Levels of cAMP, cGMP, 17 beta-estradiol and progesterone in the follicular fluid of the largest follicle during the follicular phase of the estrus cycle and after induced superovulation with gonadotropins in cows]

The objective of this study was to compare the concentrations of 17 beta-estradiol, progesterone, cAMP and cGMP in the follicular fluid of the largest cow follicle from the follicular phase of physiological sexual cycle and of follicles after synchronization of fut by cloprostenol (PGF2 alpha) and superovulation treatment with serum gonadotrophin (PMSG), in dependence on steroidal dominance of follicles. 2 x 25 cows, Slovak Pied x Lowland Black-Pied crossbreds with active corpus luteum, were subjected to superovulation treatment on the basis of rectal examination. Rut synchronization was achieved by cloprostenol of Czechoslovak provenience (Oestrophan Spofa), administered at the amount of 500 micrograms per dose. Serum gonadotrophin (Bioveta Concern, Ivanovice na Hané) at the amount of 2500 I. U. was administered forty-eight hours before the second dose of closprostenol. The animals were killed in slaughterhouses 48 hours later, or 72 hours later, since administration of the second dose of cloprostenol. The phase of the sexual cycle of control animals was determined by the method after Ireland et al. (1980) on the basis of morphological appearance of corpus luteum, presence of large preovulation follicle and by means of average concentrations of progesterone in blood serum. Aspirated follicular fluid was centrifuged using a cooling centrifuge at 3000 G. After separation, the supernatant was stored in a freezer at -18 degrees C until further treatment. 17 beta-estradiol and progesterone in the follicular fluid were determined by means of kits under the brand-names RIA-test-ESTRA (SI-125-9), or RIA-test-Prog (SI-125-6). Concentrations of cyclic nucleotides were determined by the RIA kits from the Institute vor Radioisotope Research, Production and Use (Prague), cAMP by 125J RIA kit (RIO12) and cGMP by 125J RIA (RIO42).(ABSTRACT TRUNCATED AT 250 WORDS)     

Colour Doppler sonography of cystic ovarian follicles in cows

The goals of the present study were to investigate whether colour Doppler sonography can be used to differentiate temporary from persistent ovarian follicles and follicles with luteal tissue from follicles without luteal tissue and to assess the response of follicular cysts to administration of a gonadotropin releasing hormone (GnRH) analogue. Fifty-four cows having ovarian follicular structures with a diameter of >15 mm but no corpus luteum were included. These cows were examined via B-mode and colour Doppler sonography. The same examinations were repeated 10 to 12 days later, and the cows with follicular cysts (n=17) received a GnRH analogue. Blood flow was measured before and 30 min after treatment. Ten to 12 days later, the response to treatment was assessed using B-mode sonography. While 31 of 54 follicles disappeared spontaneously (temporary follicles), 23 follicles persisted and were diagnosed as cystic ovarian follicles (COFs). There was no difference between temporary follicles and COFs in regard to total area, wall thickness or the perfused area. In the luteinized follicles (n=13), based on the plasma progesterone concentration, total area was twice as large, wall thickness was three times greater and the perfused area was 4.5 times larger than those of the non-luteinized follicles (n=41). The sensitivity of diagnosing luteinized follicles was 61.5% using B-mode sonography and 92.3% using colour Doppler sonography. Twelve cows responded to GnRH, and five cows did not. There was a trend (P=0.07) toward higher (59.3%) blood flow in the cyst wall 30 min after treatment in the responding cows compared with the non-responding cows. Our results showed that the perfused area more accurately reflects active luteal tissue than wall thickness. Thus, colour Doppler sonography is superior to B-mode sonography for differentiating follicular and luteal cysts and aids in the selection of treatment. However, exact prediction of COFs destined to regress or persist and the response of COFs to treatment with a GnRH analogue were not possible using colour Doppler sonography.     

Effects of Twice-Weekly Follicular Punctures of Ovaries With or Without the Corpus Luteum on Follicular and Luteal Dynamics

We investigated the effects of twice-weekly follicular punctures of ovaries with or without corpus luteum (CL) on follicular and luteal dynamics. A cross-over design was used, with each cow (seven Japanese Black beef cows) being assigned to one of the three groups at 2-month intervals. Follicular punctures were performed twice weekly for three consecutive weeks until day 20 (oestrus = day 0). All visible follicles (diameter >3 mm) in the ovaries bearing CL (ipsilateral group) or those in the contralateral ovaries (contralateral group) were aspirated. As a control, all visible follicles in both ovaries were aspirated (bilateral group). Follicular development, CL formation and progesterone concentrations in each cow were monitored from days 0 to 30. Follicular growth profiles in the punctured ovaries during/after puncture treatment were similar, irrespective of the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries. After puncture, two cows (28.6%) each in the ipsilateral and bilateral groups did not exhibit behavioural oestrus until day 30, whereas all cows in the contralateral group exhibited oestrus. CL growth and increase in progesterone concentrations after the last follicular puncture in the bilateral group were delayed when compared with those in the ipsilateral group. Our results indicate that the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries does not significantly influence follicular growth in punctured ovaries during/after puncture treatment. However, follicular puncture in ovaries bearing CL may disturb or delay oestrus occurrence after treatment.     

Comparison of leptin levels in serum and follicular fluid during the oestrous cycle in cows

Leptin is mainly synthesised in white adipose tissue. Besides its effects on body weight and metabolic homeostasis, leptin also has effects on puberty, sexual maturation and reproduction. In this study the relationship between leptin, IGF-1, oestradiol (E2) and progesterone levels were investigated in serum and follicular fluid from cows. This study included 72 healthy, Brown Swiss cows aged 4-5 years. Samples from the jugular vein and follicular fluids were collected. Phases of the oestrus cycle of cows were classified according to their serum progesterone levels (< 3.18 nmol/l, follicular phase and the others as luteal phase). Follicles were grouped as large (> or = 8 mm) or small (< 8 mm). Leptin, IGF-1, oestradiol and progesterone levels were measured from serum and follicular fluid. Leptin concentrations were found to be significantly higher in luteal-phase follicular fluid of small follicles (P < 0.05). These were classified as atretic follicles. There was a positive correlation between serum and follicular fluid leptin levels in the luteal phase. Serum leptin was found to have a positive correlation with follicular fluid progesterone level (P = 0.01) in the preovulatory follicles. The present study shows that there is a relationship between the concentration of leptin in follicular fluid and atresia in small follicles.     

Radioimmunoassay of milk progesterone as a tool for fertility control in smallholder dairy farms

This study focused on the use of radioimmunoassay of progesterone in milk for the diagnosis of post-partum ovarian cyclicity and accurate detection of oestrus and non-pregnancy in cows in the artificial insemination (AI) programme in Bangladesh. In Investigation 1, milk samples were collected on day 0 (day of AI), day 9–13 and day 21–24 from 444 milking cows of various breeds presented for the first post-partum insemination by 413 farmers living at 182 villages/regions in Mymensingh District from 6 AI centres and sub-centres. Each cow was then examined three times after each AI until it stopped returning to oestrus. Sixty to 90 days after the last AI, the cows were examined per rectum to confirm the pregnancy. Milk progesterone data on day 21–24 contributed to a clear diagnosis with respect to non-pregnancy in 100% cows, indicating a possible use of this progesterone assay for identifying non-pregnant cows in AI programmes. In Investigation 2, milk progesterone was monitored two times in a month with a 10-day interval in 88 cows. The samples were taken between 10 days after calving and the first detected oestrus, followed by two more samples 10 days apart. The proportion of cows accurately detected in oestrus was 30%. Another 30% were stated to be in oestrus when they were not (false positive) and 40% were not detected when they were in oestrus (false negative). The mean intervals between calving and oestrus and between calving luteal activity were 40 to 362 days (median = 120, n = 82) and 34 to 398 (median = 111, n = 64) days, respectively. The body condition scores at calving and at the initiation of luteal activity influenced the interval between calving and luteal activity (p < 0.05). Cows suckled twice daily initiated luteal activity earlier than their counterparts suckled several times daily (p < 0.05). Determination of progesterone in milk on day 21–24 is a good means for detecting non-pregnant cows.     

A survey of the postpartum reproductive performance of dairy cows with fertility problems in southern Iceland

The purpose of this survey was to evaluate the reproductive performance of dairy cows on problem farms in southern Iceland. In all 229 cows on 6 farms were studied. The animals were examined clinically by rectal palpation, once a month. Blood samples were taken 2–5 and 7–10 weeks after calving. The blood samples were analysed for the contents of glucose, urea, inorganic phosphate, calcium and magnesium. Milk samples for progesterone profiles were taken, by the farmer, every 4th day from day 10 postpartum until first oestrus. Because of this sampling method, 128 cows had no rise in progesterone levels, when milk sampling was stopped. These 128 animals were excluded from the study. The results are based on 101 animals.There was a large variation between cows in postpartum reproductive performance. In the total material 1st ovulation occurred later than reported in many other countries. Fifty percent of the cows had ovulated 35 days after calving and 90 percent 70 days after calving.The first luteal phase was short in about 60 % of the cows. The progesterone values assayed from those short cycles were lower than the values assayed from the following cycle. First artificial insemination (ai) was on the average 77 days postpartum (pp). The conception rate to first service was 49 %. Of 100 milk samples taken at the time of ai, 20 had high progesterone value. This indicates a high frequency of luteal phase inseminations.Clinical ketosis was diagnosed in 35 cows. Of these, 31 had a low glucose value. Cows with clinical ketosis ovulated, on the average, later than other animals and 24 ovulated later than 40 days pp. The results indicated that the fertility problems of these cows studied were late ovulations, low conception rate, probably in part, due to luteal phase inseminations, and a high frequency of ketosis which could be caused by low quality feedstuff.     

Sequential Hormonal Changes in the Postpartum Dairy Cow

Peripheral plasma levels of 15-keto-13,14-dihydro-PGF_2α, progesterone, Cortisol, LH and prolactin were studied in 6 primiparous postpartum dairy cows. The cows were followed by hormone measurements and clinical examinations from parturition until pregnancy was established. Blood was collected 3 times per day. The cervix, uterus and ovaries were examined by rectal palpation at 6–10 days intervals. The cows were observed for signs of oestrus twice daily and were additionally teased with a bull to provoke standing heat.Four cows had a normal parturition and dropped their fetal membranes shortly afterwards. (NR group). The remaining 2 retained their fetal membranes for more than 24 h following parturition (RFM group). One out of 6 cows showed standing oestrus at the first ovulation, 4 animals were in oestrus at the second ovulation and all cows showed signs of oestrus at the third ovulation. Although the length of the first luteal phase varied from 9 to 22 days a corpus luteum was in all cases palpated. The secretion of progesterone during the first luteal phase was terminated by a PGF_2α release.A significant difference in 15-keto-13,14-dihydro-PGF_2α levels between the 2 groups was found on days 0–4 (2.39 vs 6.87 nmol/1 at Ρ < 0.06). Postpartum prostaglandin F_2α release as reflected by the level of 15-keto-13,14-dihydro-PGF_2α lasted shorter in the NR group than in the RFM group (15–17 vs 21 days). Significant positive correlations between 15-keto-13,14-dihydro-PGF_2α and Cortisol as well as between prolactin and Cortisol during the first 24 days postpartum were noted only in cows having normal parturition. The most pronounced daily prolactin variations occurred during the second luteal phase (NR group), when a significant difference between the times 8.00, 12.00 and 15.00 was recorded (14.7, 31.5 and 19.7 μg/l, respectively). Moreover, a partial negative correlation between log value of prolactin and arithmetical value of LH was found in these cows only during the first luteal phase after parturition.     

Characteristics of developing prolonged dominant follicles in cattle

In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.     

Comparison of oestrus synchronisation programmes in dairy cattle using oestradiol benzoate, short-acting progesterone and cloprostenol, or buserelin and cloprostenol

AIM: To evaluate the efficacy of a programme using oestradiol benzoate, progesterone and the prostaglandin-F2 (PG) analogue, cloprostenol, to synchronise oestrus and ovulation in dairy cows, compared with a programme using a gonadotropinreleasing hormone (GnRH) agonist, buserelin, and cloprostenol. METHODS: Twenty non-lactating dairy cows, at random stages of the oestrus cycle, were randomly assigned to 1 of 2 treatments. In Treatment 1 ( OPPG; n=10), cows were injected with 2 mg oestradiol benzoate intramuscularly (IM) plus 200 mg progesterone subcutaneously (SC) on Day 0, followed by 500 microg cloprostenol IM on Day 9 and 1 mg oestradiol benzoate on Day 10. In Treatment 2 (GPG; n=10), cows were injected with 10 microg buserelin IM on Day 0, 500 microg cloprostenol IM on Day 7 and 10 microg buserelin on Day 9. The ovaries of all cows were examined by ultrasonography, using an 8 MHz probe, from 5 days before the initial treatment until ovulation. Cows were observed for oestrus 3 times daily for 7 days after cloprostenol treatment. Blood samples were collected daily for determination of progesterone, and 6-hourly for 36 h after the second oestradiol or buserelin injection for the determination of follicle stimulating hormone (FSH) and luteinising hormone (LH) concentrations. RESULTS: The percentage of cows observed in oestrus was higher in the OPPG group than in the GPG group (100% vs 55.6%, p=0.018). Treatment with either short-acting progesterone plus oestradiol benzoate or buserelin was followed by atresia or ovulation of the dominant follicle. Emergence of a new follicular wave occurred earlier (p>0.001) in the GPG group (2.2+/-0.2 days) than in the OPPG group (3.6+/-0.2 days). There was no significant difference between treatment groups in the variation of time of follicular wave emergence or size of the largest follicles at either the time of initial treatment (10.8+/-1.4 mm vs 11.1+/-0.8 mm), cloprostenol treatment (13.8+/-0.7 mm vs 14.0+/-1.3 mm) or of ovulation (15.4+/-0.7 mm vs 17.6+/-1.1 mm; p=0.10). The LH surge occurred sooner after the second injection of buserelin (4.0+/-1.0 h) than after the second injection of oestradiol benzoate (22.8+/-1.2 h; p>0.001). The interval between the second injection of oestradiol benzoate or buserelin and ovulation did not differ significantly between treatment groups (1.7+/-0.3 days vs 1.6+/-0.2 days; p=0.69). CONCLUSIONS: The use of short-term progesterone treatment, combined with oestradiol benzoate for follicular wave synchronisation, and cloprostenol to cause lysis of residual luteal tissue, is a promising alternative to established methods of oestrus synchronisation in cows.     

Steroids and cAMP in follicles of postpartum beef cows treated with norgestomet.

To determine time of occurrence of follicular changes that may be associated with the length of the subsequent luteal phase, follicles were collected at different times before ovulation from cows expected to have corpora lutea of short (control) or normal (norgestomet-treated) life span. Beginning on d 20 to 23 postpartum (d 0 of study), 34 crossbred beef cows received either a 6-mg implant of norgestomet for 9 d or served as untreated controls. Ovaries were removed from norgestomet-treated cows on d 6 (N6; n = 9), d 8 (N8; n = 8), or the day after implant removal (N10; n = 8). Control cows were ovariectomized on d 6 (C6; n = 4) or d 10 (C10; n = 5). The largest and second largest follicles greater than 8 mm (F1 and F2, respectively) were dissected from the ovaries. Granulosal and thecal layers and follicular fluid were separated and assayed for estradiol-17 beta, progesterone, androstenedione, and testosterone. Cyclic 3'5'adenosine monophosphate (cAMP) was determined in thecal and granulosal tissue. Diameters of the F1 (14.6 +/- .4 mm) and F2 (10.6 +/- .4 mm) did not differ due to treatment. A greater proportion (P less than .05) of the F1 (20/33) than of the F2 (4/27) had estradiol:progesterone ratios of greater than 1 in follicular fluid. Contents of estradiol, androstenedione, and testosterone in theca and granulosa and follicular fluid, androstenedione in theca, and testosterone in theca and follicular fluid (all P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effect of Energy Balance on Ovarian Activity in a Herd of Norwegian Cattle

The study involved 34 primiparous cows fed ad libitum grass silage and fixed amounts of concentrate per cow and stage of lactation. It revealed that number of days from calving to maximum progesterone concentration in first luteal phase was negatively related to (p<0.05) energy balance summarized over weeks 3-12 post-partum. One standard deviation improvement of the summarized energy balance relative to the mean reduced the length of the anovulatory period by 12 days. Similarly, an improved energy balance enhanced progesterone secretion during the oestrus cycle and early pregnancy, as measured by 3 variables; 1) maximum progesterone concentration in first luteal phase, 2) cumulative progesterone secretion bounded by the maximum concentrations in first and in third luteal phase and 3) cumulative progesterone secretion in the first month of pregnancy. All results were supported by the estimated regression coefficients of the 4 ovarian activity variables on summarized non-estrified fatty acids and ac-etoacetate variables.     

Plasma progesterone concentrations in dairy cows with cystic ovaries and clinical responses following treatment with fenprostalene

Sixty-two dairy cows diagnosed as having cystic ovarian degeneration were used to study the correlation between rectal palpation findings and plasma progesterone concentrations and the response of cysts to treatment using fenprostalene, a luteolytic agent. Rectal palpation accurately determined the presence of luteal cysts as confirmed by plasma progesterone concentrations of 3 ng/mL or more. Treatment with fenprostalene was very effective for luteal cysts: a high percentage of treated cows exhibited estrus within seven days after treatment. The conception rate following artifical insemination during the induced estrus was 87.5% (21/24). Rectal palpation was much less accurate for the diagnosis of follicular cysts. Cows diagnosed as having follicular cysts had wide variations in plasma progesterone concentrations. Response to fenprostalene treatment was poor in cows with nonluteinized cystic follicles associated with low progesterone concentrations. However, cows diagnosed as having follicular cysts, but with progesterone concentrations of 1 ng/mL or more, responded better to fenprostalene treatment than cows with low progesterone concentrations.

It was concluded that, if correctly diagnosed, luteal cysts can be successfully treated with fenprostalene, and conception rates following treatment can be expected to be normal.

     

Effects of intermittent injections of LHRH on specific binding of 125I-labeled gonadotropins to granulosa and theca, and concentrations of steroids in serum and ovarian follicles during postpartum anovulation in suckled beef cows

To examine ovarian follicular response to low-dose injections of luteinizing hormone-releasing hormone (LHRH), 32 anovulatory, suckled beef cows were allotted to one of four treatment groups and injected with either saline or 500 ng LHRH every 2 h for 48 or 96 h, starting 21.4 +/- .4 d after parturition. Two hours after the last injection of LHRH, cows were ovariectomized and 10 to 15 ovarian follicles per pair of ovaries were removed and categorized by diameter as small (1.0 to 3.9 mm), medium (4.0 to 7.9 mm) or large (greater than or equal to 8.0 mm). Injections of LHRH did not affect (P greater than .10) steroid levels in small follicles or numbers of gonadotropin receptors in small and medium follicles. Concentrations of progesterone in fluid of medium follicles increased 1.5-fold (P less than .05) after 96 h of LHRH, whereas concentrations of estradiol and androstenedione were unchanged. In fluid of large follicles, concentrations of progesterone were fourfold greater (P less than .05) in LHRH-treated than in control cows at 48 h, but by 96 h progesterone was twofold greater (P less than .05) in control than LHRH-treated cows. In large follicles, concentrations of estradiol were unchanged (P greater than .10) after 48 h of LHRH injections but after 96 h estradiol was twofold greater (P less than .05) in LHRH-treated than control cows. Increased concentrations of estradiol in large follicles coincided with increased numbers of binding sites for human chorionic gonadotropin (hCG) but not follicle stimulating hormone (FSH) in granulosa and theca.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incidence of Error in Oestrus Detection Based on Secondary Oestrus Signs in a 24-h Tie-Stalled Dairy Herd with Low Fertility

Oestrus detection error and conception rates after AI based only on secondary oestrus signs were evaluated in a high yielding, 24-h tie-stalled dairy herd with low fertility, using milk progesterone profiles. Oestrus detection was based on the secondary oestrus signs such as restlessness, swelling, congestion of vulva and clear mucus discharge. Sixty eight AI conducted after observing the secondary oestrus signs in 44 animals were included in the study. Of the 68 AI, 53 (77.9%) were conducted in the follicular phase, and 13 (19.1%) and 2 (2.9%) were carried out in the luteal phase and during pregnancy, respectively. The overall error in oestrus detection based on milk progesterone profiles was 22.1%. The oestrus detection error did not differ significantly among different secondary oestrus signs. None of the AI conducted in the luteal phase resulted in conception, whereas 20.8% of AI conducted in the follicular phase resulted in conception. No significant difference in the conception rates among the groups of cows with different secondary oestrus signs was shown. The high incidence of oestrus detection error in this study might have been caused by the detection of cows in oestrus based only on secondary oestrus signs due to the confinement of animals. In conclusion, there was a high incidence of heat detection error in the 24-h tie-stalled dairy herd and oestrus detection based only on secondary oestrus signs resulted in low conception rate.     

Luteal activity and effect of dietary energy restriction on follicular development in lactating cows

The aim of this research has been to evaluate the presence of anomalies in the ovarian cycle activity during postpartum and to verify whether 72‐hr dietary fasting during the dominance phase, the phase before ovulation, might modify the ovarian follicle population. The presence of anomalies in ovarian cycle activity has been evaluated in 30 Italian Friesian cows starting from 20 days postpartum until 211 days of lactation. Long oestrus and brief dioestrus or scarce luteal activity have been the main anomalies found through measuring progesterone concentrations in the whey. Until 100 days of lactation, the BCS values of the problematic animals have been significantly lower than those in animals with normal ovarian activity. After 100 days of lactation, the ovarian anomalies continued to appear despite the fact that all the animals have reached comparable BCS values. Starting from the results of this trial, the effect of 72‐hr dietary fasting on dominant follicles has been studied in six cows. Ultrasonography revealed that the diameter of the follicles at 71 days postpartum has been significantly lower than at 181 days. A 72‐hr dietary restriction at 101 and 211 days postpartum did not affect the size of the dominant follicle. However, at 101 days postpartum, half of the animals presented follicular cysts. The effect of fasting differed if the animal has been in early postpartum or 211 days of lactation. Further researches are necessary to understand how different metabolic conditions can modify the follicular population but on the other hand the study shows the utility for farmers and field veterinarians of monitoring the resumption of the ovarian cycle postpartum through the whey progesterone concentrations.     

Effects of application in spring of urea fertiliser on aspects of reproductive performance of pasture-fed dairy cows

AIMS: To assess if raising concentrations of crude protein (CP) in pasture in spring by the frequent application of urea fertiliser would affect ovarian follicular dynamics, luteal function, onset of oestrus and reproductive performance of dairy cows under farming conditions in New Zealand. METHODS: Spring-calved dairy cows were grazed for 101 days in paddocks that were either not fertilised (Control; n=20) during the course of the study, or were fertilised with 40-50 kg nitrogen (N)/ha every 4-6 weeks (High-N; n=20). Similar generous pasture allowances were offered to both groups. Concentrations of CP in pasture, urea in serum and progesterone in milk were measured. Ovarian follicular and luteal dynamics were determined using ultrasonography. Oestrous behaviour and the number, time and outcome of inseminations were also recorded. RESULTS: Mean concentrations of CP in pasture and urea in serum was higher in the High-N than the Control group (25.2 vs 21.6 and 8.3 vs 5.4 mmol/L for CP and urea, respectively; p<0.001). Intervals between calving and first oestrus, first insemination and conception, the time of first emergence of a dominant follicle, milk progesterone concentration, and the diameter of the corpus luteum (CL) in the first luteal phase did not differ significantly between groups. The interval from calving to first ovulation tended (p=0.10) to be lower and the diameter of the dominant follicle of the oestrous cycle at which cows conceived was greater (p=0.02) in Control than High-N cows. CONCLUSIONS: The use of large amounts of urea fertiliser during spring and the consequent increases in concentrations of CP in pasture and urea in serum did not negatively affect any of the parameters of reproductive performance of pasture-fed dairy cows that were assessed in this study.     

The levels of 5alpha-dihydrotestosterone in follicular fluid in healthy and atretic ovine follicles

Dihydrotestosterone (DHT) induces follicular atresia under experimental conditions. However, whether it causes any antagonistic effect under natural condition is not known. In the present study, we investigated concentrations of DHT in follicular fluid and correlated them with concentrations of estradiol-17beta (E2) and its androgen substrates, androstenedione (A4) and testosterone (T), in healthy and atretic follicles of sheep. Merino ewes were treated twice with PGF2alpha (PG) to synchronize estrus. The ovaries were recovered at 14 days after the second PG (luteal phase) or 24h after the third PG given 14 days after the second PG (follicular phase). Follicles were dissected and their size and appearance were recorded. Follicular fluid was collected from follicles larger than 3.5mm and concentrations of E2, progesterone (P4), A4, T and DHT were determined by RIA. The inhibitory effect of DHT on conversion of T to E2 was tested in cultured granulosa cells. Appreciable levels of DHT were observed in the follicular fluid of ovine preovulatory follicles. The levels of DHT were much lower than those of E2, A4 and T, irrespective of physiological conditions of follicles. No difference was found in DHT concentration between healthy and atretic follicles. Dihydrotestosterone marginally inhibited aromatization of T in granulosa cells but this effect was only observed when the levels of DHT were 10 times higher than that of T in culture medium. These results indicate that DHT is present in ovine preovulatory follicles but its levels are not sufficient to exert any antagonistic effect on follicular development.     

Comparison of different methods of diagnosis of cystic ovarian disease in cattle and an assessment of its treatment with a progesterone-releasing intravaginal device

The aims of this study were to assess the accuracy of different common methods of differentiating between follicular and luteal ovarian cysts, and to monitor the response of the cysts to 12 days treatment with a progesterone-releasing intravaginal device (PRID). On the basis of agreement between the different methods, 25 of the 46 cases examined were diagnosed as follicular and 14 as luteal cysts; for the other seven cases the methods disagreed. The use of ultrasound was more accurate in diagnosing follicular cysts than luteal cysts, and combined with plasma progesterone concentrations gave the most accurate assessment of cyst type (92 per cent for follicular cysts and 82 per cent for luteal cysts). The mean (se) plasma progesterone concentration was lower in the cows with follicular cysts than in those with luteal cysts (0.29 [0.05] v 3.90 [0.63] ng/ml; P<0.05). Luteal cysts had thicker walls (5.3 [0.04] v 2.5 [0.2] mm; P<0.0001), and the wall thickness of all the cysts was positively correlated with plasma progesterone concentration (r=0.52, P<0.0004). Cows with luteal cysts had more additional follicles greater than 5 mm in diameter (P<0.01). In cows with follicular cysts and other follicles greater than 5 mm in diameter, the mean oestradiol concentration was 7.9 (1.8) pg/ml compared with 24.2 (3.1) pg/ml (P=0.002) in cows without other follicles greater than 5 mm in diameter on either ovary. At the time of PRID removal, plasma progesterone concentration had increased in the cows with follicular cysts to 1.59 (0.06) ng/ml (P<0.05) and decreased in the cows with luteal cysts to 0.87 (0.01) ng/ml (P<0.05), although there was no change in original cyst structure in 45 per cent of the cases. However, new ovarian structures were frequently observed during the treatment. The overall pregnancy rate for cows with both types of cyst after treatment was 50 per cent after three inseminations, but the first service pregnancy rate was only 18 per cent for cows with follicular cysts and 28 per cent for cows with luteal cysts. After treatment, the fertility of cows with follicular cysts was similar to that of paired herdmates, whereas cows with luteal cysts took 40 days longer to calve again than healthy herdmates. However, the culling rate was higher for cows with follicular cysts (41 v 11 per cent).     

Changes in surface follicles and ovarian weight in testing for responsiveness in dairy cows after induction of superovulation using PMSG and cloprostenol

We evaluated the ovaries of 34 cows cross-bred of the Slovak Pied and Lowland Black-pied breeds which were culled and intended for slaughter during the winter type of feed rations. For superovulation treatment we used PMSG in the preparation Serum Gonadotropin (Bioveta, Nat. Ent., Ivanovice na Hané) and cloprostenol in the preparation Oestrophan inj. Spofa. We weighed the excised ovaries, fixed them in formalin 10% and made a quantitative evaluation of the surface follicles and differentiated them into recruited and selected or dominant follicles. We determined the concentration of 17 beta-estradiol and testosterone with the aid of the 3H RIA set from the firm Sorin after extraction by diethylether with separation of free and bound hormone with active charcoal. 72 hours after the giving of cloprostenol 43% reacted positively to superovulation treatment and after seven days a 50% positive response was recorded. After a dose of 2000 i. u. of PMSG (n = 6) embryo was obtained, whereas after 3000 i. u. of PMSG (n = 8) we flushed out eight embryos, of which four zygotes were suitable for transfer. After a higher dosage of PMSG there was an increase in the average weight of the ovaries, in right-hand ovaries significantly with P less than 0.05. After super-ovulation treatment the concentration of 17 beta-estradiol (E2) in the follicular fluid from follicles seven days after insemination was found to be so low as to be below the limit of detection, with the exception of four samples (mean = 8.60 nmol.l-1). The greatest concentration of E2 was from animals (n = 5) 72 hours after the giving of cloprostenol--1099.61 nmol.l-1) of follicular fluid. The concentration of testosterone was lower in the follicles of untreated cows in the follicular phase (mean = 5.92 nmol.l-1) compared with the follicles of super-ovulated animals the seventh day after insemination (mean = 14.12 nmol.l-1). The number of recruited and especially selected surface antral follicles 72 hours (n = 7) after the giving of cloprostenol and seven days (n = 8) after insemination was significantly higher in the group of brood cows reacting positively to superovulation in comparison with the animals which did not respond. It appears that the simultaneous monitoring of hormonal and morphological changes in the follicular system will help in objectivising the evaluation of the functional activity of stimulated follicles.     

Plasma progesterone and gonadotrophin concentrations following norgestomet treatment with and without cloprostenol in beef cows

Twelve Hereford cross Friesian cows received subcutaneous implants containing 6 mg norgestomet and intramuscular injections of 5 mg oestradiol valerate and 3 mg norgestomet. Six of the cows also received 0.5 mg cloprostenol eight days later and all implants were removed on day 9. When treatment was commenced between days 3 and 5 of the ovarian cycle, luteal function was not prevented although the luteal phase was shortened in some cases. When treatment was commenced between days 8 and 14 of the cycle progesterone concentrations remained above basal levels for five to seven days. Cows with corpora lutea that were given cloprostenol underwent rapid luteolysis. It is concluded that oestradiol valerate does not control luteal function adequately, particularly if administered early in the cycle, and this may explain failure of oestrus synchronisation in some cases. Administration of prostaglandin 24 hours before norgestomet implant removal may improve the degree of oestrus synchronisation in groups of cyclic cows.