Offering excess oocyte aspiration and vitrification to patients undergoing stimulated artificial insemination cycles can reduce the multiple pregnancy risk and accumulate oocytes for later use.

BACKGROUND: The prevention of multiple pregnancies remains a major challenge in patients treated with ovarian stimulation prior to intrauterine insemination (IUI). The pilot study presented here investigates whether multiple pregnancies can be minimized by a microscopically confirmed aspiration of oocytes from supernumerary follicles immediately before intrauterine insemination and evaluates the benefit of concomitant excess oocyte cryopreservation for future use. METHODS: Thirty-four aspirations of supernumerary follicles were performed immediately prior to IUI in 31 patients undergoing ovarian stimulation. sIUI was only performed if cumulus-oocyte complexes were microscopically observed in the aspirated follicular fluid. All collected mature excess oocytes were cryopreserved using the vitrification technique. RESULTS: Only four sIUI procedures had to be cancelled due to failed oocyte retrieval or premature ovulation. IUI treatment resulted in a clinical pregnancy rate of 23.5% per cycle. All were singleton pregnancies. A total of 111 oocytes were cryopreserved. Patients with polycystic ovary syndrome (PCOS) had an average of 6.07 oocytes vitrified, whereas patients without PCOS had 1.3 oocytes vitrified per cycle. CONCLUSION: Microscopically confirmed collection of excess oocytes prior to stimulated IUI reduced cancellation rates, further reduced the risk for multiple pregnancy and may lead to future additional pregnancies because, based on current information, approximately 5% of the vitrified oocytes could potentially establish a pregnancy.

Relationship between leptin content, metabolic hormones and fat deposition in three beef cattle breeds.

The aim of the study was to determine if cattle breeds differing in their carcass characteristics also differ in the profiles of their leptin and metabolic hormones. Three breeds, Belgian Blue (BB) (n=12), Limousin (L) (n=12) and Aberdeen Angus (AA) (n=12) with varying ability to deposit fat and protein were compared. Blood, muscle and subcutaneous (SC) adipose tissue were sampled. Animal performance, carcass and meat characteristics were determined as well as plasma leptin concentration, leptin gene expression in SC adipose tissue, leptin-receptor gene expression in SC adipose tissue and plasma concentration of insulin, tri-iodothyronin (T3), thyroxin (T4) and cortisol. The BB bulls showed the lowest values of leptin gene expression (P<0.05). Values of plasma leptin concentration and of leptin-receptor gene expression tended to be lower in BB than in the other breeds. For a similar amount of adipose tissue (after normalisation), BB bulls showed a higher ratio of plasma leptin (P<0.05), whereas normalised leptin gene and leptin-receptor gene expressions did not significantly differ between breeds. Belgian Blue bulls also differed in their metabolic hormone profile, tending to show lower values of insulin, T3 and T4 than the two other breeds. Cortisol levels were significantly lower (P<0.05) in BB than in L and AA animals.

Pre-ovulatory follicular characteristics and ovulation rates in different breed crosses, carriers or non-carriers of the Booroola or Cambridge fecundity gene.

Terminal follicular dynamics and ovulation rates (OR) were compared in different local breeds after introducing fecundity genes of different origin. Crossbred ewes which were carriers (F+) or non-carriers (++) of Booroola (BFec) or Cambridge genes (CFec) were included: CambridgexCambridge (CC), CambridgexSuffolk (CS), CambridgexTexel (CT), BooroolaxTexel (BT) and BooroolaxGerman Mutton Merino (BGM). The numbers of small (diameter 2-3.5 mm), medium (diameter >3.5-5.0 mm) and large (diameter >5.0 mm) growing follicles, the maximum diameter before ovulation and the regression and artesia rates of ovarian follicles >/=2 mm in diameter were studied laparoscopically and repeatedly during the last 5 days of an induced oestrous cycle. The ORs were determined one cycle before and two cycles after the repeated laparoscopy. BFec and CFec significantly enhanced the OR of all crossbreeds. Carriers of BFec or CFec did not have significantly different ORs due to any crossbreeding effect. The same observation was made for non-carriers of both Fec gene types. Whatever the crossbreed, the number of small, medium and large growing follicles were similar between carriers and non-carriers in spite of a higher number of ovulating follicles in carriers of both Fec gene types. The diameter of ovulatory follicles did not differ among crossbreds, or between carriers and non-carriers except in the BT (5.2+/-0.2 vs. 6.5+/-0.8 mm, respectively) and CC (6.6+/-0.2 vs. 5.6+/-0.3 mm) ewes.The higher OR in the presence of the Booroola gene was associated with a low atresia rate of large follicles in all crossbreeds (BT: 52+/-8% (F+) vs. 61+/-7% (++); BGM: 51+/-6% vs. 75+/-5%). The high OR of the carriers of the CFec gene seemed to be associated with a lower number of large growing follicles with a lower (P<0.05) atresia rate as compared with Booroola crossbreeds. In conclusion, follicular features were similar between purebred Cambridge and its crossbred CS and CT. In ewes carrying the BFec or CFec gene, the reduction in follicular atresia seemed to be one of the main follicular features implicated in the higher OR.

Control of ovarian follicles activity in the ewe.

During the ovine estrous cycles, three waves of follicular growth, closely associated with the FSH secretion pattern, were observed. The parameters of these follicular waves and the ability of follicles to produce steroids in vitro were studied in various conditions. In vivo, the follicular events were similar between the breeding season and the anestrus, except for the lack of ovulation; but at the end of the breeding season and in anestrus, the follicles lose a big part of their aromatization ability. In ewes carrying the Booroola fecundity gene or Cambridge fecundity gene, the reduction in follicular atresia seems to be one of the main follicular features implicated in the control of high ovulation rate. In vitro, the most relevant difference is an early acquisition of estrogen production ability of small follicles in Booroola fecundity gene barring ewes. Fluoro-gestone-acetate (FGA) pessaries reduced the number of growing follicles; despite this effect disappearing after the sponge withdrawal, the ovulation rate is significantly reduced. But an equine chorionic gonadotrophin (eCG) treatment restores the ovulation rate (OR) by reducing the atresia rate of pre-ovulatory follicles. In similar conditions, a pretreatment of the ewes with melatonin again reduced the atresia rate of large follicles and resulted in an increased ovulation rate. In vitro, FGA blocked aromatization ability, and melatonin inhibited both androstenedione and estradiol production, but a further treatment with eCG partly restores the steroid secretion. Immunization against androstenedione leads to a higher OR, owning to a reduced atresia of large follicles. Daily growth hormone injections for a hole cycle resulted in an increased follicular population and ovulation rate, while FSH plasma levels decreased and the follicle sensitivity to gonadotrophins was reduced.

Effects of season and phase of the estrous cycle on steroidogenesis and LH-FSH sensitivity of large ovine follicles perfused in vitro.

The aims of this study were to compare steroïdogenesis (progesterone, androstenedione and estradiol production) and response to LH and FSH challenge by whole perifused follicles 4 to 5.5 mm in diameter, obtained at different periods of the breeding season (onset, middle, end), during anestrus and the luteal phase. We have observed that all follicles do not have the same steroïdogenetic potential and do not respond with the same intensity to LH and FSH. At the middle of the breeding season, LH and FSH supplementation was ineffective in increasing progesterone secretion by follicles (0.19+/-0.05 vs. 0.20+/-0.03 ng/mL). In contrast, gonadotrophin challenge elicited significant (P<0.05) increases in androstenedione (0.94+/-0.34 vs. 0.35+/-0.09 ng/mL) and estradiol (120+/-11 vs. 49+/-10 pg/mL) production immediately after its administration. At the onset of the breeding season, steroidogenesis was identical under both basal and gonadotrophin-stimulated conditions unlike that in middle of the breeding season. However follicles were more sensitive to the gonadotrophin challenge in terms of estradiol production than those collected at the middle of the breeding season (220+/-45 vs. 120+/-11 pg/mL). Follicles obtained at the end of the breeding season featured higher progesterone (2.61+/-0.81 vs. 0.19+/-0.05 ng/mL; P<0.05) and lower estradiol production (10+/-3 vs. 49+/-10 pg/mL; P<0.05) that was not influenced by LH and FSH. Basal androstenedione secretion was comparable to that observed at the middle of the breeding season (0.42+/-0.10 vs. 0.35+/-0.09 ng/mL), but the response to stimulation was significantly higher (1.82+/-0.61 vs. 0.94+/-0.34 ng/mL; P<0.05). In anoestrus and the luteal phase, follicles presented higher progesterone and androstenedione and lower estradiol concentrations (P<0.05) compared with those obtained during the follicular phase at the middle of the breeding season. In the luteal phase, follicles remained capable of responding to LH-FSH challenge by increasing estradiol secretion (9+/-1 before and 21+/-6 pg/mL after LH-FSH; P<0.05). In contrast, in the luteal phase, estradiol production was not increased by LH-FSH challenge (7+/-2 vs. 12+/-4 pg/mL).

Body weight, oestrous and ovarian activity in local Burundian ewes and goats after parturition in the dry season.

To compare body weight losses and post-partum anoestrus, twelve local Burundian ewes and twelve goats which had lambed or kidded at the middle of the dry season were followed from parturition until eight months. Each dam was weighed every month and bled three times a week to assess the ovarian activity resumption by plasma progesterone (P4) profiles. For the two species, the body weight decreased significantly (P < 0.05) compared to the body weight at parturition. The mean time during which the body weight decreased was longer (P < 0.05) for the ewes (3.2 +/- 1.2 months) than for the goats (2.3 +/- 1.4 months), however, the relative body weight losses did not significantly differ. The time to oestrous and ovarian activity resumption was highly correlated to the body weight losses for the ewes (r = 0.81; P < 0.01 and r = 0.58; P < 0.05) not for the goats. The first oestrus preceded the first ovarian activity by 4 days in the goats, but occurred 36 days later in the ewes. Nevertheless, after the first luteal function, the subsequent ovarian activity was regular and normal in the ewes but not in the goats and in some cases a return to anoestrus could be observed. These data indicate that in the ewe, not in the goat, the body weight losses are one of the major limiting factors of the oestrous and ovarian resumption after parturition during the dry season. Moreover, the occurrence of luteal function does not initiate a regular reproductive cyclicity in the goat in contrast to the ewes.

Effects of FGA and PMSG on follicular growth and LH secretion in Suffolk ewes.

The effects of fluorogestone acetate (FGA) and/or pregnant mare serum gonadotrophin (PMSG) on follicular growth and LH secretion in cyclic ewes were determined. Suffolk ewes (n=40), previously synchronized with cloprostenol were divided into 4 experimental groups (n=10 ewes per group). Group I served as the control, while groups II, III and IV received FGA, PMSG, FGA and PMSG respectively. Four ewes of each group underwent daily laparascopy for 17 d. All the ovarian follicles>or=2 mm were measured, and their relative locations were recorded on an ovarian map in order to follow the sequential development of each individual follicle. Comparisons were made of the mean day of emergence and the mean number of small, medium and large follicles, the atresia rate and the ovulation rate. For each group, 3 waves of follicular growth and atresia were observed during the cycle. During luteal phase, FGA treatment accelerated the mechanisms of follicular growth but reduced the number of large follicles and increased the atresia rate. In the follicular phase, FGA treatment was detrimental to both the number of large follicles and the ovulation rate. By contrast, PMSG enhanced recruitment of small follicles and the ovulation rate. Serial blood samples were collected during the luteal and follicular phases to study LH secretion. None of the treatments had any effect on LH secretion patterns.

Ovarian follicular dynamics in Suffolk ewes at different periods of the year.

Five Suffolk ewes underwent daily laparoscopy for 18 days at the onset, middle and end of the breeding season (August, November and February, respectively) and during anoestrus (May) to examine their ovaries. Ovarian follicles > or = 2 mm were measured and their relative locations reported on an ovarian map to follow the sequential development of each individual follicle. Some aspects of follicular growth (mean day of emergence and mean number of small, medium-sized and large follicles, maximum size before regression and ovulation rate) were also studied and compared. Three waves of follicular growth and atresia were observed during the oestrous cycle; two waves occurred during the luteal phase and one during the follicular phase. This cyclic pattern of growth and atresia was not influenced by the stage of the breeding season. A similar turnover of follicles occurred during anoestrus. Features of follicular growth were similar at all stages studied except for the size of the large follicles which were smaller during the two waves of the luteal phase (P < 0.05). The number of growing follicles did not vary and the mean ovulation rate was 1.2 +/- 0.4 at all stages of the breeding season.

Effects of hormonal manipulation on the resumption of postpartum reproductive activity in Texel ewes.

Three experiments were conducted on Texel ewes to study the influence of prostaglandin F(2,alpha) (PGF(2 alpha)), prolactin (PRL), estradiol (E(2)), and gonadotrophin releasing hormone (GnRH) on postpartum reproductive activity. In Experiment 1, oral administration of indomethacin (25 to 50 mg/day/ewe) from Day 3 post partum to the first detected estrus inhibited plasma 13, 14-dihydro-15-keto, PGF(2 alpha) (PGFM) concentrations (P < 0.0001). This treatment resulted in an earlier rise in the frequency and amplitude of luteinizing hormone (LH) pulses and a resumption of estrous behavior (P < 0.05), while ovarian activity estimated by progesterone (P(4)) concentrations resumed to the same extent in treated ewes and controls. Bromocriptine treatment (2.5 mg/day/ewe) reduced plasma PRL levels (P < 0.0001) but had no effect on ovarian activity as evidenced by P(4) and resumption of estrus or on either the frequency or amplitude of the LH pulse. In Experiment 2, a single injection of GnRH agonist (42 mcg of buserelin/ewe) on Day 16 post partum resulted in an abrupt elevation of plasma LH concentrations; mean LH values were 18 to 27 times higher when compared with those of the control ewes. Two days after this treatment, ovulations occurred in 5 of the treated ewes and in 2 of the control ewes. This induced ovarian activity was not associated with estrous behavior; however, after an adequate subsequent luteal phase all the treated ewes displayed estrus, the resumption of estrus thus being earlier in treated than in control ewes (P < 0.01). In Experiment 3, E(2) supplementation from Day 16 to Day 28 post partum increased the number of LH pulses per 6 hours in suckling ewes (P < 0.05) and induced earlier resumption of estrus in dry ewes but not in suckling ewes (P < 0.01). Luteal function was detected about 5 and 8 days after the insertion of E(2) implants in 4 dry ewes and in 2 suckling ewes, respectively.

Changes in the pattern of prolactin secretion in the postpartum ewe and daily rhythms of prolactinemia during different reproductive states.

In order to study the relationship between plasma prolactin (PRL) levels and the secretion pattern of this hormone, blood samples were performed every 10 minutes for six hours on dry, restricted and unrestricted suckling Texel ewes on Days 7 and 30 after October or November lambings. On Day 7 post partum, mean plasma PRL concentration, frequency and amplitude of PRL pulses were higher in unrestricted suckling ewes than those in other groups (P < 0.01). Moreover, mean plasma PRL concentrations irrespective of the managing mode were positively related to PRL pulse frequency on Day 7 (R = 0.74, P = 0.0001) and Day 30 (R = 0.43, P = 0.087) post partum. In a second experiment, hourly blood sampling was performed for a period of 24 consecutive hours on cyclic ewes at the luteal or estrual phase (in December), and on seasonal anestrius (in March) or pregnant (in May) ewes in order to study daily variations in PRL secretion. Day/night variations in prolactinemia were only detected in cyclic ewes in December (P < 0.05) not in seasonal anestrous or pregnant ewes in March or May. During the estrual phase of the cycle, the results clearly showed a circahemidian rhythm in PRL secretion with a significant increase at the beginning of daylight or darkness (P < 0.01).

Effects of suckling mode on endocrine control of reproductive activity resumption in Texel ewes lambing in July or November.

To study the effects of the overlapping of seasonal and lactation anestrus and the influence of the suckling mode on the resumption of reproductive activity in Texel ewes, two experiments were carried out after the July and November lambings. The frequency and the duration of suckling decreased with the age of the lambs, and the suckling intensity in the lambs that were allowed to suck unrestrictedly was three times higher than in lambs restricted to sucking three times a day. The overlapping of seasonal anestrus and lactation delayed the resumption of ovarian and estrus activity, but no difference was observed between dams suckling single lambs and twins. The resumption of ovarian and estrus activity was much earlier after the November lambing than after the July lambing. Weaning after the November lambing shortened the interval between parturition to first estrus but not to the first luteal function. The reduction of suckling intensity by suckling management had no effect on the resumption of ovarian and estrus activity. In early postpartum, suckling inhibited the luteinizing hormone (LH) pulsatile secretion and consequently the first LH discharge. However, the earlier restoration of gonadotropin stimulation in dry ewes was not immediately followed by ovarian activity. The suckling inhibition may be due to a temporary disturbance in hormonal balance, the rise in prolactin (PRL) and cortisol secretions. Plasma estradiol 17beta (E2) concentrations were higher in dry than in suckling ewes in early postpartum. Follicle stimulating hormone (FSH) secretion was not involved in the process of delaying the resumption of reproductive activity after lambing.

Effects of continuous long-term intravenous infusion of long-chain fatty acids on feeding behaviour and blood components of adult sheep.

Albumin complexes of palmitic, stearic and oleic acids were continuously infused intravenously into adult sheep to investigate their effects on feeding behaviour and blood components. They were compared with saline infusions as during a preliminary test albumin alone showed no influence on voluntary food intake. Oleic and palmitic acid infusions induced a significant decrease of the voluntary food intake, relative to saline infusion. Food intake also decreased in experimental sheep when stearic acid was used, though not significantly. For all the tested fatty acids, blood beta-hydroxybutyrate and acetoacetate concentrations, plasma lipid content and the distribution of fatty acids in triacylglycerol remained unchanged. Oleic acid infusion induced the following significant changes of blood composition between control and experimental sheep: increased free fatty acid (FFA) concentration, increased oleic acid and decreased palmitic acid proportions in FFA and an increased amount of oleic acid balanced by a decreased linoleic acid content in phospholipids and cholesteryl esters. The only significant changes observed with the palmitic acid infusion were: decreased linoleic acid content in phospholipids and cholesteryl esters and increased oleic acid content in cholesteryl esters. The stearic acid infusion induced a decrease of stearic acid quantity in phospholipids and a corresponding increase in oleic acid in phospholipids and cholesteryl ester. It is suggested that these long-chain fatty acids might affect the long-term control of voluntary food intake in ruminants.

Fluctuations in the plasma levels of the follicle-stimulating hormone during estrous cycle, anestrus, gestation and lactation in the ewe: evidence for an endogenous rhythm of FSH release.

Two experiments were carried out to analyse FSH secretion in the ewe. The first was a long-term study during which four ewes under controlled photoperiods were checked for plasma concentrations of FSH twice daily for a period of 16 months. They were successively anestrous, cycling, gestating and lactating. The results suggested that an endogenous secretion rhythm of FSH persisted throughout each of the physiological states of the ewes. The periodic cycles of FSH production lasted about 5 days during anestrus and gestation but extended to about 6 days during estrus. One of the three waves of secretion we noted during one cycle was represented by the two periovulatory surges, the first coincident with the LH peak, the second occurring 30-40 h later. Plasma levels of FSH were similar during estrous cycles and anestrus, whereas the FSH secretion decreased gradually throughout gestation. During lactation, large differences were observed among animals before the recovery of cyclic ovarian activity. The second experiment consisted of frequent blood sampling (every ten minutes) of eight ewes for 6 hours during anestrus. FSH was secreted differently compared to LH. No pulsatile production of FSH was demonstrated and no increase in FSH levels was seen at the time of the episodic LH surge.

Effects of long-term ad libitum feeding on plasma lipid components and blood glucose, beta-hydroxybutyrate and insulin concentrations in lean adult sheep.

The long-term changes in plasma lipid composition and blood glucose, beta-hydroxybutyrate and insulin concentrations in experimental ad libitum fed adult Suffolk sheep were studied and compared with those of control sheep receiving a maintenance diet. The results were interpreted in relation to changes in food intake, body weight and body composition. Plasma lipid distribution was slightly influenced by the intake level and the fattening state. Compared to the maintenance diet, ad libitum feeding induced a decrease in the concentration of plasma free fatty acids (FFA); when the food intake decreased, this FFA concentration increased gradually. The fatty acid composition of the lipid fractions was subject to variation in the long-term ad libitum fed sheep. Myristic and palmitic acids in FFA, palmitoleic acid in triglycerides and linoleic acid in all the lipid fractions showed decreasing values associated with reduced food intake in the fattening sheep. Stearic and oleic acids in FFA, phospholipids and cholesterol esters increased when body weight and body fat increased and food intake decreased; they showed stable values by the end of the experiment, when food intake and body weight and composition also stabilized. Throughout the test, blood glucose concentration remained unaffected. A stable blood beta-hydroxybutyrate concentration, 2 times the control level, was observed from the 4th to the 10th experimental week in the ad libitum fed sheep (phase 1). From the 14th to the 26th experimental week (phase 2), decreased values were associated with increasing body weight and decreasing food intake. From the 30th week on (phase 3), beta-hydroxybutyrate concentration stabilized at a level 50 p. 100 higher than in the control sheep. During the first 6 weeks of ad libitum feeding, the experimental sheep achieved the same plasma insulin concentration as the control sheep. Thereafter a 30-fold increase was noted. Moreover, fluctuations of between 20 and 1 600 microU/ml were observed in fattening phases 2 and 3. This experiment demonstrated that feeding adult sheep ad libitum induces complex interrelationships between food intake and fattening state on the one hand and several blood substances on the other. Other experiments are needed to clarify these interrelationships.

Influence of diet and prolonged fasting on blood lipids, ketone bodies, glucose and insulin in adult sheep.

In order to study changes in blood composition during feeding and prolonged fasting, six adult Suffolk ewes received three different diets during three periods of four weeks. Two intake levels (one and three times maintenance) and two roughage/concentrate ratios (100 and 14 p. 100 roughage) were compared according to a twofold latin square. Afterwards the animals were fasted for an 11-day period. The concentration of free fatty acids in the blood decreased when concentrate was added to the diet and when energy intake level, insufficient to meet maintenance requirements, was increased to just sufficient and then to excessive. Blood lipid and lipoprotein composition was not influenced by a change in diet. Supplying concentrate instead of roughage produced increases in the proportions of stearic acid in the triglycerides and free fatty acids and of linoleic acid in the phospholipids and cholesteryl esters and decreases in the proportions of oleic acid in the free fatty acids and of oleic and palmitic acids in the triglycerides and cholesteryl esters. When increasing intake level, the proportion of linoleic acid in the phospholipids and cholesteryl esters was further increased. Increased insulinemia was also observed with concentrate intake, particularly when the concentrate was fed ad libitum. Differences in blood composition resulting from the dietary treatments rapidly disappeared with fasting. After an initial decrease, blood ketone bodies increased and the beta-hydroxybutyrate/acetoacetate (BHB/AA) ratio decreased. Glycemia decreased initially and then stabilized; the concentration of free fatty acids increased from the first day onwards and the proportion of free fatty acids in total blood lipids also increased. An increased proportion of oleic acid in the free fatty acids and triglycerides was compensated for by a decrease of saturated acids. A decrease in the level of polyunsaturated fatty acids in the phospholipids and cholesteryl esters was balanced by increased levels of saturated and oleic acids. The high density lipoprotein/low density lipoprotein ratio (HDL/LDL) decreased and insulinemia showed a strong decrease during the first day. Blood composition seemed to stabilize after-5-6 days of fasting, except for the ketone bodies which increased again after 10 days of the fast and for glycemia which increased after 5-6 days of the fast. The influence of previous dietary treatment on changes in blood composition during fasting clearly depended on the body conformation of the animal. When animals showed similar body conformation, no differences in the blood changes were observed during fasting. But when lean and fat animals were compared, extensive differences were found in the blood composition after the food was removed.