Short communication: Chronology of different sexual behaviors and motion activity during estrus in dairy cows.

In studying the efficiency of a variety of methods for estrus detection in a large dairy herd, we suspected a definite sequence of estrus signs. Consequently, we observed a subset of animals continuously between 0400 and 2400 h, making a note of the precise timing and frequency of each sexual behavior. Sixteen Holstein-Friesian cows, >20 d postpartum, were equipped with motion activity-sensing neck collars and had milk progesterone profiles monitored simultaneously. The duration between the first and last observed estrus behavior was (mean ± SE) 14.0 ± 1.9 h, with a range 8.5 to 28.75 h. The duration of standing to be mounted (STBM) was 4.68 ± 1.49 h, with a range of 0.25 to 18.25 h. Sniffing the vulva of another cow occurred on average 5.5 ± 1.3 h (range = 0.25-18.25 h) before the first STBM. By ranking the first appearance of each behavior, we established that sniffing was followed by the active behaviors of mounting another cow and not accepting a mount, as well as the passive behaviors of being sniffed and STBM by another cow. Chin resting occurred before not accepting a mount and STBM. All these behaviors were observed in the reverse order after the last STBM. The mean profile of motion activity revealed an increase in motion activity with the onset of exploratory behaviors, and highest values occurred within the period of STBM. Such distinct behavioral sequences may be controlled by changes in peripheral progesterone and estradiol concentrations, as well as by subtle independent mechanisms via pheromones in differing concentrations or divergent composition.

Association of production diseases with motor activity-sensing devices and milk progesterone concentrations in dairy cows.

We have previously established that the efficiency of identifying oestrus with activity-sensing devices can be compromised by common production diseases; the present study was undertaken to determine how these diseases may affect device readings. A total of 67 Holstein-Friesian cows, >20 days postpartum, were equipped with activity-sensing neck collars and pedometers, and simultaneous milk progesterone profiles were also monitored twice a week. The influences of common production stressors on maximum activity and progesterone values were analysed. Approximately 30% potential oestrus events (low progesterone value between two high values) remained unrecognised by both activity methods, and progesterone values in these animals were higher on the potential day of oestrus when both activity methods did not detect an event (0.043 ±â€¯0.004 versus 0.029 ±â€¯0.004 ng/mL; P = 0.03). Data from a subset of 45 cows (two events each) were subjected to mixed models and multiple regression modelling to investigate associations with production diseases. Cow motor activity was lower in lame cows. Maximum progesterone concentrations prior to oestrus increased as time postpartum and body condition score (BCS) increased. There were also fewer days of low progesterone prior to oestrus associated with increases in BCS and maximum progesterone concentrations prior to oestrus. In conclusion, lameness was associated with lower activity values, but this suppression was insufficient to account for lowered oestrus detection efficiency of either device. However, associations were identified between production diseases and progesterone profiles.

KNDy neurone activation prior to the LH surge of the ewe is disrupted by LPS.

In the ewe, steroid hormones act on the hypothalamic arcuate nucleus (ARC) to initiate the GnRH/LH surge. Within the ARC, steroid signal transduction may be mediated by estrogen receptive dopamine-, ß-endorphin- or neuropeptide Y (NPY)-expressing cells, as well as those co-localising kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy). We investigated the time during the follicular phase when these cells become activated (i.e., co-localise c-Fos) relative to the timing of the LH surge onset and may therefore be involved in the surge generating mechanism. Furthermore, we aimed to elucidate whether these activation patterns are altered after lipopolysaccharide (LPS) administration, which is known to inhibit the LH surge. Follicular phases of ewes were synchronised by progesterone withdrawal and blood samples were collected every 2 h. Hypothalamic tissue was retrieved at various times during the follicular phase with or without the administration of LPS (100 ng/kg). The percentage of activated dopamine cells decreased before the onset of sexual behaviour, whereas activation of ß-endorphin decreased and NPY activation tended to increase during the LH surge. These patterns were not disturbed by LPS administration. Maximal co-expression of c-Fos in dynorphin immunoreactive neurons was observed earlier during the follicular phase, compared to kisspeptin and NKB, which were maximally activated during the surge. This indicates a distinct role for ARC dynorphin in the LH surge generation mechanism. Acute LPS decreased the percentage of activated dynorphin and kisspeptin immunoreactive cells. Thus, in the ovary-intact ewe, KNDy neurones are activated prior to the LH surge onset and this pattern is inhibited by the administration of LPS.

Kisspeptin, c-Fos and CRFR type 2 co-expression in the hypothalamus after insulin-induced hypoglycaemia.

Normal reproductive function is dependent upon availability of glucose and insulin-induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c-Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p < 0.05 for both). In the arcuate nucleus (ARC), insulin-treated ewes had an increased expression of c-Fos. Furthermore, the percentage of kisspeptin cells co-expressing c-Fos increased in the ARC (from 11 to 51%; p < 0.05), but there was no change in the medial pre-optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.

Identification of inadequate maternal progesterone concentrations in nulliparous dairy heifers and treatment with human chorionic gonadotrophin.

Plasma progesterone concentrations were estimated for 381 nulliparous dairy heifers from eight farms on days 4-6 after insemination. Heifers were synchronised using two treatments of prostaglandin F2α (PGF2α) 11 days apart. Alternate heifers were treated with 1500 IU human chorionic gonadotrophin (hCG) at the time of sampling, and the pregnancy status assessed ~35 days after insemination. Heifers with no corpus luteum (CL; n-30) at the second PGF2α had lower progesterone concentrations (P<0.001) and pregnancy rates (PR; P=0.001) compared with heifers with CL (n=351, defined as cyclic). The risk of no CL increased in heifers <15 months of age (OR=7.5, P<0.001) and with body condition score <2.5 (OR=4.5, P=0.001). Those with no CL were removed from further analysis. The 20 per cent cyclic control heifers with progesterone concentrations ≤2 ng/ml on days 4-6 had lower PRs compared with those with higher concentrations (42 per cent vs 64 per cent, OR=0.3, P=0.01). There was no overall effect of hCG on PR compared with controls (51 per cent vs 60 per cent, P=0.10). However, heifers with high progesterone on days 4-6 (>2 ng/ml) had a lower PR after treatment (51 per cent vs 64 per cent, OR=0.6, P=0.02). Heifers inseminated with sexed (n=18) compared with conventional (n=333) semen had a decreased PR (33 per cent vs 57 per cent, OR=0.2, P=0.01). This study demonstrates that higher plasma progesterone concentrations as early as days 4-6 after insemination have a positive effect on first insemination PR, but there was no beneficial effect of hCG treatment on these days in dairy heifers.

Effects of high somatic cell counts in milk on reproductive hormones and oestrus behaviour in dairy cows with special reference to those with concurrent lameness.

The present investigation aims to establish the reason(s) why dairy cows with high somatic cell counts (SCCs; >100,000 cells/ml) are less fertile than cows with low SCCs alone. The objective of Study One was to determine whether differences in steroid hormone profiles could explain the low incidence of ovulation in cows with combined High SCC and lameness. Between 30 and 80 days post-partum, animals were scored for SCC and lameness and three groups were formed: Healthy (n=22), High SCC alone (n=12) or High SCC + Lame (n=9). The ovarian follicular phases of all cows were synchronised by administering gonadotrophin releasing hormone (GnRH) followed seven days later by prostaglandin F2alpha (PG). Milk samples were collected daily throughout the entire study period; twice daily during the follicular phase, blood samples were taken and the ovaries were monitored using ultrasonography. Progesterone concentrations were similar in all three groups during each of five specific time periods, i.e. throughout the five days before PG injection, the peri-ovulatory period, on Day 5 and on Day 7, and during the mid luteal phase 12-17 days after ovulation (P>0.13). Mean plasma oestradiol concentrations monitored every 12h during the 36h period before ovulation were similar in all groups (Healthy, 2.80±0.30pg/ml; High SCC alone, 3.82±0.48pg/ml; High SCC+Lame 2.94±0.51pg/ml; P=0.175). The objective of Study Two was to establish whether cows with High SCC (scored and synchronised as above) display different behaviours, especially the intensity and timing of oestrus. Intervals from PG to the onset of oestrus or to the first stand-to-be-mounted (STBM) were longer for the High SCC cows than the Low SCC animals (n=8 and 20; P=0.011 and 0.002, respectively). Also, cows with High SCC tended to have a less intense oestrus and a lower maximum oestrus score per 30-min period than Low SCC cows (P=0.063 and 0.066, respectively). In conclusion, High SCC±lameness did not affect progesterone or twice daily oestradiol profiles but the onset of oestrus was delayed and oestrus tended to be less intense in cows with High SCC. These factors could explain low fertility associated with High SCC.

Kisspeptin, c-Fos and CRFR type 2 expression in the preoptic area and mediobasal hypothalamus during the follicular phase of intact ewes, and alteration after LPS.

Increasing estradiol concentrations during the late follicular phase stimulate sexual behavior and the GnRH/LH surge, and it is known that kisspeptin signaling is essential for the latter. Administration of LPS can block these events, but the mechanism involved is unclear. We examined brain tissue from intact ewes to determine: i) which regions are activated with respect to sexual behavior, the LH surge and LPS administration, ii) the location and activation pattern of kisspeptin cells in control and LPS treated animals, and iii) whether CRFR type 2 is involved in such disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and control animals were killed at 0 h, 16 h, 31 h or 40 h (n=4-6/group) after progesterone withdrawal (time zero). At 28 h, other animals received endotoxin (LPS; 100 ng/kg) and were subsequently killed at 31 h or 40 h (n=5/group). LH surges only occurred in control ewes, during which there was a marked increase in c-Fos expression within the ventromedial nucleus (VMN), arcuate nucleus (ARC), and medial preoptic area (mPOA), as well as an increase in the percentage of kisspeptin cells co-expressing c-Fos in the ARC and mPOA compared to animals sacrificed at all other times. Expression of c-Fos also increased in the bed nucleus of the stria terminalis (BNST) in animals just before the expected onset of sexual behavior. However, LPS treatment increased c-Fos expression within the VMN, ARC, mPOA and diagonal band of broca (dBb), along with CRFR type 2 immunoreactivity in the lower part of the ARC and median eminence (ME), compared to controls. Furthermore, the percentage of kisspeptin cells co-expressing c-Fos was lower in the ARC and mPOA. Thus, we hypothesize that in intact ewes, the BNST is involved in the initiation of sexual behavior while the VMN, ARC, and mPOA as well as kisspeptin cells located in the latter two areas are involved in estradiol positive feedback only during the LH surge. By contrast, disruption of sexual behavior and the LH surge after LPS involves cells located in the VMN, ARC, mPOA and dBb, as well as cells containing CRFR type 2 in the lower part of the ARC and ME, and is accompanied by inhibition of kisspeptin cell activation in both the ARC and mPOA.

Effects of stress on reproduction in ewes.

Stressors, such as poor body condition, adverse temperatures or even common management procedures (e.g., transport or shearing) suppress normal oestrus behaviour and reduce ewe fertility. All these events are co-ordinated by endocrine interactions, which are disrupted in stressful situations. This disruption is usually temporary in adult ewes, so that, when prevailing conditions improve, normal fertility would resume. Imposition of an experimental stressor (shearing, transport, isolation from other sheep, injection of endotoxin or insulin or cortisol infusion) suppresses GnRH/LH pulse frequency and amplitude. Part of the cause is at the pituitary, but effects on GnRH/LH pulse frequency and the GnRH/LH surge are mediated via the hypothalamus. It is not yet clear whether delays in the surge are caused by interruption of the oestradiol signal-reading phase, the signal transmission phase or GnRH surge release. Stressors also delay the onset of behaviour, sometimes distancing this from the onset of the pre-ovulatory LH surge. This could have deleterious consequences for fertility.

Estrous behavior, luteinizing hormone and estradiol profiles of intact ewes treated with insulin or endotoxin.

Acute insulin administration causes a disparity between the onset of estrous behavior and the LH surge in ovary-intact ewes. To examine the considerable variation in responses, in the present study we used a large number of animals to confirm findings with insulin, and examine whether endotoxin has the same effect. During the breeding season, follicular phases of intact ewes were synchronized with progesterone vaginal pessaries and received saline vehicle (n=22; controls), insulin (4 IU/kg; n=21 ewes) or endotoxin (LPS; 100 ng/kg; n=10) at 28 h after progesterone withdrawal (time zero). In controls, the LH surge onset occurred at 36.5±5.7 h and were first mounted by a ram at 38.2±1.8 h, but there was a delay of 17.6 h (P<0.001) and 7.2 h (P<0.05), respectively, in half the insulin-treated animals ('insulin-delayed') but not in the other half; and a delay of 22.5 h (P<0.001) and 20.7h (P<0.001), respectively, in all LPS-treated animals. Plasma estradiol concentrations decreased after both stressors, and remained low for a period of time equivalent to the LH surge delay (P<0.001; Rs-q=78%). Cortisol increased for 12h after treatment in both insulin subgroups and the LPS group (P<0.05); whereas progesterone increased in the insulin-delayed and LPS groups from 4.0±0.5 ng/ml and 5.3±1.0 ng/ml to a maximum of 5.7±0.3 ng/ml and 8.8±1.6 ng/ml, respectively (P<0.05 for both comparisons). Plasma triglycerides were measured to assess insulin resistance, but concentrations were similar before and after treatment (0.25±0.01 mmol/l versus 0.21±0.01 and 0.25±0.01 mmol/l versus 0.26±0.01 mmol/l in the insulin-non delayed and insulin delayed subgroups, respectively). Therefore, we hypothesize that a) when an acute stressor is applied during the late follicular phase, the duration of the LH surge delay is related to the duration of estradiol signal disruption b) cortisol is not the key disruptor of the LH surge after insulin, c) insulin (but not LPS) can separate the onsets of LH surge and estrus by approximately 10h, providing a model to identify the specific neuronal systems that control behavior distinct from those initiating the GnRH surge.

Comparison of oestrus detection methods in dairy cattle.

Sixty-seven Holstein-Friesian cows, from 20 days postpartum, were recruited into the study and fitted with both a pedometer (SAE Afikim) and a Heatime neck collar (SCR Engineers) and allocated a heat mount detector (either scratchcard [Dairymac] or KaMaR [KaMaR]) or left with none, relying only on farm staff observation. Common production stressors and other factors were assessed to determine their impact on the ability of each method to accurately detect oestrus and to investigate effects on the frequency of false-positive detections. Only 74 per cent of all potential oestrus periods (episodes of low progesterone) were identified by combining information from all methods. There was no difference between the methods in terms of sensitivity for detecting 'true oestrus events' (approximately 60 per cent), with the exception of scratchcards, which were less efficient (36 per cent). Pedometers and KaMaRs had higher numbers of false-positive identifications. No production stressors had any consequence on false-positives. The positive predictive values for neck collars or observation by farm staff were higher than those of other methods, and combining these two methods yielded the best results. Neck collars did not detect any of the nine oestrus events occurring in three cows with a body condition score (BCS) of less than 2, and the efficiency of correctly identifying oestrus was also reduced by high milk yield (odds ratio [OR]=0.34). Pedometer efficiency was reduced by lameness, low BCS or high milk yield (OR=0.42, 0.15 or 0.30, respectively).

Influence of lameness on follicular growth, ovulation, reproductive hormone concentrations and estrus behavior in dairy cows.

The objective of this study was to examine the effect of a chronic stressor, lameness, on reproductive parameters. Seventy cows 30-80 days post-partum were scored for lameness and follicular phases synchronized with GnRH followed seven days later by prostaglandin (PG). Fifteen Lame animals did not respond to GnRH ovarian stimulation. Milk progesterone for 5 days prior to PG was lower in the remaining Lame cows than Healthy herdmates. Fewer Lame cows ovulated (26/37 versus 17/18; P = 0.04) and the interval from PG to ovulation was shorter in Lame cows. In Subset 1 (20 animals), the LH pulse frequency was similar in ovulating animals (Lame and Healthy) but lower in Lame non-ovulators. An LH surge always preceded ovulation but lameness did not affect the interval from PG to LH surge onset or LH surge concentrations. Before the LH surge, estradiol was lower in non-ovulating cows compared to those that ovulated and estradiol concentrations were positively correlated with LH pulse frequency. In Subset 2 (45 cows), Lame ovulating cows had a less intense estrus than Healthy cows, although Lame cows began estrus and stood-to-be-mounted earlier than Healthy cows. In conclusion, we have identified several parameters to explain poor fertility in some chronically stressed animals. From 30 to 80 days post-partum, there was a graded effect that ranged from 29% Lame cows with absence of ovarian activity, whereas another 21% Lame cows failed to express estrus or ovulate a low estrogenic follicle; in 50% cows, many reproductive parameters were unaffected by lameness.

Effects of lameness, subclinical mastitis and loss of body condition on the reproductive performance of dairy cows.

A total of 318 cows were monitored in the pre-breeding postpartum period for the presence of three production stressors: lameness, subclinical mastitis and body condition score (BCS) loss. For each stressor, cows were given a classification of severely, moderately or non-affected based on mobility scores, somatic cell counts and BCS change. The number of days from calving to onset of the first luteal phase was greater in cows that had one severe production stressor (median 44 days) or two moderate production stressors (41 days) compared with cows that had no stressors (31 days) (P=0.02 and P=0.04, respectively). More than one severe stressor increased the interval further. There was no difference between cows with one moderate stressor (median 38 days) and those with none (P=0.13). The delay to the first luteal phase was significantly longer in cows with two moderate stressors if the onset of one stressor occurred at the time when resumption of ovarian activity was expected. The presence of these production stressors in early lactation had no effect on the interval from calving to establishment of the next pregnancy or the number of inseminations required despite the negative effect on the onset of the luteal phase.

Neuronal responses in the brainstem and hypothalamic nuclei following insulin treatment during the late follicular phase in the ewe.

The aim of this study was to determine the neuronal responses following insulin administration during the late follicular phase. Intact ewes were given either saline or insulin (5 IU/kg, i.v.) at 35 h after progesterone withdrawal and killed 3 h later. There was a marked increase in the number of Fos-positive noradrenergic neurones in the caudal brainstem of insulin-treated ewes. In the hypothalamic paraventricular nucleus, insulin treatment increased the presence of Fos-positive corticotrophin-releasing hormone neurones (from 2% to 98%) and Fos-positive arginine vasopressin parvocellular neurones (from 2% to 46%). Interestingly, after insulin treatment, despite a general increase in Fos-positive neurones in the arcuate nucleus (ARC), there was a marked reduction (from 47% to 1%) in Fos-positive ß-endorphin neurones. Similarly, colocalized Fos and oestradiol receptor (ER) α-positive neurones decreased in the ARC after insulin (from 7% to 3%). Conversely, in the ventromedial nucleus, ERα-positive neurones with Fos increased (from 7% to 22%) alongside a general increase in Fos-positive neurones. Overall, a complex system of neurones in brainstem and hypothalamus is activated following insulin administration during the late follicular phase.

The effect of a chronic stressor, lameness, on detailed sexual behaviour and hormonal profiles in milk and plasma of dairy cattle

The objectives of the present study were to quantify the effects of a biological chronic stressor (lameness) on the duration and frequency of different oestrous behaviours in parallel with milk hormone profiles. Dairy cows 51.8 +/- 1.4 days postpartum (n = 59), including 18 non-lame control cows, were scored for lameness and closely observed for signs of oestrus having had their follicular phases synchronized by administration of gonadotrophin-releasing-hormone (GnRH) followed by prostaglandin F(2alpha) (PG) 7 days later. Lameness shortened the period when herd-mates attempted to mount the lame cows (1.83 +/- 0.69 h vs 5.20 +/- 1.53 h; p = 0.042) but did not affect the overall duration of total behaviours (lame 12.3 +/- 1.3 h vs non-lame 15.2 +/- 1.3 h). Lameness also lowered the intensity of oestrus [1417 +/- 206 points (n = 18) vs 2260 +/- 307 points (n = 15); p = 0.029]. Throughout the synchronized oestrous period, lame cows mounted the rear of herd-mates less frequently (p = 0.020) and tended to chin rest less (p = 0.075). Around the period of maximum oestrous intensity, lameness also diminished the proportion of cows mounting the rear of another cow and chin resting (p = 0.048, p = 0.037, respectively). Furthermore, lame cows had lower progesterone values during the 6 days before oestrous (p < or = 0.05). Fewer lame cows were observed in oestrus following PG (non-lame 83%, lame 53%; p = 0.030); however, if prior progesterone concentrations were elevated, lame cows were just as likely to be observed in oestrus. In conclusion, following endogenous progesterone exposure, lameness shortens the period when herd-mates attempt to mount lame cows but does not affect the incidence of oestrous. However, lame cows are mounted less frequently and express oestrus of lower intensity. This is associated with lower progesterone prior to oestrus but not with abnormal oestradiol or cortisol profiles in daily milk samples.

Effect of insulin on the relationship of estrous behaviors to estradiol and LH surges in intact ewes.

The objective of this study was to determine whether acute stress alters the frequency of spontaneous estrous behaviors and temporal relationships with preovulatory increases in peripheral plasma estradiol and LH. Follicular phases of intact ewes were synchronized with prostaglandin administered at progesterone pessary withdrawal (PW). Twelve ewes served as controls and 12 were acutely stressed (insulin 4 IU/kg given at 30 and 32 h after PW). Ewes being near to ram(s) 21.3+/-1.9h after PW was the first precopulatory behavioral sign and rams nosed the perineal region of ewes after a further 9.0+/-2.0 h (P<0.01), with ewes being nudged and mounted by rams 6.8+/-2.3h later still (P<0.01). Insulin did not affect the frequency or timing (relative to PW) of each behavioral sign of estrus. However, within each animal, estradiol values were more than 2 pg/ml lower (P<0.05) for 6h following insulin, and the onset of the LH surge was delayed in insulin-treated ewes compared to controls (49.5+/-3.3 versus 38.2+/-2.6h; P=0.01). Consequently, the interval between the onset of being mounted and the LH surge was longer in insulin-treated ewes compared to controls (10.4+/-3.0 versus 2.3+/-0.7 h; P<0.01). Maximum LH values were also 15 ng/ml lower after insulin (P<0.01). Thus, acute stress did not alter the timing or frequencies of estrous behaviors but it did reduce estradiol concentrations and delayed the onset and magnitude of the LH surge.

The effect of a chronic stressor, lameness, on detailed sexual behaviour and hormonal profiles in milk and plasma of dairy cattle.

The objectives of the present study were to quantify the effects of a biological chronic stressor (lameness) on the duration and frequency of different oestrous behaviours in parallel with milk hormone profiles. Dairy cows 51.8 +/- 1.4 days postpartum (n = 59), including 18 non-lame control cows, were scored for lameness and closely observed for signs of oestrus having had their follicular phases synchronized by administration of gonadotrophin-releasing-hormone (GnRH) followed by prostaglandin F(2alpha) (PG) 7 days later. Lameness shortened the period when herd-mates attempted to mount the lame cows (1.83 +/- 0.69 h vs 5.20 +/- 1.53 h; p = 0.042) but did not affect the overall duration of total behaviours (lame 12.3 +/- 1.3 h vs non-lame 15.2 +/- 1.3 h). Lameness also lowered the intensity of oestrus [1417 +/- 206 points (n = 18) vs 2260 +/- 307 points (n = 15); p = 0.029]. Throughout the synchronized oestrous period, lame cows mounted the rear of herd-mates less frequently (p = 0.020) and tended to chin rest less (p = 0.075). Around the period of maximum oestrous intensity, lameness also diminished the proportion of cows mounting the rear of another cow and chin resting (p = 0.048, p = 0.037, respectively). Furthermore, lame cows had lower progesterone values during the 6 days before oestrous (p < or = 0.05). Fewer lame cows were observed in oestrus following PG (non-lame 83%, lame 53%; p = 0.030); however, if prior progesterone concentrations were elevated, lame cows were just as likely to be observed in oestrus. In conclusion, following endogenous progesterone exposure, lameness shortens the period when herd-mates attempt to mount lame cows but does not affect the incidence of oestrous. However, lame cows are mounted less frequently and express oestrus of lower intensity. This is associated with lower progesterone prior to oestrus but not with abnormal oestradiol or cortisol profiles in daily milk samples.

Follicle and oocyte morphology in ewes after treatment with insulin in the late follicular phase.

Stress reduces fertility in ruminants. Various experimental models, such as insulin-induced hypoglycaemia, have been used to investigate the mechanisms involved, and have revealed abnormal LH profiles (both pulse and surge secretion). This disruption affects follicular function and it is proposed there may be negative consequences on subsequent oocyte morphology. Insulin (5iu/kg), administered to ewes in the late follicular phase, induced hypoglycemia for 10h, decreased estradiol concentrations for 8-12h and delayed the LH surge by 15h. Although the diameters of dominant follicles just before ovulation were not affected, granulosa cells had fewer pycnotic nuclei, less apoptosis and increased proliferation 16-17h after the LH surge. Nevertheless, we did not observe gross ultra-structural differences in nuclear, cytoplasmic or cumulus maturity between oocytes from insulin-treated and control animals. This suggests that reduced LH pulsatility and a delay in the LH surge may only produce very subtle changes in gross oocyte morphology, imperceptible by electron microscopy.

Influence of somatic cell count, body condition and lameness on follicular growth and ovulation in dairy cows.

The objective of this study was to investigate the effect of somatic cell count (SCC), body condition score (BCS) or lameness score on ovarian follicular growth and ovulation in dairy cows. Seventy four animals 30-80 days post-partum were monitored for all three conditions before synchronization of ovarian follicular phases by administration of gonadotrophin releasing hormone (GnRH) followed seven days later with prostaglandin F2alpha (PG). Ultrasonography of both ovaries twice daily throughout the follicular phase revealed that fewer animals with combined high SCC and lameness (4/9) ovulated compared to healthy animals (19/21; P=0.006) or animals with only high SCC (11/11; P=0.004) or only lameness (21/27; P=0.06). Overall, regardless of the presence of other concurrent conditions, fewer lame cows ovulated than Non Lame animals (30/42 and 30/32; P=0.015). Mean follicular growth and maximum follicular diameter were unaffected by any of the three conditions. However, dominant follicle growth and maximum diameter were greater in the 60 animals that ovulated compared to the 14 that did not; 1.83+/-0.16 versus 0.96+/-0.26mm/day (P=0.014) and 19.4+/-0.4 versus 16.4+/-1.2mm (P=0.003), respectively. In conclusion, lameness reduced the proportion of cows that ovulated and the synergistic effect of high SCC and lameness reduced that proportion further. However, follicular growth and maximum follicular diameter were unaffected by high SCC, low BCS or lameness.

Lameness, activity time-budgets, and estrus expression in dairy cattle.

The aim of the present study was to identify specific behavioral patterns that contribute to diminished estrus expression in lame cows. Behavioral scan and focal sampling were used to examine the effect of lameness on daily activity budgets, sexual behavior, feeding activities, and body condition score. A total of 59 milking cows (51.8 +/- 1.4 d postpartum) were monitored on a commercial dairy farm for 5 d following estrus synchronization. Overall, lame cows (n = 39) spent proportionately less time elevated on their feet and more time lying down compared with nonlame cows (n = 20). This included lame cows spending less time walking or standing. Overall, the total proportion of scans in which an estrous behavior was observed was very small but tended to be smaller for lame compared with nonlame cows. Throughout a day, lame cows displayed a lower proportion of estrous behaviors in the early morning. Lameness did not affect durations of drinking, grazing, or ruminating, or how these behavioral states fluctuated throughout the day. Similarly, rumination chewing rates were the same for lame and nonlame cows, and there was no association between lameness and dominance/displacement while feeding at a feed-fence. Lame cows did, however, have a slower bite rate at pasture and had a lower body condition score. Lame cows were also nearer the rear of the herd, both as they left the field and when entering the milking parlor. In conclusion, lame cows have longer lying times and spend less time standing, walking, and expressing an estrous behavior. Lame cows also have a lower bite rate at pasture and are more likely to be of lower body condition score.

Chronic stress, hormone profiles and estrus intensity in dairy cattle.

The objectives of the present study were to determine if lameness, a model for a natural chronic stressor, affects hormone concentrations in milk prior to estrus and/or the subsequent expression of estrus in the postpartum period. Dairy cows>20 days postpartum were scored for lameness and observed for estrus intensity using a weighted scoring system (>100 points=estrus=Day 0). Increasing lameness score was not associated with daily profiles of milk progesterone (throughout Days -18 to 0), estradiol (Days -6 to 2) or cortisol (Days -18 to 2) around estrus, maximum estradiol values or estradiol concentrations on Day 0. However, post hoc pair wise comparisons revealed that prior to estrus, severely lame cows had lower maximum progesterone concentrations compared to nonlame cows (1.3+/-0.1, 1.2+/-0.2, 0.7+/-0.1 ng/ml milk; P=0.042). Furthermore, severely lame cows expressed behavioral estrus with lower intensity (284+/-128 points, n=9) compared to moderately lame (662+/-310 points, n=9) or nonlame animals (583+/-275 points, n=18; P=0.05 and P=0.02, respectively). Resting concentrations of cortisol (Days 20-80 postpartum) did not vary between days postpartum or lameness score. The incidence of behavioral estrus was not affected by increasing lameness score, as 54.2%, 56.2% and 50.0% periods with low progesterone were associated with spontaneous estrus expression, respectively. Concluding, in this biological model of chronic stress, lameness did not affect the incidence of behavioral estrus but did reduce estrus intensity once ovarian cyclicity had resumed after calving. This reduced intensity of estrus was associated with lower maximum progesterone values prior to estrus but not abnormal daily cortisol or estradiol values in milk.