimated by transrectal ultrasonographic scanning of the ovaries. In this study, 100 cows were detected in estrus visually, using a scoring system. Standing behavior was observed in 50% of those cows, although there was more than one cow in estrus at a time in most (85%) cases. There was no correlation between follicular size and ovulation time or estrous behavior score. Levels of 305-d milk yield and parity were also not correlated with estrous behavior scores. However, a significant correlation (0.31) was found between detection score and day of ovulation. A retrospective analysis revealed that cows that ovulated 0 to 24 h after artificial insemination (AD) scored almost three times the number of estrous behavior points compared to the ones that ovulated 24 to 48 h after AI. Ovulation more than 48 h after AI resulted in pregnancy in only 15% of the cows.     

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.     

Influence of energy balance and body condition on estrus and estrous cycles in Holstein heifers

The objective was to determine effects of energy balance and body condition on estrous behavior and estrous cycles in Holstein heifers. Before the experiment heifers were fed so body condition remained moderate or they became fat. During the 2 x 2 factorial experiment, moderate and fat heifers were in positive or negative energy balance. Heifers were fed individually twice daily, and energy balance was calculated daily. Heifers were observed for 30 min every 3 h, and all standing and mounting events were recorded for three consecutive estrous cycles. When progesterone in serum was less than 1 ng/ml, standing and mounting activity were profiled. Area, peak, and duration of activity profiles were examined among groups of heifers. Negative energy balance or fat body condition did not reduce peak or duration of estrous behavior and thus did not reduce detectability of estrus. Onset of diestrus was delayed in heifers that were fat and in negative energy balance relative to other heifers. We suggest that fat body condition, coincident with negative energy balance, may reduce accuracy of timing artificial insemination relative to ovulation and may consequently reduce fertility in cattle.     

Relationships of sexual activities in estrous cows to different frequencies of observation and pedometer measurements

Sexual activities were observed continuously for 120 h in Holstein dairy cows during moderate climate. Pedometer measurements of motor activity were correlated with total activities initiated, total activities received, mounts received, and time in estrus. Twelve of 14 individual sexual activities also were correlated on a within-cow basis with pedometer-measured increases in motor activity; disoriented mounts and licking front were not correlated to pedometer increases. Although observations of four 1-h and four or six 30-min observations accounted for greater variation, pedometer increases in activity accounted for as much variation in total sexual activities as periodic observations of 30 min or 1 h for 1 to 3 X /d. Combining periodic observations with pedometer measurements resulted in greater coefficients of determination for analyses of sexual activities than with each individual factor. Sexual activities and pedometer increases also were correlated positively with body condition score and day postpartum, and they were correlated negatively with milk production and body weight change since calving. Although mounting and total sexual activities were shifted significantly from expected on a quartile basis, shifts were probably not of practical significance and provided little evidence for more frequent occurrence of estrous activity during nocturnal periods. Rather, the most marked decrease in mounting and total sexual activities occurred at milking and feeding times, indicating that dairy farmers should be more concerned with disruptions of activities by management practices rather than nocturnal occurrence of estrus.     

Comparison of ovarian function and circulating steroids in estrous cycles of Holstein heifers and lactating cows

Ovarian function was compared between nulliparous heifers (n = 29; 10 to 16 mo old) and lactating Holstein cows (n = 31; 55.9 +/- 3.5 d postpartum). Follicular dynamics, corpus luteum growth, and regression, and serum steroid concentrations were evaluated through ultrasonography and daily blood sampling. Most heifers (27 of 29) but only 14 of 31 cows had typical spontaneous estrous cycles after cycles were initiated. Twelve cows had atypical cycles, and 5 became anovulatory during the study. The 12 cows with atypical estrous cycles had low serum estradiol after luteolysis and failed to ovulate the dominant follicle present at luteolysis. Heifers and cows with typical cycles were compared directly. Interovulatory intervals were similar between heifers (22.0 +/- 0.4 d) and cows (22.9 +/- 0.7 d). Those animals had estrous cycles with either 2 (15 heifers; 11 cows), 3 (9 heifers; 2 cows), or 4 follicular waves (3 heifers; 1 cow). Cows ovulated later after luteolysis than heifers (5.2 +/- 0.2 vs. 4.6 +/- 0.1 d, respectively), and had more multiple ovulations (17.9 vs. 1.9%). Maximal serum estradiol concentration preceding ovulation was lower in cows than in heifers (7.9 +/- 0.8 vs. 11.3 +/- 0.6 pg/mL) even though ovulatory follicles were larger in cows (16.8 +/- 0.5 vs. 14.9 +/- 0.2 mm). Similarly, maximal serum progesterone concentration was lower for cows (5.6 +/- 0.5 vs. 7.3 +/- 0.4 ng/mL), whereas maximal volume of luteal tissue was larger for cows than heifers (11,120 +/- 678 vs. 7303 +/- 308 mm3). Thus, higher incidence of reproductive anomalies in lactating cows, such as low conception rate, ovulation failure, delayed ovulation, and multiple ovulations, may be due to lower circulating steroid concentrations in spite of larger ovulatory follicles and luteal structures.