Spontaneous cyclic embryonic movements in humans and guinea pigs.

Motility assessment before birth can be used to evaluate the integrity of the nervous system. Sideways bending (SB) of head and/or rump, the earliest embryonic motility in both humans and guinea pigs, can be visualized sonographically. We know from other species that early embryonic motility is cyclic. This study explores the distribution of SB-to-SB intervals in human and guinea pig embryos before the appearance of more complex movements such as general movements. We hypothesized that the activity in both species is cyclic. We made 15-min sonographic recordings of SBs between 5 weeks and 0 days (5wk0d) and 7wk0d conceptional age (CA) in 18 human embryos of uncomplicated IVF pregnancies (term 38 weeks) and in 20 guinea pig embryos between 3wk4d and 4wk0d CA (term 9 weeks). SB-to-SB interval durations were categorized as long (≥10 s) or short (<10 s) intervals. For human embryos, the median values for long and short intervals were 61 s (range, 10-165 s) and 3 s (range, 1-9 s) respectively; for guinea pigs 38 s (range, 10-288 s) and 5 s (range, 1-9 s), respectively. During development, the duration of long intervals decreased while the number of short intervals increased for both species. The earliest embryonic motility in the human and guinea pig is performed cyclically with distinct developmental milestones. The resemblance of their interval development offers promising possibilities to use the guinea pig as a noninvasive animal model of external influences on motor and neural development.

Addition of chromic oxide to creep feed as a fecal marker for selection of creep feed-eating suckling pigs.

OBJECTIVE: To determine whether the addition of chromic oxide (Cr(2)O(3)) to creep feed could be used as a visual marker in feces for selection of creep feed-eating suckling pigs. ANIMALS: 20 suckling pigs. PROCEDURES: Via syringe, 5 pigs (2 to 3 days old on day 0; 1 pig/treatment) from each of 4 litters received oral administrations of 10, 20, 30, or 40 g of creep feed containing 10 g of Cr(2)O(3)*kg(1) on each of 2 consecutive days (days 20 and 21) or 30 g of creep feed containing 10 g of Cr(2)O(3)*kg(1) on day 20 and 30 g of Cr(2)O(3)-free creep feed on day 21. On days 21 through 24, 6 fecal samples were collected from each pig at regular intervals between 8:00 AM and 6:00 PM. Green-colored feces were considered indicative of creep feed consumption (eaters). Data analyses were based on single and multiple fecal samples. RESULTS: On day 22, evaluation of 1 fecal sample/pig and multiple fecal samples per pig resulted in identification of as many as 40% and only 15% of the feed-treated pigs wrongly as noneaters, respectively. Repeated sampling over multiple days would identify 99% of eaters accurately. Pigs erroneously identified as noneaters were those administered either low amounts of Cr(2)O(3)-supplemented creep feed for 2 days or Cr(2)O(3)-supplemented creep feed on only 1 day. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that addition of Cr(2)O(3) to creep feed enables selection of individual creep feed-eating suckling pigs via examination of feces, provided that repeated fecal samples are evaluated.

EMG activity of the muscular and stromal layer of the cervix in relation to EMG activity of the myometrium and cervical dilatation in PGF2alpha induced parturition in the cow.

The goal of this study was to quantify and characterize the electromyographic (EMG) activities in the cervical outer muscular layer and in the cervical stromal layer, and to characterize their relationship with myometrial EMG activity and cervical dilatation during PGF2alpha-induced parturition in term pregnant cows. We continuously measured the EMG activity of the uterine myometrium and cervical outer muscular layer as well as the cervical stromal layer in five cows using bipolar electrodes while at the same time measuring changes in the cervical diameter with ultrasound cervimetry. This we did from the moment a prostaglandin analogue was injected until the expulsion of the calf. In contrast to the cervical stromal layer, the cervical outer muscular layer showed distinct EMG activity, which began to increase at about the same time as the EMG activity of the myometrium, i.e. some 12 h before the start of cervical dilatation. However, the rate of this increase was lower than in the myometrium and it was not characterized, like in the myometrium, by an increase in maximum EMG amplitude. Although the cervical outer muscular layer showed contracture and contraction like EMG activity in unison with in the myometrium, it was also characterized by a more irregular EMG activity, which occurred independently from the myometrium. These data suggest that while the outer muscular layer of the cervix may be considered to be a caudal continuation of the myometrium, it also displays activity independently from the myometrium. The physiological relevance of this activity remains to be explored.

High iNOS mRNA and protein levels during early third trimester suggest a role for NO in prelabor cervical ripening in the bovine.

Nitric oxide (NO) plays a key role in the processes leading to cervical softening prior to labor. Inducible nitric oxide synthase (iNOS) contributes most to the increased production of NO during labor, as demonstrated in the rat cervix, or at term pregnancy in women. Changes in expression of iNOS during late gestation have not yet been studied longitudinally in any species, because repeatedly taking biopsies could not be performed. iNOS mRNA (n = 6) and protein expression (n = 3) in serial cervical biopsies of pregnant pluriparous cows taken around days 225, 250, and 275 of pregnancy and within 1.5 hr after calving (d225, d250, d275 and parturition biopsies, respectively) were measured using quantitative RT-PCR and Western blotting. iNOS mRNA expression decreased from the d225 biopsy onwards, differences being significant between the d250 and d275 (P < 0.05) and between the d275 and parturition biopsies (P < 0.05). iNOS protein expression decreased from d225 to d250 onwards. Immunohistochemical analysis of biopsies showed, besides positive staining in endothelium and epithelium, which remained unchanged at different time points, that iNOS expressing cells in the connective tissue cells of early biopsies were predominantly spindle shaped (mostly smooth muscle cells and some fibroblasts). In the parturition biopsies, iNOS reactivity was mainly found in mononuclear leucocytes. These results lead us to suggest that iNOS from spindle shaped cells is involved in prepartum cervical ripening, while iNOS in mononuclear inflammatory cells may be important for local tissue repair mechanisms during postpartum cervical involution.

Cortisol administration to pregnant sows affects novelty-induced locomotion, aggressive behaviour, and blunts gender differences in their offspring.

Several behavioural effects of prenatal stress are reported in literature, and these seem to depend, among other factors, on the gender studied and the period of gestation in which prenatal stress is applied. In the present study, oral administration of hydrocortisone-acetate (HCA) to 41 pregnant sows was used as a model for prenatal stress, since corticosteroids are considered a key mediator in the effects of prenatal stress. HCA was orally administered to pregnant sows during three periods of gestation: 21-50 (period 1, P1, n = 10), 51-80 (period 2, P2, n = 10) and 81-110 (period 3, P3, n = 10) days after insemination (term 115 days). Control sows (n = 11) received vehicle from 21 to 110 days after insemination. Between days 9 and 48 after birth, treatment effects on male and female piglet behaviour were determined in the home pen and in four different behavioural tests. During the backtest, no gender differences were observed in vocalisations in HCA-piglets, while control males vocalised more than control females. In the home pen at 14 days of age, HCA-piglets spent less time in social interactions than control piglets. During the novel environment test, P1- and P3-piglets walked more than control piglets, but this was not observed during the novel object test, four days later. At weaning, P2- and P3-piglets performed less individual play. Prior to mixing with an unfamiliar piglet (male piglets only), HCA-piglets had lower salivary cortisol concentrations than control piglets, but no difference was observed after mixing. P1-, P2-, and P3- piglets had fewer non-aggressive encounters, and P2-piglets continued fighting longer than control piglets. The present study demonstrates that elevated maternal cortisol concentrations during gestation affect piglet behaviour, and effects do differ between male and female piglets. In addition, effects depend on the period of cortisol administration.

Comparison of accuracy of transabdominal ultrasonography, progesterone and pregnancy-associated glycoproteins tests for discrimination between single and multiple pregnancy in sheep.

The aim of the present study was to evaluate and, compare the accuracy of transabdominal ultrasonographic (US) and the progesterone (P4-RIA) and ovine pregnancy-associated glycoprotein (ovPAG-RIA) tests for the discrimination between single and multiple pregnancy in sheep. One hundred pregnant AwassixMerino ewes were scanned by transabdominal ultrasonography (3.5 MHz linear-array transducer) at Days 43-56 and 81 of these ewes were scanned at Days 76-87 of gestation. The ewes were scanned in dorsal recumbency at the bare area of the inguinal regions (without pre-scanning shaving of the ventral abdominal wall). After each scan, blood samples were withdrawn from the jugular vein to estimate the levels of P4 and ovPAG by radioimmunoassay. At lambing, 61 ewes gave birth to single lambs and 39 ewes gave birth to multiples. The sensitivity of the transabdominal US, the P4-RIA and the ovPAG-RIA tests for determining ewes carrying multiples was 54, 64.1 and 64.1% at Days 43-56. At Days 76-87 of gestation these accuracies were 60.0, 66.7 and 76.6% for the US, P4-RIA and PAG-RIA tests, respectively. The specificity of the transabdominal US, the P4-RIA and the ovPAG-RIA tests for determining ewes carrying singles, was 78.6, 60.7 and 62.3% at Days 43-56 and 78.4, 64.7 and 70.6% at Days 76-87 of gestation, respectively. It is concluded that the accuracy of transabdominal ultrasonographic (without pre-scanning shaving of the ventral abdominal wall), the P4- and the ovPAG-RIA tests for determination of the fetal numbers in AwassixMerino crossbred ewes is too low to be used in the field.

Continuous fetal heart rate monitoring during late gestation in cattle by means of Doppler ultrasonography: reference values obtained by computer-assisted analysis.

Continuous fetal heart rate (FHR) monitoring using transabdominal Doppler ultrasonography can be assumed to provide information about the viability of the bovine fetus during late gestation, as has been found in humans. To be able to recognize unfavourable fetal conditions, first the normal ranges of FHR parameters in cattle should be established. Therefore, in this study we aimed to determine the normal ranges of computerized FHR parameters, like basal fetal heart rate (BHR), number of accelerations and decelerations per hour and short and long term variation (STV and LTV) during the last 3 weeks before calving (n = 21 cows). Each cow had one recording in each of three episodes of 7 days before parturition. As recording time in the cow is limited, we also studied whether these FHR parameters differ between recordings of 30 and 60 min duration (n = 31 pairs of recordings). The outcomes of FHR recordings with a duration of 30 or 60 min did not differ significantly, except for a higher percentage of signal loss in the 60 min recordings. Therefore, determination of normal ranges was performed in 30 min recordings. BHR decreased from 3 to 2 weeks (114 to 109 bpm; P < 0.0001) before parturition and then remained constant until 2 days before calving. The mean number of accelerations per hour ranged between 4.4 and 5.0 h(-1) and did not change significantly with time. Compared to 3 weeks before parturition, STV was significantly higher at 2 weeks (P < 0.05), but not at 1 week before parturition (8.1, 10.0, and 9.2 ms, respectively). Changes in LTV showed a time course comparable to that of STV, but significance was not reached (51.4, 58.6, and 58.4 ms for respectively 3, 2 and 1 weeks before parturition). No decelerations were found during the period understudy. In conclusion, this study has provided normal ranges of bovine computerized FHR parameters during the last 3 weeks of gestation, allowing a comparison with data from cows with compromised gestations in future.

Lower birth weight and attenuated adrenocortical response to ACTH in offspring from sows that orally received cortisol during gestation.

Prenatal stress is known to affect several offspring characteristics, but its effects depend among other factors on the period of gestation in which it is applied. In the present study, oral administration of hydrocortisone-acetate (HCA) was used to elevate cortisol concentrations in pregnant sows to levels also observed after psychological stress. HCA was administered during three different periods of gestation (115 days in pigs): period 1: 21-50 (P1, n = 10), period 2: 51-80 (P2, n = 10) and period 3: 81-110 (P3, n = 10) days after insemination. Control sows (n = 11) received vehicle from 21-110 days after insemination. When P1-, P2- and P3-sows did not receive HCA, they also received vehicle. During gestation, weekly saliva samples were taken from the sows to determine salivary cortisol concentrations. Treatment effects on sow, litter and piglet characteristics were determined. In addition, two female piglets per litter were subjected to an ACTH-challenge test at 6 weeks of age to determine the adrenocortical response to ACTH. Pigs were slaughtered at 6 months of age and slaughter weight, back fat thickness and percentage of lean meat were analysed. During the period of treatment with HCA, salivary cortisol concentrations were increased in P1-, P2- and P3-sows compared to control sows (P < 0.01). The total number of piglets born per litter did not differ among treatment groups (P > 0.30), but pooled HCA-litters had a higher percentage of live born piglets (P < 0.05) and fewer mummies than control litters (P < 0.05). Gestation length did not differ among treatment groups (P = 0.21), but did affect treatment effects on birth weight. Overall, HCA-piglets weighed less at birth, and remained lighter until weaning (P < 0.05). The salivary cortisol concentrations after i.m. injection of ACTH (2.5 IU/kg) were lower in P1- and P3-piglets compared to control piglets. At slaughter, HCA-treatment indirectly decreased lean meat percentage and increased back fat thickness. In conclusion, elevated peripheral cortisol concentrations in pregnant sows affect both litter characteristics and piglet physiology, the latter depending on the period of gestation during which concentrations were elevated. Underlying mechanisms require further investigation.

The effect of a single oxytocin or carbetocin treatment on uterine contractility in early postpartum dairy cows.

The uterotonic characteristics and effectiveness of a single treatment with either oxytocin or carbetocin were quantified in early postpartum dairy cows after normal, uncomplicated calvings. Both the short-term (within 4 h), and the long-term effects (between 12 and 36 h) of the two treatments were compared. Between 14 and 16 h after parturition, 27 multiparous Holstein-Friesian dairy cows, without fetal membrane retention, were selected and divided into three groups. The first group (n = 9) was administered 50 IU oxytocin intramuscularly, the second group (n = 10) received 0.35 mg carbetocin, while animals of the third group (n = 8), serving as a control, were administered 5 mL saline solution. A transcervically introduced open tip catheter system was used for the non-invasive acquisition of the intrauterine pressure (IUP) recording. After digitalization, the signals were analyzed, using a specially adapted graphical software program. A significant short-term effect was found both in the oxytocin and carbetocin treated groups from the analysis of the contraction frequencies (FREQ) and of the total area under the curve (TAUC). After significant peaking during the first post-treatment hour, the values of the parameters for these two groups remained higher during the second hour, returning to the initial levels again during the third hour and reaching the level of the control group by the 12th hour. Mean amplitude (AMP), duration (DUR) and area under the curve (AUC) of pressure cycles were not significantly affected by any of the treatments. Although mean FREQ and TAUC significantly declined from the initial values to 12, 24 and 36 h in all groups, mean AMP and AUC in the oxytocin and carbetocin treated groups, and mean DUR only in the carbetocin treated group to 12 and 36 h, the long-term analysis revealed no significant treatment differences for any IUP parameters. Because treatment with either oxytocin, or carbetocin elicited similar uterotonic effects in healthy, early postpartum cows, it cannot be expected, that using carbetocin in preference to oxytocin, will result in a more beneficial clinical effect on uterine involution during this period.

Validation of pressure measurements and electromyographic results for the uterus of cattle during the early postpartum period.

OBJECTIVE: To evaluate methods for on-farm measurements of uterine contractility in postpartum dairy cows by comparing data simultaneously recorded by use of 2 intrauterine pressure (IUP) devices and quantified electromyographic (EMG) signals. ANIMALS: 5 cows during the first 48 hours after parturition. PROCEDURE: 2 EMG electrodes were implanted on the surface of the gravid uterine horn. Parturition was induced by injection of a prostaglandin F2alpha analogue at day 274 of gestation. An open-tip catheter and pressure microtransducer were transcervically inserted and affixed to a caruncle immediately after calving. Changes in IUP were recorded concurrent with EMG recordings during 2-hour periods at 2, 6, 12, 18, 24, 36, and 48 hours after parturition. Novel acquisition and analysis software programs were used with a digital data-filtering capability for evaluation of IUP and EMG signals. RESULTS: The method for intrauterine fixation of the 2 pressure measurement instruments was effective and allowed easy, externally guided removal of the devices 48 hours after parturition. There was a high correlation between the data obtained by the 2 pressure measuring systems. Good correlation was also found between pressure data obtained by the open-tip catheter system and EMG signals. Although the quantified IUP and EMG signals were highly comparable, synchronization was not always evident during visual inspection of these signals. CONCLUSIONS AND CLINICAL RELEVANCE: The open-tip IUP catheter system with a special fixation method is suitable for use in on-farm studies. It will enable investigators to record natural and pharmacologically influenced uterine contractility in early postpartum dairy cows.

Ultrasonographic appearance of the conceptus, fetal heart rate and profiles of pregnancy-associated glycoproteins (PAG) and prostaglandin F2alpha-metabolite (PGF2alpha-metabolite) after induction of fetal death with aglepristone during early gestation in cattle.

A higher incidence of fetal losses, especially after the use of artificial reproduction techniques, asks for more intensive monitoring of bovine pregnancies. In this study, a model for fetal death (FD) was created by administering the antiprogesterone aglepristone twice, at Day 47 and 48 of gestation (n=5). Control heifers received the solvent (n=5). The temporal relationships between changes in ultrasonographic appearance of fetal fluids and membranes, fetal heart rate (FHR) and peripheral plasma levels of pregnancy-associated glycoprotein (PAG) and PGF2alpha-metabolite as determined by radioimmunoassay associated with FD were monitored at eight hour intervals around treatment. For the analysis of plasma levels the period under study was divided into five epochs (T1: before injection of aglepristone/solvent; T2: from first to second injection; T3: from second injection to FD; T4: from diagnosis of FD to 56 h later; T5: from 56 h to 104 h after diagnosis of FD). Control heifers produced healthy calves at term, but in treated heifers, FD occurred on average at 58 (range 48-80) h after first injection of aglepristone. Fetal death was always preceded by a visible reduction of the amount of allantoic fluid and by segregation of the allantochorionic membrane from the endometrium. FHR remained rather constant in both groups, but a (non-significant) drop in FHR around 8h before FD was diagnosed in four of five treated animals. All fetuses were expulsed after FD. Levels of PAG remained constant or even slightly increased in controls, but decreased in treated animals from T2 onward: levels during T4 and T5 significantly differed from those during T1 and from values in controls during T4 and T5 (P<0.01). PGF2alpha-metabolite levels did not change in the controls, but in the treated group they were significantly higher during T3 when compared to T1 (P<0.05). After this increase, a sharp decrease in PGF2alpha-metabolite level occurred, reaching a significantly lower level at T5 when compared to control animals (P=0.01). It is concluded, that FD induced by aglepristone is preceded by ultrasonographic visible changes in fetal membranes and fluids and a rise in PGF2alpha-metabolite and is followed by a drop in PAG and PGF2alpha-metabolite.

Characteristics of bovine early puerperal uterine contractility recorded under farm conditions.

A non-invasive, digital technique was used to measure and quantify intrauterine pressure (IUP) changes in early postpartum dairy cows kept under farm conditions in order to document physiological changes in uterine contractility after uncomplicated calvings. In addition, possible relationships between characteristics of uterine contractility and blood ionized calcium (Ca(2+))-concentrations were investigated. Recordings of uterine contractility were made by using a transcervically inserted open tip catheter in 12 healthy cows during their first 48h after calving. The IUP recording technique appeared easily applicable under farm conditions. Although mean frequency (FREQ), amplitude (AMP) and area under the curve (AUC) of the myometrial contractions significantly decreased due to time, untreated early postpartum cows showed a high variability in characteristics of uterine contractility. There was no correlation between blood Ca2+ -concentrations and any of the contractility parameters.

Ultrasonography of the female reproductive organs in farmed ostriches (Struthio camelus spp.).

The main objective of this study was to determine whether transcutaneous ultrasonography could be used as a diagnostic tool to monitor the reproductive organs of female breeding ostriches. An additional aim was to investigate whether the use of this technique could facilitate prediction of the start or end of the egg production season. A technique for on-farm ultrasound scanning is described and examples of ultrasonographic images of different ovarian structures, developing ova and eggs within the genital tract are presented. These data were obtained by scanning mature female breeding ostriches (n=8). In vivo scanning took place a day prior to slaughter, and immediately after slaughter the reproductive organs were scanned in vitro in a water bath. By comparing ultrasonographic images with post mortem ovarian morphology, it appeared that the following morphological structures can be identified using ultrasonography: ovarian follicles of different sizes (diameter (phi) 1-9 cm), atretic follicles, post-ovulatory follicles (POFs), ova at different stages of development and eggs within the genital tract. Of the number of follicles counted during post mortem investigation, 58% (95% confidence interval 0.41-0.79) had been detected during previous in vivo examination. In the second part of our study, ultrasonographic scans were made at weekly intervals in two farmed female ostriches (n=2) during the breeding season in order to determine the predictive value of the technique. By comparing the images of ovarian activity with individual egg production of these hens, preliminary evidence was obtained that scanning might be of value in predicting egg production, especially at the start and the end of the breeding season. It is concluded that transcutaneous ultrasound scanning in mature female breeding ostriches is an easy, noninvasive technique for the monitoring of ovarian (in)activity, for visualization of different functional ovarian structures, for following the development of individual ova and for visualization of an egg with calciferous shell within the oviduct, and that this technique will be a valuable tool in future research on reproductive physiology and pathology, and the development of more advanced reproductive technologies, such as artificial insemination.

The monitoring of bovine pregnancies derived from transfer of in vitro produced embryos.

Both an increased rate of embryonic, foetal and perinatal losses, and the occurrence of deviations in foetal and placental development are associated with bovine pregnancies obtained from in vitro produced embryos. This thus requires for a more accurate and frequent monitoring of foetal and maternal functions during pregnancies. Such approaches will enable to establish the period during which these losses and deviations in development occur and to plan possible clinical interventions. This paper reviews some recent data on return rates, late embryonic and foetal losses in recipients after the transfer of either MOET, IVF or nuclear transfer embryos. Special attention is paid to the diagnostic value of measurements of pregnancy specific/associated proteins and progesterone in maternal plasma. Possibilities to measure foetal body sizes, size of placentomes and foetal heart rate by means of transrectal or transabdominal ultrasonography are illustrated with data from the literature and with recent results from our own large field study with MOET, IVP-co-culture and IVP-SOF embryos.