Mesquite pod extract modifies the reproductive physiology and behavior of the female rat.

Phytoestrogens are non steroidal compounds that can bind to estrogen receptors, mimicking some effects of estradiol (E(2)). These compounds are widespread among legumes, which are used as pasture, and their importance in animal agriculture has increased. Mesquite (Prosopis sp) is a widespread legume, widely used to feed several livestock species in Mexico. The main product of mesquite is the pod, which is considered high quality food. As a legume, it could be assumed that mesquite contains some amounts of phytoestrogens which might induce potential estrogenic effects. However, to our knowledge, there are no reports regarding the possible estrogenic activity of this legume either in livestock or in animal models such as the rat. Therefore, in this study, we evaluated the potential estrogenic effects of mesquite pod extract on several aspects of behavior and reproductive physiology of the female rat. The effects of the extract were compared with those of E(2) and two isoflavones: daidzein (DAI) and genistein (GEN). The following treatments were given to groups of intact and ovariectomized (OVX) female rats: vehicle; mesquite pod extract; E(2); GEN; DAI. Compared to vehicle groups, mesquite pod extract, DAI, GEN, and E(2) increased uterine weight and induced growth in vaginal and uterine epithelia. In intact rats, mesquite pod extract, GEN and DAI altered estrous cyclicity, decreased lordotic quotient and intensity of lordosis. In OVX rats, mesquite pod extract, DAI and GEN induced vaginal estrus, increased vaginal epithelium height, and induced lordosis, although its intensity was reduced, compared with intact rats in estrus and E2-treated rats. These results suggest that mesquite pod extract could have estrogenic activity. However, the presence of phytoestrogens in this legume remains to be confirmed.

Sexually active bucks are able to stimulate three successive groups of females per day with a 4-hour period of contact.

Bucks rendered sexually active by a photoperiod treatment of long days can induce fertile ovulation in a group of goats with only 4 h of contact daily with a male:female ratio of 1:10. Here we tested whether such bucks could induce fertile ovulations when stimulating successively three different groups of anovulatory goats when interacting 4 h per day during 15 consecutive days. Control males (n=3) were introduced in the control group (n=25) of does at 8:00 h and were removed at 12:00 h. Experimental males (n=3) were in contact with the experimental groups of does: from 8:00 h to 12:00 h with a first group (n=27), from 12:00 h to 16:00 h with a second group (n=26) and with a third one (n=27) from 16:00 h to 20:00 h. Bucks were then placed until next day in another pen. Both in the control and the experimental groups, more than 85% of females ovulated, and the proportions did not differ between the control and experimental groups (P≥0.67) or between the three experimental groups (P≥0.67). Moreover, the ovulation rate did not differ significantly between the control and the experimental females nor between the three experimental groups. Bucks were able to fertilize more than 72% of does independently of the number of females they were exposed to (P≥0.17). Finally, more than 58% of females kidded and fertility did not differ between the control and experimental groups (P=1) nor among experimental groups (P≥0.77). We conclude that sexually active bucks are able to induce fertile ovulation in three successive groups of anovulatory goats even when the period of contact between sexes is reduced to 4 h per day.

The ovulation rate in anoestrous female goats managed under grazing conditions and exposed to the male effect is increased by nutritional supplementation.

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.

The ovarian expression of mRNAs for aromatase, IGF-I receptor, IGF-binding protein-2, -4 and -5, leptin and leptin receptor in cycling ewes after three days of leptin infusion.

An experiment was carried out to determine the pattern of follicular expression of mRNAs for aromatase, IGF-I receptor (IGF-IR), IGF-binding protein (IGFBP)-2, -4 and -5, leptin and the long form of the leptin receptor (Ob-Rb) in ten ewes infused with human recombinant leptin (n = 5; 1 microg/h) or saline (n = 5) for 72 h in the luteal phase of the oestrous cycle. At the end of infusion a follicular phase was induced with a luteolytic dose of a prostaglandin F2alpha analogue and the ovaries were collected 32 h later. One ovary from each ewe was serially sectioned at 10 microm using a cryostat at -20 degrees C. All follicles >1 mm in diameter were counted and probed with specific oligoprobes for aromatase, IGF-IR and IGFBP-2, -4 and -5 and specific riboprobes for leptin and Ob-Rb. Leptin mRNA was detected in theca and granulosa cells and Ob-Rb mRNA was detected only in granulosa cells, of some, but not all antral follicles. Leptin doubled the number of follicles with a diameter >/=3.5 mm (1.0 +/- 0.36 (s.e.m.) vs 2.4 +/- 0.24; control vs leptin; P < 0.02) but had no effect on the number of >/=1 < 3.5 mm follicles. Leptin had no effect on the number of follicles expressing aromatase mRNA but it decreased significantly the number of follicles expressing mRNA for IGF-IR (10.7 +/- 0.79 vs 7.4 +/- 0.81; control vs leptin; P < 0.05), IGFBP-2 (10.0 +/- 0.82 vs 5.2 +/- 0.87; control vs leptin; P < 0.05) and IGFBP-5 (5.2 +/- 1.60 vs 1.2 +/- 0.30; control vs leptin; P < 0.05). Leptin increased the diameter of IGFBP-2 mRNA-positive follicles (1.5 +/- 0.15 vs 2.2 +/- 0.31 mm; control vs leptin; P < 0.05) and increased follicular mRNA expression for IGFBP-2 (0.30 +/- 0.021 vs 0.39 +/- 0.027 arbitrary units; control vs leptin; P < 0.05) and IGFBP-5 (0.46 +/- 0.019 vs 0.25 +/- 0.053 arbitary units; control vs leptin; P < 0.05). The mRNA for IGFBP-4 was detected in the theca of only two follicles from the control group. Leptin increased the number of follicles expressing Ob-Rb mRNA (0.25 +/- 0.25 vs 1.40 +/- 1.17; control vs leptin; P < 0.05) but had no effect on the number expressing leptin mRNA. Leptin decreased plasma concentrations of oestradiol (P < 0.05) and increased concentrations of FSH (P < 0.001) and insulin (P < 0.001), with no effect on glucose concentrations. These data show that: (i) ovine granulosa cells express mRNA for Ob-Rb and leptin and (ii) leptin increased the number of follicles >/=3.5 mm. Furthermore, the data suggest that suppression of oestradiol production by leptin is not mediated by inhibition of aromatase gene expression. Finally, the data indicate that the action of leptin in ovarian follicles is mediated by the IGF system, because leptin increased mRNA expression of IGFBP-2 and -5. Leptin also decreased the number of follicles expressing IGF-IR and IGFBP-2 and -5. We suggest that these actions of leptin on the IGF system decrease the bioavailability of IGF-I, resulting in decreased oestradiol production.

Ovarian follicular expression of mRNA encoding the type I IGF receptor and IGF-binding protein-2 in sheep following five days of nutritional supplementation with glucose, glucosamine or lupins.

The IGF system is associated with ovarian folliculogenesis. The effect of the IGFs mediated through the type I receptor (IGF-IR) and IGF-binding protein-2 (IGFBP-2), is to regulate the growth and atresia of follicles. To test if the mRNAs for IGF-IR and IGFBP-2 are differentially regulated in the follicle we used nutritional treatments that stimulate folliculogenesis and measured, by in situ hybridisation, their mRNAs expression. Groups of five anoestrous Merino ewes were fed wheat straw (control) or the control diet supplemented with lupins (500 g/day). Other ewes were fed the control diet and infused with glucose (50 mmol/h) or with glucosamine (3.5 mmol/h). Intravaginal progestagen sponges were inserted for 12 days, and nutritional treatments were started 5 days before progestagen removal. Follicular development was studied after an artificial follicular phase, simulated by progestagen for 12 days and a regime of GnRH pulses given for 36 h following progestagen withdrawal, when the animals were killed. The ovaries were collected and stored at -80 degrees C until sectioning at 10 microm. Every 25-28th and 29-32nd section was probed for IGF-IR and IGFBP-2 using 35S-labelled oligonucleotide probes. None of the nutritional treatments affected the number or size of follicles positive for IGF-IR, but glucose (P < 0.001) and lupin (P < 0.001) treatments reduced the follicular concentration of mRNA. The nutritional treatments all increased the number of follicles positive for IGFBP-2 (P < 0.05) and reduced their mean diameter (P < 0.05) and with the exception of lupin feeding, the concentration of mRNA (P < 0.05). The results show that all treatments affected the intrafollicular IGF system and suggest that IGF-IR and IGFBP-2 are nutritionally regulated in the follicle. However, the effects of treatments were variable and suggest the existence of multiple regulatory mechanisms that allow for normal variation in composition and balance of the ruminant diet.

Folliculogenesis and ovarian expression of mRNA encoding aromatase in anoestrous sheep after 5 days of glucose or glucosamine infusion or supplementary lupin feeding.

Improved nutrition increases ovulation rate in sheep and there is evidence that intra-ovarian pathways mediate responses to nutrition. An experiment was conducted to examine the effect of dietary energy on folliculogenesis. Anoestrous Merino ewes were fed a diet of wheat straw alone (control, n = 5), or wheat straw supplemented with lupins (500 g day(-1), n = 5). Other ewes were fed wheat straw and infused with glucose (50 mmol h(-1), n = 5) or with glucosamine (3.5 mmol h(-1), n = 5). Intravaginal progestagen sponges were inserted for 12 days, and nutritional treatments were started 5 days before sponge removal. At sponge removal, the ewes were injected with a regimen of GnRH pulses (500 ng every 4 h from 0 to 12 h; 250 ng every 2 h from 14 to 24 h; and 200 ng every 1 h from 25 to 36 h) to simulate normal follicular development. Thirty-six hours after sponge removal, the animals were killed and the ovaries were collected and stored at -80 degrees C. The ovaries were sectioned serially every 10 microm. Every 20th section was stained (to estimate number and diameter of follicles) and every 17-19th section was probed by in situ hybridization for P(450) aromatase. Data were analysed using ANOVA and chi-squared tests. There was an effect of treatment (P < 0.05) on the number of follicles 2-3, 3-4 and 6-7 mm in diameter. Aromatase-positive follicles (1.6-7.9 mm) were detected in 31 follicles from 15 ewes across all four groups. In ten animals, the largest follicle was aromatase-positive. The diameters of aromatase-positive follicles were larger (P = 0.004) in lupin fed compared with glucose-infused ewes (4.9 +/- 0.5, 3.6 +/- 0.7, 5.3 +/- 0.5 and 4.2 +/- 0.5 mm for control, glucose-infused, lupin-fed and glucosamine-infused groups, respectively). Treatment did not affect the plasma concentration of FSH when compared with controls, indicating that the energy supplements were modifying recruited (2-3 mm and 3-4 mm) and selected follicles (> 6 mm) directly. In conclusion, dietary energy can directly stimulate folliculogenesis in recruited and selected follicles, and this effect may be mediated by changes in systemic leptin concentrations and the hexosamine energy-sensing pathway in the follicle.

Ovulation and luteal characteristics following removal of the ovine corpus luteum (lutectomy) at four times during the oestrous cycle.

The life span of the corpus luteum (CL) may depend on follicular development. To provide evidence relating to this hypothesis, each of 32 ewes was randomly assigned to have its CL removed on day 2, 3, 4 or 10 after oestrus. Twenty ewes were treated with 1000 IU of human chorionic gonadotrophin (hCG) 36 h after CL removal to induce ovulation; the other 12 ewes were not treated with hCG. Blood samples were collected daily to monitor the ovulatory response and the characteristics of the next cycle at the first sign of oestrus and up to day 21 after surgery or hCG administration. Every animal ovulated within 7 days of hCG administration, regardless of when its CL had been removed. It was concluded that the follicles found in the ovary as early as the second day after oestrus respond to endogenous or exogenous ovulatory stimuli affecting the life span of resulting CL.