Follicular deviation and acquisition of ovulatory capacity in bovine follicles.

Selection of dominant follicles in cattle is associated with a deviation in growth rate between the dominant and largest subordinate follicle of a wave (diameter deviation). To determine whether acquisition of ovulatory capacity is temporally associated with diameter deviation, cows were challenged with purified LH at known times after a GnRH-induced LH surge (experiment 1) or at known follicular diameters (experiments 2 and 3). A 4-mg dose of LH induced ovulation in all cows when the largest follicle was > or =12 mm (16 of 16), in 17% (1 of 6) when it was 11 mm, and no ovulation when it was < or =10 mm (0 of 19). To determine the effect of LH dose on ovulatory capacity, follicular dynamics were monitored every 12 h, and cows received either 4 or 24 mg of LH when the largest follicle first achieved 10 mm in diameter (experiment 2). The proportion of cows ovulating was greater (P < 0.05) for the 24-mg (9 of 13; 69.2%) compared with the 4-mg (1 of 13; 7.7%) LH dose. To determine the effect of a higher LH dose on follicles near diameter deviation, follicular dynamics were monitored every 8 h, and cows received 40 mg of LH when the largest follicle first achieved 7.0, 8.5, or 10.0 mm (experiment 3). No cows with a follicle of 7 mm (0 of 9) or 8.5 mm (0 of 9) ovulated, compared with 80% (8 of 10) of cows with 10-mm follicles. Thus, follicles acquired ovulatory capacity at about 10 mm, corresponding to about 1 day after the start of follicular deviation, but they required a greater LH dose to induce ovulation compared with larger follicles. We speculate that acquisition of ovulatory capacity may involve an increased expression of LH receptors on granulosa cells of the dominant follicle and that this change may also be important for further growth of the dominant follicle.     

Regulation of cytochrome P450scc synthesis and activity in the ovine corpus luteum

The rate-limiting step in luteal biosynthesis of progesterone consists of cleavage of the side chain of cholesterol by mitochondrial cytochrome P450 side-chain cleavage enzyme (P450_scc) to form pregnenolone. Luteal mRNA encoding P450_scc, quantitated on selected days of the 16-day ovine estrous cycle, was similar on days 3 and 6, increased by 2-fold on day 9 (P < 0.05) and remained elevated on day 15. Levels of P450_scc mRNA on day 15 of pregnancy were not different from those found on any day of the cycle (P < 0.05). To determine whether levels of mRNA encoding P450_scc are hormonally regulated, ewes on day 10 of the estrous cycle were injected with hCG or prostaglandin F₂ (PGF₂). P450_scc mRNA was not increased for up to 36 h after injection of hCG, nor decreased within 8 h after injection of PGF₂ (P < 0.05). An assay for P450_scc activity was developed which utilized ovine small and large luteal cells in the presence of 22R-hydroxycholesterol and ovine high density lipoprotein. Enzyme activity was quantitated by measurement of progesterone production. In small luteal cells activation of the protein kinase A (PKA) second-messenger system by treatment with LH resulted in 910% increase in progesterone production without altering activity of P450_scc. Activation of the protein kinase C (PKC) second-messenger system with phorbol 12-myristate 13-acetate caused a 51% reduction in progesterone secretion from large luteal cells but did not alter activity of P450_scc. These findings suggest that in mature luteal tissue steady state levels of mRNA encoding P450_scc, and enzyme activity are independent of acute regulation by activation of PKA or PKC second-messenger systems.     

Estrus and pregnancy in beef heifers following use of the Syncro-Mate-B treatment (SMB)

Five studies were conducted in yearling heifers which were either puberal or prepuberal utilizing the Syncro-Mate-B (SMB) treatment (G. D. Searle & Co., Chicago, Ill.). In trial 1 the pregnancy rate in Brahman crossbred heifers after 21 days of breeding was 56% and 28% greater (P<.005) in the SMB treated groups than in the nontreated groups. Pregnancy rate in 3 4 Simmental heifers (trial 2) was increased 39% (P<.05) after 21 days of breeding. Hereford and crossbred heifers (trial 3) weighing less than 250 kg failed to respond to SMB treatment whereas heifers weighing greater then 250 kg had increased reproductive performance. Conversely the results obtained in trial 4 using Brahman crossbred heifers was not influenced by weight or SMB treatment. Crossbred Santa Gertrudis heifers (trial 5) weighing greater then 250 kg and treated with SMB had improved reproductive performance compared to the control groups. While in treated heifers weighing less than 250 kg reproductive performance was far from optimum.     

Peripheral progesterone levels in pregnant and non-pregnant heifers following use of HCG.

Progesterone concentration in jugular blood of bred beef heifers was determined on days 9 and 16 in two trials. Human chorionic gonadotrophin (HCG) was administered to some of the heifers in each trial in an attempt to improve pregnancy percentage.In Trial 1, 183 heifers were divided into a control group and three groups of animals which were subjected to a 9-day estrous synchronization treatment prior to breeding. The treatment consisted of an ear implant containing 17 α acetoxy-11-beta-methyl 17 nor preg 4-ene 3, 20 dione (norgestomet) left in place for 9 days and an injection of 5 mg estradiol valerate (EV) and 3 mg of norgestomet given at the time of implantation. The heifers in one group received .25 mg estradiol-17β at time of implant removal; heifers in the 2nd group received 1500 IU of HCG in 5% beeswax and 95% sesame oil at breeding time, while heifers in the 3rd group received a placebo injection containing 5% beeswax and 95% sesame oil at breeding time. No differences in serum levels of progesterone were observed (P>0.5) between treatments or between pregnant and non-pregnant heifers on day 9 or 16 (P>.05). Pregnancy percentage in heifers receiving HCG was similar to that noted in the control heifers or the placebo injected heifers while injection of estradiol 17β decreased the proportion of heifers which became pregnant.In trial 2, 58 heifers which had been bred 1 or 2 times without becoming pregnant were divided into a control group and a group in which heifers received 1000 IU of HCG 96 hr. after observed estrus. In heifers receiving HCG, serum levels of progesterone were higher (P<.01), on day 9 and 16 post estrus than in controls but no difference in serum progesterone was noted (P>.05) between pregnant and non-pregnant heifers on day 9 or 16. The proportion pregnant did not differ (P>.05) between heifers receiving HCG and control heifers.     

Calving difficulty and calf survival in beef cows fed two energy levels

Angus and Hereford yearling heifers (469) were bred to either Hereford or Angus bulls and fed either a high level (13.3 Mcal of ME) or a low level of nutrition (7.3 Mcal of ME) for the last 116 days of pregnancy. The level of calving difficulty did not differ between heifers receiving the high (H) and low (L) level of nutrition the first year (36% and 34%) but was significantly higher (P < .01) in heifers receiving H the second year (33% vs. 16%). Calves born to heifers receiving H weighed at birth 2.7 kg more (P < .01) both years than calves born to heifers receiving L. In the first year of the study the size of the pelvic opening was 10 cm larger 1 week before calving (P < .01) in heifers receiving H than in heifers receiving L. Calf survival was not related to level of nutrition, breed of dam or breed of sire either year of the study.     

Follicular-fluid factors and granulosa-cell gene expression associated with follicle deviation in cattle

Intrafollicular changes in the largest follicle (F1) and second-largest (F2) follicle were examined in relation to follicle diameter deviation. Deviation is characterized by continued growth of the largest follicle and the cessation of growth of the smaller follicles. Granulosa cells and follicular fluid were obtained from slaughterhouse ovaries (n = 95 pairs, experiment 1), and follicular fluid was collected in vivo (n = 28 heifers, experiment 2). Several ranges in the diameter of F1 were used to represent the progressive growth of the follicle. The diameter range with the first significant increase in the difference between F1 and F2 was determined for each end point and was used as an indicator of the sequence of events associated with diameter deviation. An increased difference for diameter and for estradiol concentration occurred (P: < 0.05) simultaneously at the 8.5- to 8.9-mm range in both experiments. In experiment 1, the increased difference between F1 and F2 in LH receptor (LHr) mRNA expression occurred (P: < 0.05) at the 8.0- and 8.4-mm range. In F2 of experiment 2, there was a progressive decrease (P: < 0.05) in free insulin-like growth factor (IGF)-1 and a progressive increase (P: < 0.05) in IGF binding protein (BP)-2 across the follicle-diameter ranges (7.5-11.2 mm). No differences were detected between F1 and F2 for 3beta-hydroxysteroid dehydrogenase mRNA expression in experiment 1 and testosterone, total inhibin, and dimeric inhibin-A concentrations in experiment 2. The results indicated that the acquisition of granulosa cell LHrs by F1, as indicated by increased LHr mRNA expression, occurred one diameter range before an increased difference between F1 and F2 for diameter or estradiol concentrations. On a temporal basis, it is concluded that LHr acquisition plays a role in the establishment of diameter deviation. In addition, the reduced growth of F2 may have involved the reduced bioavailability of IGF-1 in association with elevated IGFBPs.     

Reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate

We hypothesized that reducing the size of the ovulatory follicle using aspiration and GnRH would reduce the size of the resulting CL, reduce circulating progesterone concentrations, and alter conception rates. Lactating dairy cows (n=52) had synchronized ovulation and AI by treating with GnRH and PGF2alpha as follows: Day -9, GnRH (100 microg); Day -2, PGF2alpha (25 mg); Day 0, GnRH (100 microg); Day 1, AI. Treated cows (aspirated group; n=29) had all follicles > 4 mm in diameter aspirated on Days -5 or -6 in order to start a new follicular wave. Control cows (nonaspirated group: n=23) had no follicle aspiration. The size of follicles and CL were monitored by ultrasonography. The synchronized ovulation rate (ovulation rate to second GnRH injection: 42/52=80.8%) and double ovulation rate of synchronized cows (6/42=14.3%) did not differ (P > 0.05) between groups. Aspiration reduced the size of the ovulatory follicle (P < 0.0001; 11.5 +/- 0.2 vs 14.5 +/- 0.4 mm), and serum estradiol concentrations at second GnRH treatment (P < 0.0002; 2.5 +/- 0.4 vs 5.7 +/- 0.6 pg/mL). The volume of CL was less (P < 0.05) for aspirated than nonaspirated cows on Day 7 (2,862 +/- 228 vs 5,363 +/- 342 mm3) or Day 14 (4,652 +/- 283 vs 6,526 +/- 373 mm3). Similarly, serum progesterone concentrations were less on Day 7 (P < 0.05) and Day 14 (P < 0.10) for aspirated cows. Pregnancy rate per AI for synchronized cows was lower (P < 0.05) for aspirated (3/21=14.3%) than nonaspirated (10/21=47.6%) cows. In conclusion, ovulation of smaller follicles produced lowered fertility possibly because development of smaller CL decreased circulating progesterone concentrations.     

Regulation of the corpus luteum by protein kinase C. I. Phosphorylation activity and steroidogenic action in large and small ovine luteal cells

The activity and steroidogenic action of protein kinase C were evaluated in small and large steroidogenic ovine luteal cells. Protein kinase C activity (per mg protein) was threefold greater in large than in small luteal cells, whereas protein kinase A activity was similar in the two cell types. Phorbol 12-myristate 13-acetate (PMA) activated protein kinase C in luteal cells as demonstrated by membrane association of 91% of available protein kinase C within 15 min of PMA treatment. Longer treatments with PMA produced cells with low protein kinase C activity (protein kinase C-deficient cells) but did not affect cellular viability or protein kinase A activity. Activation of protein kinase C caused an acute, dose-dependent inhibition of progesterone production in unstimulated large and luteinizing hormone (LH)-stimulated small luteal cells. This inhibition by PMA appeared to be specific for protein kinase C since it was greatly attenuated in protein kinase C-deficient cells and since an inactive phorbol ester, 4 alpha-phorbol, had no effect on luteal progesterone production. The inhibitory locus of protein kinase C action in small luteal cells appeared to be distal to the adenylate cyclase enzyme because progesterone production was inhibited similarly in cells stimulated with LH, forskolin, or dibutyryl cyclic adenosine 3',5'-monophosphate. Cholesterol side-chain cleavage activity, as measured by metabolism of 25-hydroxycholesterol, was inhibited by PMA in large, but not in small, luteal cells. These data indicate that activation of protein kinase C specifically inhibits progesterone production in both large and small ovine luteal cells, although the intracellular mechanisms invoked appear to differ in the two cell types.