Search

Short-term nutritional supplementation stimulates folliculogenesis in ewes probably by insulin-mediated actions of glucose in the follicle. The aim of this study was to determine the effect of glucose on follicle number and granulosa levels of Aromatase P450 and phosphorylated Akt and AMPK. Twelve Ile-de-France ewes were allocated to two groups; one (n=7) infused with saline and the other (n=5) with glucose (10mM/h) for 72h in the luteal phase. At the end of infusion, ovaries were collected and all follicles >1mm in diameter were dissected to recover granulosa cells. Aromatase P450 and phosphorylated Akt and AMPK were analysed by Western blotting of granulosa cell lysates. Blood plasmas collected before and during the infusions were analysed for progesterone, oestradiol, LH, FSH, glucose, insulin and IGF-I. The infusion of glucose significantly increased follicle number but, significantly reduced Aromatase P450 and phosphorylated Akt and AMPK in granulosa cells. The circulating concentration of glucose rose significantly 3h after the start of the glucose infusion and remained elevated until 27h then fell; the circulating concentration of insulin rose significantly by 3h and remained elevated. The circulating concentration of oestradiol fell significantly by 32h and remained low; the circulating concentrations of LH and FSH were unaffected. These data show that short-term infusion of glucose stimulated follicular growth but decreased Aromatase P450 in granulosa cells. The reduced levels of phosphorylated Akt and AMPK suggest that the phosphatidylinositol 3-kinase pathway has been inhibited by high concentrations of glucose. These data also suggest that there may be functional cross-talk between FSH and insulin signalling in granulosa cells., (Copyright © 2011. Published by Elsevier B.V.)

Two experiments were conducted to determine if nutritional supplementation improved ovulation and pregnancy rates in female goats managed under grazing conditions and submitted to the male effect. In Experiment 1, one group of does did not receive nutritional supplementation, while the other group was supplemented daily for 7 days starting at the time when the males were introduced to the females. The ovulation rate at the second male-induced ovulation was greater (P<0.05) in supplemented (2.0+/-0.1) than in non-supplemented (1.6+/-0.1) does. For Experiment 2, female goats were supplemented for 0, 7, 14 or 28 days, starting 9 days following buck introduction. The proportion of does that were pregnant in the group supplemented for 28 days was greater (P<0.05) than in the non-supplemented group, but did not differ from 14-day and the 7-day supplemented groups. The proportion of pregnant does was greater (P<0.05) in the group supplemented for 14 days compared to the group supplemented for 7 days and the non-supplemented group. These latter two groups did not differ (P>0.05). In conclusion, feed supplementation for 7 days, starting at the time when males were introduced increased ovulation rate and feed supplementation for 14 or 28 days starting 9 days after males were introduced improved pregnancy rates in goats managed under grazing conditions and exposed to males.

This experiment was conducted to determine if feed supplementation before exposure of anoestrous does to males increases ovulation rate. Does (n=50) grazing natural vegetation were divided into two groups (n=25). One group received no feed supplementation, while the other was supplemented daily, with a mixture of 950 g of alfalfa hay, 290 g of rolled corn and 140 g of soy bean per animal for 7 days before exposure to bucks. On April 7, all females were exposed to four adult sexually active bucks (two per group) for 15 days. The ovulation rate at the ovulation detected within 5 days of exposure to males, assessed by transrectal ultrasonography, was greater (P<0.05) in supplemented (1.6+/-0.2) than in non-supplemented females (1.0+/-0.2). In contrast, ovulation rate at the subsequent ovulation, detected between days 6 and 15 of contact with males, was not different (P>0.05) between supplemented (1.3+/-0.1) and non-supplemented females (1.3+/-0.2). Feed supplementation 7 days before exposure to sexually active bucks of females managed under grazing conditions increased their ovulation rate at the first male-induced ovulation but the stimulatory effect of supplementation did not persist and was not observed at the subsequent ovulation.

This study investigated the effect on follicular and embryo development of increasing quickly degradable nitrogen (QDN) intake in lactating Holstein dairy cows. Forty mature post-partum cows were fed one of two diets for a minimum of 10 weeks, starting 10 days before first insemination. The Control diet was a high production dairy ration. The High QDN diet comprised the Control ration plus 250 g urea/head/day. Both diets were formulated to ensure that the energy requirements of the cows were satisfied. The High QDN treatment resulted in a significant increase in milk urea, plasma urea and plasma ammonia concentrations. The highest plasma urea (8.2 mmol/l) and ammonia concentrations (120 micromol/l) were recorded within 7 days of the urea supplement being added to the diet. There was no effect of diet on plasma progesterone or glucose concentration. There was also no significant effect of treatment on follicular development or embryo growth. The results from this study suggest that the lactating cow can adapt to increased intakes of QDN if the increase starts at least 10 days before insemination.

The objective of this experiment was to examine the effects of an excess intake of quickly degradable nitrogen (QDN) on metabolic and reproductive parameters in lactating dairy cows. Twenty-two lactating dairy cows were fed a total mixed ration once daily. The control diet was a typical ration for high producing cows in the UK (CP = 17.5%, ME = 11.8 MJ/kg DM). The cows were randomly divided into two groups, control diet (control; n = 12) and excess QDN diet (QDN; n = 10). The QDN group was fed an additional 250 g of urea per cow per day, from 10 days before insemination (day 0) until the end of the experiment, 17 days after the second insemination. Ten days before insemination, a synchronized oestrus was induced and the cows inseminated twice, 48 and 72 h after synchronization, with commercial frozen semen from a single sire. Ovaries were scanned using B-mode ultrasonography 10 days before insemination and then daily from 3 days before insemination. Eighteen of the cows (9 control and 9 QDN) were sampled more intensively to determine the pulsatile pattern of secretion of luteinizing hormone (LH) and growth hormone (GH). Cows were slaughtered 17 days after insemination, the reproductive tracts recovered and flushed to retrieve embryos. The excess QDN diet resulted in elevated (P < 0.05) plasma urea concentrations 3 days after starting urea feeding and these were maintained until the end of the experiment. However, the excess QDN diet did not significantly affect daily milk production or plasma concentrations of insulin and IGF-I. The QDN treatment did not significantly affect pulsatile patterns of secretion of LH and GH or the number of small (< 0.5 cm diameter) and medium to large follicles (> 0.5 cm diameter). Twenty cows ovulated following synchronization (control 11/12; QDN 9/10). There were no significant differences between the control and the QDN groups in the peak concentrations of oestradiol during the follicular phase or in the post-ovulatory pattern of plasma and milk progesterone secretion. Embryos and/or foetal membranes were recovered from 10 cows (5 control and 5 QDN). The results of the current study show that feeding excess QDN, as urea, for 27 days commencing 10 days before insemination had no effect on reproductive or metabolic hormonal parameters. Ovulation and the formation and function of the post-ovulatory corpus luteum were also unaffected by excess QDN. These data suggest that the harmful effects of excess intakes of QDN on fertility occur after 17 days following ovulation.

A model designed to mimic the action of LH in preovulatory follicle development was evaluated. The model involved treatment of ewes with GnRHa (50 micrograms kg-1 24 h-1; GnRHa) at the time of luteal regression followed by exogenous LH administered as a series of bolus injections of varying frequency and amplitude. Ovarian responses to this regimen were compared to data derived from the same animals during a normal follicular phase. In experiment 1 four ewes with an autotransplanted ovary were treated with GnRHa followed by hourly LH (2.5 micrograms NIH-oLH-S25 i.v.) for 60 h at which time a further bolus of LH (25 micrograms i.v.) was given to mimic the LH surge. Plasma FSH profiles and the pattern and rate of secretion of oestradiol, inhibin, androstenedione and testosterone by the ovary in normal and GnRHa treated ewes were similar, but ovulation did not occur following the induced follicular phase. Experiment 2 examined the ability of a revised LH regimen to induce ovulation in normal intact ewes. Ewes (n = 11) were treated with GnRHa at the time of luteal regression followed by LH injections (1.25 micrograms i.v.) administered every 3 h for 12 h, every 2 h for the next 12 h and every hour for the following 36 h (3,2,1 regimen). After 60 h of LH injections the animals received an 8 h infusion of oLH (25 micrograms min-1) designed to mimic the pre-ovulatory LH surge. This treatment resulted in normal follicular phase patterns of LH and FSH and had no effect on the incidence (100%) or number (1.6 +/- 0.1) of ovulations when compared to a normal cycle (100% and 1.8 +/- 0.2). Experiment 3 examined the pattern of ovarian follicular development and hormone secretion stimulated by the 3,2,1 LH regimen in ewes with autotransplanted ovaries (n = 8). The LH regimen stimulated normal pre-ovulatory follicular development (normal 1.4 +/- 0.2; induced 1.6 +/- 0.2) and a normal pattern of ovarian oestradiol and androstenedione secretion. Pulse analysis showed, however, that baseline steroid secretion was lower in induced animals during the late follicular phase reflecting lower baseline LH at this stage. We conclude that administration of a GnRHa at the time of luteal regression in conjunction with frequent low amplitude pulses of exogenous LH induce follicular and hormonal patterns that closely mimic those observed following normal luteal regression and that this regimen represents a useful model to study the role of LH in the control of follicular selection.

The distribution of ovulation between the right and the left ovary was recorded using endoscopy, in 2806 ewes over a 5-year period. Fifteen separate tests were conducted as part of the development programme for a commercial twinning vaccine. There were significantly more ovulations on the right ovary (53.4%) compared to the left ovary (46.6%; P < 0.001). The distribution of ovulation between the ovaries was not influenced by either the breed of sheep or prior immunisation against the steroid hormones androstenedione or testosterone. These findings suggest that the hormonal control of folliculogenesis and ovulation rate is modulated by unknown local factors within the ovary and its vasculature. The site of ovulation had no effect on embryo survival, and embryos from unilateral ovulations were just as likely to survive as were embryos from bilateral ovulations. However, embryo survival was influenced by ovulation rate, and ewes with ovulation rates of four or more had reduced litter sizes and lower embryo survival.

Arterio-venous (A-V) difference techniques were used in cattle to examine ovarian energy metabolism, cholesterol uptake and steroid hormone outputs. Catheters were inserted into the ovarian vein and facial artery, and Transonic flow transducers were placed around the ovarian A-V plexus. Further, in some cows, the effects of a challenge with GnRH were examined. Glucose uptake and lactate output were significant in most individual cows. Nonesterified fatty acids (NEFA) uptake were not significant in any cow in dioestrus. Ovarian uptake of beta-Hydroxy-butyrate (3-OHB) was significant in 4 cows in dioestrus. Cholesterol uptake was significant in only 1 cow. Oxygen uptake was significant in all cows at all stages of the oestrous cycle. All cows had significant output of progesterone and oestradiol-17 beta. These data show that the bovine ovary utilises significant amounts of glucose, and Respiratory quotient (RQ) estimates demonstrated that glucose was the primary fuel used by the ovary. The significant output of lactate suggested that anaerobic pathways were mainly used for glucose oxidation. The observed uptakes of 3-OHB indicated that the ovary utilises 3-OHB as a source of energy. Cholesterol uptake was not a rate-limiting factor for steroid hormone production in the ovary. Despite the high metabolic rate in the luteal ovary, the small difference in PO2 between arterial and ovarian venous blood indicated that the ovary consumes only a small proportion of available oxygen. GnRH had no significant effect on the uptake of metabolites and energy metabolism, but it increased OBF and the output of progesterone and oestradiol-17 beta. The use of A-V methods to determine the metabolic needs of the ovary is useful in understanding the means by which nutrition can influence fertility.

The present study was undertaken to develop an experimental sheep model which could be used to investigate the abnormal follicle growth that is associated with the absence of the LH surge. On Day 10 of the oestrous cycle, 16 ewes were treated with an analogue of prostaglandin (cloprostenol; PG) and blood sampled every 4 h thereafter to determine the normal timing of the preovulatory LH surge. Three oestrous cycles later, all ewes were simultaneously treated with PG and a gonadotrophin-releasing hormone (GnRH) antagonist ([Ac-DNal1, D4C1Phe2, DTrp3, DArg6, DAla10] GnRH.HOAc; 50 micrograms kg-1 subcutaneously). Group 1 ewes (n = 6) received no further treatment. Group 2 ewes (n = 5) were additionally treated for a total of 7 days, starting at the time of PG injection, with purified ovine luteinising hormone (LH; preparation P 3 R3-5; equivalent to 1.25 micrograms NIH-oLH-S26) administered i.v. over a 2 min period. For the first 24 h, LH was given at 3 h intervals for 12 h, then every 2 h for 12 h, and thereafter hourly for 6 days. Group 3 ewes (n = 5) were treated as Group 2 but at 72 h received an additional antagonist injection (50 micrograms kg-1 subcutaneously). Mean values of LH from 24 to 96 h were significantly lower in untreated controls and Group 1 than in the other two groups (0.58 +/- 0.2 ng ml-1 and 0.55 +/- 0.2 ng ml-1 vs. 1.63 +/- 0.5 and 1.68 +/- 0.6 ng ml-1, respectively; P < 0.01). After treatment with PG alone in the untreated control group, the preovulatory LH surge began in all ewes at 59.9 +/- 2.8 h after PG. All Group 1 ewes also had an LH surge but the period from PG injection to the onset of the surge was 124 +/- 17.3 h (range 96-152 h). Only two of the Group 2 ewes had an LH surge (at 160 and 168 h, respectively) and no surge was detected in Group 3 ewes. In Group 1, mean values of follicle-stimulating hormone (FSH; 0.78 +/- 0.07 ng ml-1) were not affected by treatment with antagonist alone; however, in the two groups receiving exogenous LH pulses there was a marked decrease in FSH concentrations during the period 24-96 h. Progesterone concentrations increased 9 days after PG treatment in five out of six ewes in Group 1. In Group 2, there was evidence of a variable luteinisation response, but in Group 3 progesterone remained less than 0.08 ng ml-1 throughout. Endoscopy 112-115 h after PG confirmed that none of the 16 antagonist-treated ewes had ovulated; an event normally expected approximately 80 h after PG in sheep. The experimental protocol of Group 3 provides the basis for a model which will enable examination of the long-term functional capacity of ovarian follicles which have not been exposed to an LH surge.

Caffeine, a trimethylxanthine alkaloid, is a psycho-active drug that effects a wide range of physiological systems, including the reproductive system. Reports of infants with intra-uterine growth retardation and lowered birth weight as a result of in utero exposure to caffeine, are increasing. The drug is also known to alter steroidogenesis but it is not certain whether this is a direct and/or an indirect effect with the involvement of the central nervous system. Thus, an experiment was designed to determine the effect of acute caffeine administration on the circulating concentrations of gonadotrophins and prolactin in the ovariectomized oestradiol-implanted ewe. A single intravenous dose of caffeine (20 mg kg-1 bodyweight) did not affect circulating gonadotrophin concentrations with the parameters for the pulsatile secretion of luteinizing hormone (LH) and the mean concentration of follicle stimulating hormone (FSH) being similar in both experimental and control groups. Circulating prolactin levels, on the other hand, were significantly (P < 0.01) elevated following intravenous treatment with caffeine. The effect was immediate following caffeine administration with elevated concentrations being maintained over the next 3 h before their return to pre-treatment concentrations. The response was bi-phasic with peaks of prolactin concentrations at 1 and 3 h. The results of this experiment show that acute caffeine exposure does not affect the secretion of gonadotrophins from the anterior pituitary gland. Furthermore, they show that acute administration of caffeine stimulates prolactin secretion via an action that is independent of oestradiol feedback and which we suggest, may involve the ACTH/adrenal axis.

Dietary amino acid precursors for cathecholamineric and serotonergic neurotransmitters may be important in the mechanism of nutritional effects on ovulation rate. This paper reports the results of three experiments that examined the effect of such amino acids on ovulation rate and the concentrations of FSH and LH in sheep. In three separate experiments, groups of ewes were infused, over Days 9 to 13 of the oestrous cycle, with either tryptophan (n = 11), tyrosine (n = 11) or a mixture of tyrosine and phenylalanine (n = 11). Control ewes (n = 12 in each experiment) were infused with a vehicle over the same period. None of the amino acids infused effected ovulation rate or plasma concentrations of LH, FSH, GH or prolactin. The infusion of a mixture of tyrosine and phenylalanine increased insulin concentrations. The infusion of these amino acids was not associated with changes in gonadotrophin concentrations and therefore the effect of nutrition on ovulation rate in ewes does not seem to involve an increase in the availability of tryptophan, tyrosine or phenylalanine. Increasing the uptake of other amino acids that compete with tryptophan, tyrosine or phenylalanine for the large neutral amino acid transporter may cause a decrease in the availability of tryptophan, tyrosine or phenylalanine thereby eliciting the effects of nutrition on ovulation rate. However, this hypothesis remains to be tested.