K.L. Macmillan

Some effects of prematurely elevated concentrations of progesterone on luteal and follicular characteristics during the oestrous cycle in heifers

The aim of this study was to characterise the luteal and follicular response to artificially elevated concentrations of progesterone during the metoestrous phase of the oestrous cycle of heifers. An intravaginal controlled internal drug releasing (CIDR) device containing 1.9 g progesterone was inserted at either Day 1 of the cycle (oestrus designated Day 0) for 4 days (T1; n=12), or on Day 4 for 5 days (T4; n=11). A third group of heifers (CTRL; n=13) remained untreated. The diameters of the corpus luteum (CL) and all follicles of at least 5 mm were recorded daily in ovaries of eight heifers from each group by transrectal ultrasonography throughout the cycle. A blood sample was collected daily from every heifer to determine the concentrations of progesterone and luteinising hormone (LH) in sera. Two of the heifers with elevated progesterone levels from Day 1 had ‘short’ cycles which were characterised by ovulation of the first dominant follicle following the premature demise of the CL. These data are considered separately from the general analysis. Progesterone concentrations of heifers in both treatment groups were elevated (P<0.05) during the period of CIDR insertion, but were not different at the mid-cycle phase compared with untreated contemporaries. A sustained decline to basal concentrations of progesterone occurred earlier (P<0.05) in heifers treated from Day 1. Elevated progesterone concentrations were associated with decreased (P<0.05) mean concentrations of LH on Days 4 and 5 in heifers of the T1 group, but only on Day 5 in the T4 group. The average diameter of the corpus luteum (CL) of treated heifers from Days 8 to 18 was less than in untreated heifers (Days 11–13 and 16–18, P<0.05). Heifers in the T1 group had either one or two waves of follicle turnover, with a mean inter-oestrus interval of 8 days and 18 days, respectively. In contrast, heifers in other groups had some two-wave but mostly three-wave cycles and mean inter-oestrus intervals of about 21 days. Premature elevation of progesterone reduced (P<0.05) the size of the first dominant follicle in both treated groups of heifers. Administration of progesterone during the early and late metoestrous phase of the oestrous cycle in heifers reduced the diameters of the CL and the first dominant follicle. Elevation of progesterone from Days 1 to 5, but not from Days 4 to 9, reduced the lifespan of the CL to produce ‘short’ and ‘shortened’ cycles, with either one or two follicle waves, respectively.

The effect of level of feed intake on progesterone clearance rate by measuring faecal progesterone metabolites in grazing dairy cows

The objective of the present study was to determine the effect of level of feed intake of pasture on P4 clearance rates in dairy cows. Twelve non-lactating Holstein-Friesian cows aged 4-9 years were randomly allocated to a restricted or ad libitum group. The ad libitum group had unrestricted access to irrigated pasture, whereas the restricted group had access for only 2h per day. Each animal was drenched orally twice daily with a chromic oxide capsule to allow daily feed intake to be estimated from faecal output (FO). Endogenous progesterone (P4) production was eliminated by subcutanously implanting a capsule containing 6 mg of a potent GnRH-agonist (deslorelin) into the ear of each animal 3 weeks before inserting a CIDR device containing 1.9 g P4 into the vagina. Two luteolytic PGF2alpha were given 10 days later. Each device was removed after 11 days and residual P4 measured. Daily plasma samples were assayed for P4. Faecal samples were also taken daily and assayed for pregnanes (FP4M) containing a 20-oxo-, a 20alpha- or a 20beta-OH group with EIAs. The average daily dry matter (DM) intake of pasture was higher for cows in the ad libitum group (15.9 versus 6.3 kg DM, P=0.001). Their plasma P4 concentrations were lower (1.08 versus 1.71 ng/ml, P=0.05), even though the average residual P4 content of the used CIDR devices was not affected by feed intake (1.20 versus 1.25 g, P>0.05). The concentrations of FP4M were not affected by level of feed intake (20-oxo-: 3.3 versus 1.7, 20alpha-: 3.5 versus 3.7, 20beta-: 2.1 versus 3.2 microg/g DM). Daily excretion rates of 20-oxo- and 20alpha- were higher in ad libitum cows (20-oxo-: 17.8 versus 4.3mg per day, P=0.05; 20alpha-: 18.2 versus 8.9 mg per day, P=0.001), but daily yield of faecal 20beta- was not affected by feed intake (11.9 versus 8.6 mg per day, P=0.5). These results show that there was a negative relationship between feed intake and plasma P4 concentrations in these CIDR-treated GnRH-downregulated Holstein cows. Concentrations of FP4M were not affected by level of feed intake or FO, but daily excretion rate of FP4M was associated with the volume of faeces.

Insulin regulates milk protein synthesis at multiple levels in the bovine mammary gland

The role of insulin in milk protein synthesis is unresolved in the bovine mammary gland. This study examined the potential role of insulin in the presence of two lactogenic hormones, hydrocortisone and prolactin, in milk protein synthesis. Insulin was shown to stimulate milk protein gene expression, casein synthesis and 14C-lysine uptake in mammary explants from late pregnant cows. A global assessment of changes in gene expression in mammary explants in response to insulin was undertaken using Affymetrix microarray. The resulting data provided insight into the molecular mechanisms stimulated by insulin and showed that the hormone stimulated the expression of 28 genes directly involved in protein synthesis. These genes included the milk protein transcription factor, ELF5, translation factors, the folate metabolism genes, FOLR1 and MTHFR, as well as several genes encoding enzymes involved in catabolism of essential amino acids and biosynthesis of non-essential amino acids. These data show that insulin is not only essential for milk protein gene expression, but stimulates milk protein synthesis at multiple levels within bovine mammary epithelial cells.

Effect of treatment with progesterone and oestradiol benzoate on ovarian follicular turnover in postpartum anoestrous cows and cows which have resumed oestrous cycles

Two experiments were carried out to determine the effect of a low dose of progesterone (P) with and without the addition of an injection of oestradiol benzoate (ODB) on ovarian follicle dynamics, oestradiol production and LH pulsatility in postpartum anoestrous cows, compared with cows which had resumed oestrous cycles (cycling cows). In the first experiment, anoestrous Jersey cows were treated with (AN+P, n=8) or without (AN-3, n=3) a previously used intravaginal progesterone releasing (CIDR) device for 10 days, commencing 3 or 4 days after emergence of a new dominant follicle (DF1) as determined by transrectal ultrasonography. Contemporary cycling cows (CYC+P, n=8) were similarly treated with used CIDR devices and injected with prostaglandin F(2alpha) (PGF) at the time of device insertion. Follicle turnover was monitored by daily ultrasonography and pulsatile release of LH was measured on the ninth day after device insertion. During the period of CIDR device insertion, a second dominant follicle emerged in 4/8 of the CYC+P group and 7/8 of the AN+P group (P=0.14). Maximum diameter of DF1 was greater in cows in the CYC+P compared with the AN+P group (P=0.02), but did not differ between cows in the AN+P and AN-P groups (P>0.1). Frequency of LH pulses was greater in cows in the CYC+P than AN+P group (P=0.06), and in cows in the AN+P than AN-P group (P=0.02). In the second experiment, anoestrous (n=20) and cycling (n=11) Friesian cows were treated with a new CIDR device for 6 days commencing 3 days after emergence of a new dominant follicle (DF1). Cycling cows were also injected with PGF on the day of device insertion. Half of the cows in each group were injected with 2mg ODB on the day of device insertion. Daily ultrasonography was used to monitor follicular dynamics throughout the experimental period. Follicular turnover was increased by ODB in cycling (5/5 versus 1/6; P<0.05), but not anoestrous cows (5/9 versus 4/11). Persistence of DF1 was reduced by ODB treatment in both cycling and anoestrous cows (P<0.001). Maximum diameter of DF1 was influenced by ODB treatment and reproductive status (P<0.05). In anoestrous cows in which a second dominant follicle did not emerge during the period of device insertion, the interval from emergence of DF1 to emergence of a second dominant follicle was significantly delayed by treatment with ODB (P=0.04). In conclusion, P treatment of anoestrous cows increased pulsatile release of LH, but did not induce the development of persistent follicles. Injection of ODB in association with P treatment reduced the persistence of dominant follicles in both cycling and anoestrous cows, but delayed subsequent follicular development in a proportion of anoestrous cows.

Fertility regulation in cattle.

This paper reviews the physiological, endocrinological and pharmaceutical literature pertaining to the design, development and optimisation of subcutaneous and intravaginal progestogen-containing drug delivery systems used in the control of synchrony and ovulation in cattle.

Reengineering of a commercially available bovine intravaginal insert (CIDR insert) containing progesterone

The purpose of this study was to reengineer a commercially available intravaginal insert containing 1.9 g progesterone (CIDR intravaginal insert) for a 7-day insertion period in cattle. The reengineering process resulted in a reduced initial drug load (1.38 g) and a reduction in the residual drug load following insertion, while at the same time maintaining the biological performance of the insert. The in vitro and in vivo pharmaceutical properties of the commercially available CIDR intravaginal insert were characterized initially to gain a thorough understanding of the factors that affected progesterone release from the insert. The effect of changing a selection of formulation and physical variables of the insert was also investigated (including surface area, drug load, addition of pore forming materials, silicone shore hardness and drug particle size). The knowledge gained from these studies was used to define the characteristics of the reengineered insert which was then manufactured and shown to be bioequivalent and clinically equivalent to the commercially available insert.

Conceptual and commercially available intravaginal veterinary drug delivery systems

In the veterinary area the utilization of the vagina as a route for drug delivery has focused on the systemic delivery of hormonal steroids to control synchrony, ovulation and fertility in a variety of livestock. There are several reasons for administering drugs to modify the menstrual cycle in humans and the estrous cycle in animals and this paper provides a comparison of the objectives of drug intervention to control reproduction in humans and livestock. In addition, the types of hormones and their concentrations found during the menstrual cycle in humans and during the estrous cycle in animals are compared. The paper also reviews the intravaginal drug delivery systems developed for the control of the estrous cycle in livestock and both conceptual and commercially available intravaginal drug delivery systems that have been described in the literature are described. The history of some of the delivery systems is included and the future directions of this area of research are discussed.

Characteristics of oestrus measured using visual observation and radiotelemetry

The aim of this study was to measure the level of agreement between characteristics of oestrus measured using visual observation and radiotelemetry. Oestrous cycles in 20 non-lactating Holstein cows were synchronised by treatment with an intravaginal progesterone releasing inserts and injections of oestradiol benzoate for a first oestrus (Round 1) and then re-synchronised for two subsequent and successive oestrous cycles (Rounds 2 and 3). Cows were ovariectomised following the third synchronised oestrus and then retreated to induce oestrus (Round 4). Oestrus was monitored by continuous visual observation and radiotelemetry between 24 and at least 60 h after removal of inserts at Rounds 1-4. Significant differences (P<0.001) were obtained between the mean number of mounts (42.7 versus 17.8), duration of oestrus (14.4h versus 10.9h), total duration of mounts (223.9s versus 33.1s), mean duration per mount (5.6s versus 1.9s), the number of mounts per hour (3.5 versus 2.0) and the interval to oestrus (41.2h versus 42.8h) between values determined by visual observation compared with radiotelemetry, respectively. The 95% limits of agreement for all the characteristics of oestrus measured were wide although the efficiency of detection of oestrus for both methods of recording oestrus were high (97.5% (78/80) versus 93.8% (75/80), for visual observation and radiotelemetry, respectively; P=0.25). We conclude that there is poor agreement between characteristics of oestrus measured with visual observation and radiotelemetry in Holstein cows following monitoring of synchronised oestrous cycles, although both methods were equally efficient for the detection of oestrus.

Insulin, a key regulator of hormone responsive milk protein synthesis during lactogenesis in murine mammary explants

Murine milk protein gene expression requires insulin, hydrocortisone, and prolactin; however, the role of insulin is not well understood. This study, therefore, examined the requirement of insulin for milk protein synthesis. Mammary explants were cultured in various combinations of the lactogenic hormones and global changes in gene expression analysed using Affymetrix microarray. The expression of 164 genes was responsive to insulin, and 18 were involved in protein synthesis at the level of transcription and posttranscription, as well as amino acid uptake and metabolism. The folate receptor gene was increased by fivefold, highlighting a potentially important role for the hormone in folate metabolism, a process that is emerging to be central for protein synthesis. Interestingly, gene expression of two milk protein transcription factors, Stat5a and Elf5, previously identified as key components of prolactin signalling, both showed an essential requirement for insulin. Subsequent experiments in HCll cells confirmed that Stat5a and Elf5 gene expression could be induced in the absence of prolactin but in the presence of insulin. Whereas prolactin plays an essential role in phosphorylating and activating Stat5a, gene expression is only induced when insulin is present. This indicates insulin plays a crucial role in the transcription of the milk protein genes.

Plasma Concentrations of Inhibin A and Follicle-Stimulating Hormone Differ Between Cows with Two or Three Waves of Ovarian Follicular Development in a Single Estrous Cycle

Abstract Patterns of ovarian follicle development were monitored daily in Holstein-Friesian cows that had two (n = 4) or three (n = 4) waves of ovarian follicle development during a single estrous cycle. The plasma from daily blood samples was used in assays for inhibin A, FSH, progesterone, and estradiol-17β. Mean cycle lengths for cows with two and three waves were 21.8 and 25.3 days, respectively (P < 0.02). Although the average number of follicles >3-mm diameter on each pair of ovaries was similar for two- and three-wave cows on Days 2, 3, and 4 (Day 0 = day of ovulation; 8.6 vs. 9.6 follicles), there were more follicles >6-mm diameter on the ovaries of cows with two waves on Days 3 and 4. This difference was associated with a shorter interval from wave emergence to peak concentrations of inhibin A during the first wave in two-wave cows (2.0 vs. 3.8 days; P = 0.03) and with higher peak concentrations (474 vs. 332 pg/ml; P = 0.03). Differences in peak FSH concentrations were not significant (1.7 vs. 1.3 ng/ml; P = 0.10) and were inversely related to inhibin A concentrations. The peak concentrations of inhibin A and FSH in the second nonovulatory wave in the three-wave cows were similar to the low concentrations measured in the first wave (292 vs. 332 pg/ml of inhibin A, 1.3 vs. 1.3 ng/ml of FSH; P > 0.20). Average peak concentrations of inhibin A and FSH were similar during the ovulatory wave for cows with either two or three waves in a cycle (432 vs. 464 pg/ml of inhibin A, 2.3 vs. 2.1 ng/ml of FSH; P > 0.3). The lower concentrations of FSH during the emergence of the first follicular wave in cows with three-wave cycles may have reduced the rate of development of some of the follicles and reduced the concentrations of inhibin A. This pattern of lower concentrations of FSH and inhibin A was repeated in the second nonovulatory wave but not in the ovulatory wave. Subtle differences in the concentrations of these two hormones may underlie the mechanism that influences the number of waves of ovarian follicle development that occur during the bovine estrous cycle.