Article

Number of oocytes obtained from cows by OPU in early, but not late lactation increased with plasma insulin and estradiol concentrations and expression of mRNA of the FSH receptor in granulosa cells

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Abstract

The effects of lactation stage and hormonal profile on the quality and quantity of oocytes and the gonadotrophic sensitivity of granulosa cells (GC) from small antral follicles obtained by sequential aspirations from ovaries of high producing dairy cows were examined. Cows in late lactation (263(+/- 60) days postpartum) and 98(+/- 16) days pregnant in positive energy balance (EB) showed no significant changes in plasma concentrations of estradiol, progesterone, insulin, IGF1 or expression of mRNA of the FSH receptor in GC from small antral follicles during the 49 days experimental period. There were no changes in the number and quality of oocytes obtained from each aspiration. In cows in early lactation (72.8 +/- 6 days postpartum), plasma insulin concentrations increased and were positively correlated with plasma estradiol concentration. Due to the sequential aspirations progesterone blood concentrations were low in early lactation cows. Expression of mRNA of the FSH receptor increased in GC from small antral follicles of early lactation cows together with the number of oocytes obtained with aspiration sessions. No differences were found in morphological quality or function between oocytes obtained from small antral follicles from cows in early or late lactation. In early, but not late lactation, the number of oocytes was correlated with both insulin and E2 plasma concentrations. Improved EB and sensitivity of GC to FSH may be involved in oocyte recruitment in early lactation.

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... Many studies have hypothesized that IGF-1 might mediate the effects of nutrition on reproduction (Zulu et al. 2002a;Argov et al. 2004). Circulating concentrations of IGF-1 are highly related to energy balance in postpartum cows (Beam and Butler 1997;Velazquez et al. 2008), and its concentrations during week 1-2 postpartum are related to conception rate (Patton et al. 2007). ...
... Leroy et al. (2005Leroy et al. ( , 2006 have shown that the modification of in vitro maturation conditions of culture of oocytes to mimic negative energy balance environment impaired subsequent in vitro cleavage or blastocyst rates. However, some authors failed to observe a direct relationship between oocyte quality, energy balance (Kendrick et al. 1999), diet energy level (Walters et al. 2002b) or lactation stage (Argov et al. 2004). Moreover, a direct relationship between oocyte quality on the breeding period and fertility itself on the same animals has never been investigated yet to our knowledge. ...
... The collection results are lower than those observed in commercial stations using OPU for embryo production (Merton et al. 2003), but these stations commonly use heifers or dry cows, and these animals are known to produce more oocytes than lactating cows (Rizos et al. 2005). Cleavage and blastocyst rates were similar to observations by Snijders et al. (2000; oocytes recovered after slaughter between 125 and 229 days in milk), Argov et al. (2004; OPU twice weekly between 50 and 100 days in milk) and Rizos et al. (2005; OPU twice weekly between 40 and 75 days in milk). In the present study, all the oocytes recovered (even those graded 3 and 4, scale 1-4) underwent in vitro maturation, fertilization and culture. ...
Article
The aim of this study was to determine whether postpartum variations of plasma IGF-1 and IGFBP concentrations, oocyte production and quality were related to parity and subsequent conception rate in Holstein dairy cows. Holstein dairy cows [10 primiparous (PP) and 22 multiparous (MP)] were allotted in six batches and sampled once weekly between calving and oestrous synchronization treatment started at 71.2 ± 2.0 days postpartum. During the 3 weeks before treatment, ovum pick-up (OPU) was performed twice weekly. Oocytes were scored on a 4-point scale, and oocytes from OPU1, 3 and 5 were fertilized in vitro. Seventeen cows became pregnant after first and second AI and were considered as fertile (F), while the others were considered to be subfertile (SF). Logistic regression was carried out to investigate the relationships between repeated measurements and fertility including parity and batch effects in the models. Likelihood of fertility significantly increased when plasma urea and IGFBP-3 concentrations decreased and was higher in PP compared with MP cows. There was a trend for fertility to increase when plasma IGF-1 concentrations increased (p = 0.07). In vitro cleavage and development rates were similar between SF and F cows (46.4% and 28.3% in SF vs 55.0% and 22.1% in F). Parity had an effect on plasma IGF-1 concentrations (PP: 61.65 ± 2.67 vs MP: 41.63 ± 5.81 ng/ml, p < 0.001), mean number of follicles aspirated per session (PP: 5.7 ± 1.3 vs MP: 9.5 ± 0.8, p < 0.05) and fertility (PP: 8/10 = 80% vs MP: 9/22 = 41%, p < 0.05) but not on the number of oocytes recovered per session nor their quality. In conclusion, postpartum plasma urea and IGFBP-3 concentrations, but not oocyte production and quality before breeding, were related to subsequent conception rate in our experimental design. Parity had a significant effect on energy status, follicular growth and fertility and needs to be considered when investigating relationships between nutrition and reproduction.
... Oocytes were evaluated under a stereomicroscope at a magnification of 100· and were recorded as normal or abnormal according to the morphological criteria for suitability for in vitro culture, which are based on the number of surrounding cumulus cell layers and cytoplasmic organization (Kruip et al. 1994;Agrov et al. 2004). Oocytes were defined as normal if they had a compact cumulus consisting of three or more cell layers, an intact zona pellucida and a uniformly granular cytoplasm. ...
... The oocyte quality and the measured metabolites were compared between repeat breeder and early lactation cows. This comparison was based on findings that energy deficit does not influence follicular development during the early stage of lactation (Beam and Butler 1997;Diskin et al. 2003), and that oocyte quality in normal cows did not differ between early and late stages of lactation (Agrov et al. 2004). The yield of oocytes and proportion of normal oocytes in the early lactation cow agreed with findings from normal cows in other studies (Simon et al. 1993;Gibbons et al. 1994;Bungartz et al. 1995;Hill 1995). ...
... The yield and oocyte quality were found to differ between the repeat breeders and the early lactation cows. The mean number of oocytes and the proportion of normal oocytes per repeat breeder at 347.3 ± 11.5 days postpartum were significantly lower than those in the early lactation cows, in contrast to findings from normal cows (Agrov et al. 2004). The percentage of abnormal oocytes in repeat breeders was 52.5, higher than in studies on normal cows (Simon et al. 1993;Gibbons et al. 1994;Bungartz et al. 1995;Hill 1995) and is in agreement with data from earlier studies on repeat breeder cows (Stroud et al. 1991;Dolezˇel et al. 1998). ...
Article
The objectives of the study were to evaluate the morphological quality of oocytes in repeat breeder and early lactation cows and to determine the possible associations between the quality of oocytes and a range of blood metabolites. Oocyte quality and a range of metabolites were compared between 29 repeat breeder and 13 early lactation cows. The yield of oocytes from the repeat breeders was lower than that from the early lactation cows (4.4 ± 0.2 vs 5.4 ± 0.6, p < 0.05). Percentages of abnormal oocytes for the repeat breeders and the early lactation cows were 52.5% and 37.9%, respectively (p < 0.001). An excess of abnormal oocytes to normal was found in 55.2% of the studied repeat breeders (65.8% vs 34.2%, p < 0.05). Total protein, glucose and aspartate aminotransferase did not differ (p > 0.05) between the repeat breeders with an excess of abnormal oocytes (81 ± 1.0 g/l, 3.5 ± 1.0 mmol/l and 68.5 ± 3.7 U/l), those with the prevalence of normal oocytes (84 ± 1.0 g/l, 3.6 ± 0.1 mmol/l and 73.2 ± 3.5 U/l) and the early lactation cows (83 ± 2.0 g/l, 3.7 ± 0.1 mmol/l and 74.5 ± 3.6 U/I). The repeat breeders with an excess of abnormal oocytes had higher (p < 0.05) urea (5.2 ± 0.2 mmol/l) level than in those with the prevalence of normal oocytes (4.8 ± 0.2 mmol/l) and the early lactation cows (4.7 ± 0.2 mmol/l). A trend for higher total cholesterol and lactate dehydrogenase activity was found in the repeat breeders with an excess of abnormal oocytes. In conclusion, it is suggested that possible causes of repeat breeding in dairy cows may include impaired oocytes. An excess of abnormal oocytes in the repeat breeder cows was associated with elevated blood plasma levels of urea.
... The above questions can be tested in part by treating oocyte donor cows with ROH, as those oocytes aspirated from the follicle would develop free from the effects of circulating ROH when cultured in vitro up to the blastocyst stage. We have earlier reported that the presence of RA in in vitro maturation (IVM) medium enhanced oocyte maturation, embryo development ( Duque et al. 2002a, Gó mez et al. 2003, 2004, Hidalgo et al. 2003) and pregnancy rates ( Hidalgo et al. 2003). Since ROH has no effect during IVM at physiological temperature ( Duque et al. 2002b, Lawrence et al. 2004) it was necessary to perform an overexposure of the oocytes to retinoid by using RA. ...
... The ovarian response to FSH and LH can also be modulated by RA. In response to this retinoid, stimulation of steroidogenesis (Bugavandoss & Midgley 1988, Graves-Hoagland et al. 1988, and restricted expression of FSH receptor (FSHr) in rat granulosa cells has been reported ( Minegishi et al. 1996Minegishi et al. , 2000), which could affect oocyte growth and in turn the outcome of OPU in the cow ( Argov et al. 2004). In the porcine, the effects are controversial, as exposure of immature granulosa cells to RA inhibited expression of LH receptor (LHr) by downregulation of c-Fos mRNA (Hattori et al. 2000). ...
Article
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Retinoids have been shown to enhance developmental competence of the oocyte in cattle, sheep and pigs. In this study we investigated whether exogenous retinol stimulates the bovine oocyte during its intrafollicular growth and the time limits of exposure to exogenous retinol. In addition, we also determined the efficiency of ovum pick-up techniques in combination with retinol treatment and the viability of embryos after transfer to recipients. In Experiment 1, heifers were injected with retinol or vehicle, and concentrations of retinol in the blood were analysed on Day 0 (prior to injection), Day 1 and, together with follicular fluid, Day 4. Blood retinol increased by Day 1 and cleared on Day 4, but retinol remained higher within the follicle. In Experiment 2, oocyte donors were injected weekly with retinol or vehicle four times during a twice-per-week cycle of eight recovery sessions (starting 4 days before the first session), followed by a second eight-session cycle without treatment. Oocytes recovered were fertilized and cultured in vitro. Retinol treatment yielded higher numbers of low-quality oocytes throughout, although retinol measured during cycles did not change. Total oocytes, and morulae and blastocyst rates, increased during the first five sessions following treatment with retinol. As previously shown with oocytes from slaughterhouse ovaries, retinoic acid stimulated blastocyst development. Following transfer to recipients, blastocysts from oocytes exposed to retinol were unable to establish pregnancy. Our study confirms the existence of an effect of retinol on the intrafollicular oocyte in the cow and provides evidence regarding the teratogenic effect of retinol.
... Oocyte quality deteriorates due to postpartum nutritional depression [5], with impairments potentially being one of the main causes of low fertility in high-producing cows [6]. Previous studies reported no significant differences in oocyte morphology [7] or developmental competence to the blastocyst stage [8,9] between different lactation stages, while another showed impaired oocyte morphology at approximately 100 days in milk (DIM) than at 30 DIM [10]. These findings suggest that differences in the oocyte quality of cows between different lactation stages may not be readily detectable by morphology and developmental competence to blastocysts. ...
Article
Impaired oocyte quality is one of the main causes of low fertility in modern high-yielding dairy cows. One of the potential factors of the impaired oocyte quality is the effects of free fatty acids (FFA). In fact, high FFA supplementation to culture media exacerbated oocyte developmental competence in vitro. Meanwhile, artificially induced high blood FFA levels in heifers did not affect the lipid composition of oocytes in vivo; however, the oocyte lipid profile of postpartum cows has not yet been investigated. Therefore, the profile of lipids involved in energy metabolism, including FFA and triacylglycerols (TAG), and their relationship between plasma and oocytes were compared among cows at different lactation stages. Heifers were used as a control group that was not affected by lactation. Plasma and oocytes were collected from heifers (n = 4) and 14 Holstein cows categorized to the early lactation stage: 25–47 days in milk (DIM) (n = 6), peak lactation stage: 61–65 DIM (n = 4), and middle lactation stage: 160–202 DIM (n = 4). The FFA and TAG profiles of plasma and oocytes were examined by liquid chromatography mass spectrometry. Plasma FFA positively correlated with oocyte TAG (P < 0.05). Plasma FFA and oocyte TAG were significantly higher in cows in the early lactation stage than in heifers (P < 0.05), while the peak and middle lactation stage groups had intermediate levels. The proportion of oleic acid in plasma increased concurrently with elevations in total FFA, while the compositions of oocyte FFA and TAG fatty acyls were constant regardless of plasma FFA concentration or oocyte TAG content. The present results suggest that high postpartum plasma FFA concentrations affect the quantity of oocyte TAG. Taken together with the adverse effects of high FFA concentrations on oocyte developmental competence in vitro, oocyte quality in postpartum cows may be impaired due to high circulating FFA concentrations. These results provide a more detailed understanding of the effects of postpartum high circulating FFA concentrations on the low fertility of cows.
... e process of follicular growth, the physiochemical properties of the blood–follicle barrier transform thoroughly, suggesting that the oocyte's environment undergoes compositional changes (Edwards, 1974; Wise, 1987; Gosden et al., 1988). In addition, the active transport mechanisms through the follicular wall may also alter during follicular growth. Argov et al. (2004), for example, recently demonstrated that while lipoproteins are predominantly internalized by endocytosis in small follicles, this is not the case in large follicles, in which circulating lipoproteins contribute their cholesterol esters by selective uptake and without internalization of the lipoprotein as such. Hence, to further investi ...
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Unsatisfactory reproductive performance in dairy cows, such as reduced conception rates, in addition to an increased incidence of early embryonic mortality, is reported worldwide and has been associated with a period of negative energy balance (NEB) early post partum. Typically, NEB is associated with biochemical changes such as high non-esterified fatty acid (NEFA), high β-hydroxybutyrate (β-OHB) and low glucose concentrations. The concentrations of these and other metabolites in the follicular fluid (FF) of high-yielding dairy cows during NEB were determined and extensively analyzed, and then were replicated in in vitro maturation models to investigate their effect on oocyte quality. The results showed that typical metabolic changes during NEB are well reflected in the FF of the dominant follicle. However, the oocyte seems to be relatively isolated from extremely elevated NEFA or very low glucose concentrations in the blood. Nevertheless, the in vitro maturation models revealed that NEB-associated high NEFA and low glucose levels in the FF are indeed toxic to the oocyte, resulting in deficient oocyte maturation and developmental competence. Induced apoptosis and necrosis in the cumulus cells was particularly obvious. Furthermore, maturation in saturated free fatty acid-rich media had a carry-over effect on embryo quality, leading to reduced cryotolerance of day 7 embryos. Only β-OHB showed an additive toxic effect in moderately hypoglycemic maturation conditions. These in vitro maturation models, based on in vivo observations, suggest that a period of NEB may hamper the fertility of high-yielding dairy cows through increased NEFA and decreased glucose concentrations in the FF directly affecting oocyte quality. In addition to oocyte quality, these results also demonstrate that embryo quality is reduced following an NEB episode. This important observation may be linked to the typical diet provided to stimulate milk yield, or to physiological adaptations sustaining the high milk production. Research into this phenomenon is ongoing.
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Similarities between the bovine female and women in terms of reproduction and fertility, such as oogenesis, folliculogenesis and reduced fertility with advanced age make the bovine a valuable model for the study of ovarian function and dysfunction as well as reproductive aging in women. The aim of the present work was to investigate the influence of donor age on follicle numbers, yield and quality of COCs obtained by repeated OPU and on the developmental competence in vitro after oocyte maturation in vitro versus in vivo. Further, the ability of oocytes from different age classes to reprogram nuclei of bovine fetal fibroblasts was studied. Since the individual is a major factor influencing parameters of fertility and results of ART in both humans and cattle, the present study used in parts the same animals to rule out inter-individual effects on the response to one or the other approach. Experiment 1 investigated the effect of donor age in non-superstimulated German Simmental heifers (n = 12, 14 months at the beginning of the experiments), young cows in their first lactation (n = 8, 2-4 years) and old cows (n = 8, 10-15 years). A total of 38 OPU sessions were performed in two experimental periods on independent sets of animals from all age classes: 5/5/5 (32 sessions) and 7/3/3 (6 sessions). In spite of a marked influence of the experimental period, a number of parameters were also significantly affected by donor age. The total number of follicles increased with age (P<0.001) and similarly, the total number of cumulus-oocyte-complexes (COCs) recovered was higher in old cows than in young cows and heifers (P<0.01). Evaluation of the follicle size distribution revealed higher percentages of small and lower proportions of medium size follicles in old cows while the COC quality was not affected. Further, the least squares mean blastocyst rate obtained after IVM and IVF of COCs was significantly (P<0.05) higher in young cows (17.4%) than in heifers (5.1%). The culture of oocytes after SCNT resulted in similar cleavage and morula rates. Experiment 2 investigated the interaction of donor age and FSH-superstimulation using the set of animals from experimental period 1 (n = 5 per age class). During 9 OPU sessions performed in 5-week intervals proportions of in vivo matured oocytes between 65.0% and 75.1% on average were obtained. FSH-superstimulation significantly increased the numbers of follicles aspirated (P<0.001) and COCs recovered (P<0.001) as compared to non-superstimulated donors. Follicle size was shifted to less small and more medium and large follicles. Further, there was a marked positive effect of FSH-superstimulation on cleavage and blastocyst rates in all age classes (P<0.001). Importantly, the developmental deficit of heifer COCs after IVM was rescued by FSH-treatment and in vivo maturation.
Thesis
L’Ovum Pick-Up (OPU) et la production d'embryons in vitro représentent une voie d’accélération du progrès génétique par la voie femelle. Ces techniques impliquent en autre la collecte d’ovocytes de qualité pour produire des embryons transférables. La qualité des ovocytes et les taux de développement embryonnaire in vitro sont impactés par la nutrition des donneuses avant OPU, les effets étant médiés par plusieurs métabolites et hormones impliqués dans la régulation du métabolisme énergétique. Les niveaux plasmatiques d'insuline et d’IGF1 accrus sont corrélés aux niveaux des apports énergétiques chez les génisses laitières. Les régimes augmentant les concentrations d'insuline influencent négativement la qualité des ovocytes. Néanmoins, une augmentation des concentrations d'insuline stimule en quelques jours le nombre des petits follicules. Ces résultats ont conduit à formuler l’hypothèse d’une modulation transitoire des concentrations d'insuline chez les donneuses d’ovocytes basée sur l'administration orale de propylène glycol (PG). En effet, le PG augmente les concentrations d'insuline et d’IGF-1 dans le plasma chez la vache au cours du post-partum. L’hormone antimüllérienne (AMH) est un marqueur endocrinien de la réserve de follicules ovariens sensibles aux gonadotrophines chez la vache. Il a été récemment établi que les concentrations plasmatiques d'AMH aident à prédire la réponse des donneuses d’embryons collectés in vivo. Dans cette thèse, nous avons étudié l'effet de l'administration de PG à court terme sur les niveaux d'insuline, la croissance folliculaire, la réponse à la stimulation de la croissance folliculaire au traitement FSH et la production d'embryons in vitro après OPU chez les génisses avec différents profils d’AMH circulante (haut H vs bas B). La relation entre la dose de PG chez les génisses laitières et la réponse à l'insuline a été établie, ainsi que les relations avec les concentrations plasmatiques d’hormones et métabolites du métabolisme énergétique. Puis, le nombre et la qualité morphologique des ovocytes et des embryons produits ont été déterminés chez les génisses donneuses ayant une alimentation restreinte. Enfin, les profils d'expression de gènes du système IGF dans les ovocytes et les cellules du cumulus et de gènes candidats de survie embryonnaire chez des blastocystes ont été évalués.Nos données ont montré que l'administration de PG chez les génisses laitières est associée à une élévation des concentrations plasmatiques d’insuline, d'IGF1 et de glucose et à une diminution de celles de β-hydroxybutyrate (BHB) et d’urée. Les concentrations d’IGF1 dans le liquide folliculaire ont augmenté. De plus, l’administration de PG a été associée à un nombre plus élevé de petits follicules (2- 3mm) le 2ème jour du cycle œstral par rapport au lot contrôle. Cet effet positif a été maintenu sur le nombre de follicules moyens (4-8mm) le 5ème jour du cycle, après stimulation de la croissance folliculaire dans le groupe AMH H. En outre, une augmentation significative du taux de blastocystes de qualité 1 après 7 jours de culture in vitro (exprimés en pourcentage d'ovocytes fécondés) a été observée chez les génisses du lot PG par rapport au lot contrôle. L’administration de PG a conduit au développement d’un nombre plus élevé de blastocystes épanouis le 7ème jour du cycle par rapport au contrôle, uniquement dans le groupe AMH H. De plus, le PG a modifié les profils d'expression des gènes du système IGF dans les cellules du cumulus et des gènes de survie étudiés chez les blastocystes. Par conséquent, nos données montrent que l'administration de PG a amélioré la production et la qualité des embryons in vitro, peut-être en raison des modifications du système IGF1 induites par le PG en début de vague folliculaire. Celles-ci pourraient moduler l'environnement folliculaire et impacter l’expression des gènes y compris jusqu'au stade blastocyste, l’effet étant cependant plus marque chez les génisses a profil haut d’AMH.
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For about three decades, transvaginal ultrasound-guided oocyte retrieval (OPU, ovum pick-up) has been successfully adapted from human reproductive medicine to the use in cattle and later on in the horse. Over time, it turned out to be a reliable and minimally invasive method to collect (immature) oocytes from genetically high valuable donors on a repeated basis. While a large part of the success of this procedure relies on the availability of a reliable in vitro embryo production system, a major prerequisite remains the collection of good-quality oocytes. The current chapter will focus specifically on oocyte retrieval technology. Following a detailed description of OPU equipment, the technical and biological factors affecting oocyte retrieval in living donors are discussed extensively with particular interest on the need of donor preparation by hormonal stimulation. Attention will also be given to donor health issues related to repeated oocyte retrieval. Finally, a state of the art of OPU in the mare is given describing additional physiological aspects of the equine oocyte and embryo implying additional challenges both for oocyte retrieval and in vitro embryo production.
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Fertility in high yielding dairy cows is declining, and there is increasing evidence to presume that oocyte and embryo quality are major factors in the complex pathogenesis of reproductive failure. In this report we present an overview of possible mechanisms linking negative energy balance (NEB) and deficiencies in oocyte and embryo developmental competence; specifically, in the high producing dairy cow. Changes in follicular growth patterns during a period of NEB can indirectly affect oocyte quality. The endocrine and biochemical changes, which are associated with NEB, are reflected in the microenvironment of the growing and maturing female gamete, and likely result in the ovulation of a developmentally incompetent oocyte. Even after an oocyte is successfully ovulated and fertilized, a full-term pregnancy is still not guaranteed. Inadequate corpus luteum function, associated with reduced progesterone, and probably also low insulin-like growth factor concentrations, can cause a suboptimal microenvironment in the uterus that is incapable of sustaining early embryonic life. This may partly account for the low conception rates and the high incidence of early embryonic mortality in high yielding dairy cows.
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The nutritional status of a cow is a key factor in the regulation of both follicle growth and oocyte quality. In this study, the effect of diets designed to increase circulating insulin and insulin-like growth factor I (IGF-1) concentrations on steroid production by granulosa cells in vitro was examined to analyse the mechanisms through which these changes occur. Hereford X Friesian heifers (n = 24) were offered maintenance or twice maintenance diets during the experimental period 0 7 days). Circulating concentrations of FSH did not differ between the two dietary groups, whereas insulin and IGF-I concentrations showed significant diet x day of oestrous cycle interactions. Ovaries were collected on day 3 of the first follicle wave after synchronization of oestrus. Granulosa cells were isolated from small (1-4 mm) and medium-sized (4-8 mm) follicles and cultured in the presence of long R3-IGF-1 or bFSH or both. After 4 days in culture, granulosa cells isolated from small follicles, but not medium-sized follicles, collected from cattle offered the twice maintenance diet secreted significantly higher (P < 0.05) amounts of oestradiol compared with granulosa cells collected from cattle offered the maintenance diet. The effect was apparent in either the presence or absence of FSH and long R3-IGF-I. This nutritional effect on aromatase activity in granulosa cells was not apparent after day 6 of culture. There was no effect of diet on progesterone production by granulosa cells after 4 or 6 days of culture. These results support the hypothesis that dietary-induced changes in circulating insulin and IGF-I concentrations have a direct effect on the steroidogenic potential of bovine granulosa cells from small follicles. The dietary-induced increases in aromatase activity in small follicles combined with the increased concentration of metabolic hormones are possible mechanisms through which short-term changes in nutrition may affect follicle dynamics.
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Genetic improvement of dairy cows has markedly increased milk yield over the last three decades. Increased production has been associated with reduced conception rates (66% in 1951 versus 40 to 50% since 1975). Because conception rate in dairy heifers has remained higher, the metabolic demands of higher production may be related to the decline in reproductive performance in cows. During early lactation, increasing dietary intake fails to keep pace with rising milk production. The resultant negative energy balance and rate of mobilization of body reserves appear directly related to the postpartum interval to first ovulation and lower conception rate. Delays in the onset of normal ovarian activity, thus limiting the number of estrous cycles before breeding, may account for the observed decrease in fertility. Negative energy balance probably acts similarly to undernutrition and may manifest in delayed ovarian activity by impinging on pulsatile secretion of LH. Lower availability of glucose and insulin may also decrease LH pulsatility or limit ovarian responsiveness to gonadotropins. Alternatively, release of endogenous opioids in association with increasing feed intake or other lactational hormone responses may provide neural or pituitary inhibition of the pulsatile LH production that is requisite for ovarian follicular development.
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The effects were determined of dietary Ca soaps of fatty acids fed to cows to 120 DIM on milk yield, BW, peripheral concentrations of reproductive hormones, and fertility in primiparous and multiparous cows. Milk yield was increased in primiparous and multiparous cows fed Ca soaps, and milk fat and protein yields were enhanced. Body weight losses were greater for all cows fed Ca soaps, and this trend was greater and longer lasting in primiparous cows than in multiparous cows. Plasma triglyceride concentrations were elevated in multiparous cows, but FFA tended to be higher in primiparous cows fed Ca soaps. Conception rate at first AI was lower for primiparous cows fed Ca soaps (33%) than for controls (74%), but differences were not significant for later AI or between multiparous groups. No differences were apparent for plasma progesterone or estradiol in the luteal or follicular phases preceding the first AI, and differences in mean luteinizing hormone concentrations were small in a 6-h window in the follicular phase. The differences in conception rate at first AI in primiparous cows could not be explained on the basis of changes in peripheral hormone concentrations. The enhanced negative energy balance in primiparous cows fed Ca soaps apparently was related to the decrease in their conception rate.
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The euglycemic clamp technique was used to determine the effect of bovine growth hormone-releasing factor on glucose kinetics and on the response to insulin of 4 dairy cows during early lactation (mean, 35 d postpartum) and of 6 dairy cows during late lactation (mean, 194 d postpartum). Beginning 3 d prior to experiments, cows were injected twice daily with either saline or 2.5 micrograms/kg of growth hormone-releasing factor. On the day of the experiment, saline or the releasing factor (0.0119 microgram/kg per min) was infused into each cow for 5 h. After a basal period, insulin was infused at 1 and then at 6 mU/kg per min; plasma glucose concentrations were maintained at basal concentrations by the infusion of normal glucose. Growth hormone-releasing factor reduced the glucose infusion rate that was required to maintain euglycemia during the insulin infusions during late lactation but had no effect during early lactation. During the insulin infusions of the late lactation experiment only, the rates of glucose appearance, disappearance, and metabolic clearance were lower when plasma growth hormone was elevated. The results demonstrated that elevated concentrations of growth hormone decreased the responsiveness of peripheral tissues to high concentrations of insulin during late lactation but apparently had little effect during early lactation.
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Follicles were monitored daily by ultrasound and blood samples for FSH assay were collected daily from eight heifers from day 90 of pregnancy to the emergence of the first postpartum follicular wave. Follicles > or = 6 mm in diameter emerged in groups or waves in each heifer (P < 0.005). Follicular waves developed rhythmically throughout pregnancy, except that follicles > or = 6 mm were not detected during the last 21.6 +/- 2.4 (mean +/- SEM) days of pregnancy. The characteristics of the first follicular wave after day 90 were similar to previous reports for days 10-100. However, between months 4 (days 90-119) and 5, there was a decrease (P < 0.05) in monthly means for maximum diameter (mm) of largest (21.1 +/- 0.5 versus 9.5 +/- 0.5) and second largest (8.0 +/- 0.3 versus 6.9 +/- 0.2) follicles, duration of the interwave interval (8.1 +/- 0.4 versus 6.6 +/- 0.3 days), and number of follicles per wave (3.7 +/- 0.4 versus 2.5 +/- 0.4). Averaged over all follicular waves during months 4-9, the concentrations of FSH normalized to the emergence of a follicular wave increased (P < 0.05) over the 3 days before emergence, reached peak values on the day of emergence of the future dominant follicle at 4 mm, and decreased (P < 0.05) over the 3 days following emergence. Surges in FSH concentrations occurred throughout pregnancy, but during the last 30 days of pregnancy the number of surges was reduced and each heifer had one or two ineffective surges (no follicular wave detected). The temporal relationship between FSH surges and emergence of waves was closer (P < 0.01) than would be expected if the two events were independent. Surges of FSH occurred rhythmically even when there was no follicular response (no follicle > 5 mm). In association with waves in which the largest follicle reached > or = 10 mm compared with 6-9 mm, there was greater depression in the FSH nadir, longer intervals from FSH peak to nadir, and longer intervals between adjacent FSH peaks and adjacent waves.
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The objectives of this study were to develop a serum-free bovine granulosa cell culture system in which FSH-responsive estradiol production could be induced and maintained, and to use this system to evaluate the effects of FSH, insulin, and IGF-I on steroidogenesis and proliferation of bovine granulosa cells from different follicle size categories (< 4-, 4-8, and > 8-mm diameter). In the presence of FSH, granulosa cells from small follicles differentiated in vitro, and estradiol secretion increased with time (p < 0.01) so that by the end of the culture period it was similar to that of cells from large follicles. Granulosa cells from medium and large follicles secreted estradiol throughout the culture period. Cells cultured in plasma-coated culture wells had an increased proliferative response but had lower estradiol production compared to cells cultured under serum-free conditions (p < 0.01). Insulin promoted proliferation and estradiol production by granulosa cells from the three follicle-size categories (p < 0.01). Physiological concentrations of FSH induced proliferation and estradiol secretion (p < 0.01) by granulosa cells in a dose-responsive manner. The inclusion of IGF-I in the culture system enhanced proliferation and estradiol production (p < 0.01), even in the absence of gonadotropic support, demonstrating the gonadotropic characteristics of this growth factor. These results demonstrate the development of a relevant physiological culture system for bovine granulosa cells. This system will permit the detailed study of the key factors controlling the differentiation and proliferation of bovine granulosa cells.
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This study examined the mechanisms by which calcium soaps of fatty acids and bovine somatotropin (bST) affect production and reproduction of high producing cows. Calcium soaps of fatty acids were fed at 2.2% dry matter, and 500 mg of Zn-sometribove (Monsanto Inc., St Louis, MO) were injected subcutaneously every 14 d from 10 to 150 d in milk (DIM). Production of fat-corrected milk was increased by 3.5 kg/d when calcium soaps of fatty acids were fed, by 6.1 kg/d when bST was administered, and by 7.4 kg/d when calcium soaps of fatty acids were fed and bST was administered. Body weight was similar for cows on all treatments until 85 DIM after which cows that were treated with bST had lower body weights. Body condition scores decreased more for cows treated with bST and began increasing later and more slowly. Treatment with bST resulted in more cows that experienced first ovulation after 30 DIM, and more cows on the control treatment exhibited first estrus before 35 DIM. Days open were greater when bST was administered. After the first artificial insemination, conception rates were similar for cows on the control treatment and for cows fed calcium soaps of fatty acids; conception rates after the first artificial insemination were low for all cows treated with bST. Pregnancy rates at 120 and 150 DIM were decreased by bST. Number of DIM to first ovulation, number of DIM to first estrus, and days open were negatively correlated with glucose and cholesterol concentrations in plasma. Production of fat-corrected milk was correlated with days open and with concentrations of triglycerides in plasma, nonesterified fatty acids, and cholesterol. Increased production had different effects on reproduction when induced by calcium soaps of fatty acids or bST treatment. Some of the adverse effects of bST treatments were alleviated by calcium soaps of fatty acids.
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Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor beta superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development.
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The effect of fat and bovine somatotropin (bST) on preovulatory follicular hormones and lipids was evaluated by feeding cows for 150 d from parturition a control diet, a control diet plus 0.55 kg/d of calcium soaps of fatty acids, or a control diet with 500 mg of bST injected every 14 d. Fourteen days after a synchronized or natural estrus, cows were injected with a PGF2 alpha analogue; 48 h later, follicular fluid from all ovarian follicles > 8 mm was aspirated. Cows fed fat or injected with bST produced more milk and milk solids than did control cows, and cows on the bST treatment lost more body condition after calving than did cows on the other treatments. Both treatments changed the proportion of estradiol-active follicles (> 400 ng of estradiol/ml of follicular fluid) and the correlation between follicular fluid estradiol concentration and the total number large follicles per cow. In follicles aspirated between 60 and 90 DIM the percentage of estradiol-active follicles was 67, 40, and 0 for cows on the control, calcium soaps of fatty acids, and bST treatments, respectively. After 90 DIM, no differences existed between treatments in the percentage of estradiol-active follicles. Estradiol concentration in follicular fluid was correlated with DIM at follicle aspiration (r = 0.51). The proportion of oleic acid in free fatty acids in plasma at 50 DIM was lower in control cows and was lower in follicular fluid of estradiol-active follicles. Both calcium soaps of fatty acids and bST had a considerable effect on follicular development and activity and the composition of fatty acids in follicles.
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This study compared the effects of exogenous bovine somatotropin (bST) on the metabolism and ovarian activity of cows fed diets differing in ruminally degradable starch. Twenty-four multiparous and eight primiparous Holstein cows in early lactation were divided into four groups and fed diets containing 39% grain as steam-flaked sorghum or steam-rolled corn with or without bST for 90 d in a 2 x 2 factorial design. Flaked sorghum improved energy status of cows during early lactation, tending to increase plasma glucose and insulin. Administration of bST decreased plasma urea nitrogen and increased nonesterified fatty acids (NEFA). Plasma levels of beta-hydroxybutyrate (BHBA) and hepatic concentrations of triglycerides were not altered by treatments. Temporal changes in plasma glucose, urea nitrogen, NEFA, and BHBA were detected in a quadratic manner and insulin increased linearly with time, but treatments did not affect postpartum changes in these metabolites. There were greater decreases in body weight and net energy balance in cows on bST during the first 7 wk of treatment. Cows receiving bST took longer to reach the nadir of negative energy balance, and bST tended to delay the period to reach a positive energy balance. Follicular populations and incidence of cystic ovaries were not affected by treatments, but cows receiving bST had fewer double ovulations. Flaked sorghum increased plasma progesterone during the early luteal phase of the first two postpartum estrous cycles. Feeding more ruminally degradable starch improved the energy status and luteal activity of cows in early lactation.
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We conducted three experiments to test various protocols for synchronizing estrus, ovulation, or both before insemination of heifers. In experiment 1, 23 controls received two PGF2alpha injections; 23 heifers were treated like the controls plus a norgestomet implant for 8 d, with the second PGF2alpha injection 24 h before implant removal; and 23 heifers were treated like the previous group plus 100 microg of GnRH 54 h after the second PGF2alpha injection. Although norgestomet and GnRH altered some estrual characteristics, conception rates in experiment 1 (n = 69) and experiment 2 (278 heifers receiving the same treatments as those in experiment 1) generally were not different among treatments. Reproductive outcomes were not improved by adding norgestomet and GnRH to a standard PGF2alpha protocol. In experiment 3, control heifers received PGF2alpha and were inseminated after detected estrus or at 72 to 80 h after a second injection of PGF2alpha given 14 d after the first injection. Select Synch heifers, treated with GnRH either 6 or 7 d before PGF2alpha were inseminated after detected estrus, whereas Ovsynch heifers were treated like Select Synch heifers but also received a second GnRH injection approximately 36 h after PGF2alpha and were inseminated 18 h later. Estrus detection and pregnancy rates after Ovsynch were less than those of controls, whereas conception and pregnancy rates did not differ between control and Select Synch heifers. Therefore, the Select Synch protocol was equivalent to a standard PGF2alpha protocol, whereas Ovsynch was inferior to both of those protocols.
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Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.
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This review integrates information on follicular and hormonal physiology and epidemiology into a novel physiological model for regulation of the ovulation rate in lactating dairy cows. First, the basic mechanisms that produce a single ovulation are examined. Follicular deviation is a critical new concept in our understanding of selection of a single dominant follicle. Follicular deviation is characterized by an abrupt deviation in the growth rates between the two largest follicles when the future dominant follicle reaches a diameter of 8.5+/-1.2 mm (mean and SD). The mechanisms involved in this selection process are not completely defined but appear to involve acquisition of LH receptors on granulosa cells of the dominant follicle, increased estradiol production by the dominant follicle, and inhibition of circulating FSH concentrations. Second, lactation number and milk production were found to be critical epidemiological factors associated with increased ovulation rate and twinning in dairy cattle. Finally, high steroid metabolism is proposed as the critical link between high milk production and double ovulation. It is proposed that high milk production increases steroid metabolism due to increased blood flow to the digestive tract and subsequently to the liver. The liver represents the primary site of steroid metabolism, and blood entering the liver is cleared of steroids. At the time of selection of the dominant follicle, the normal increase in circulating estradiol concentrations and subsequent depression in circulating FSH is blunted due to estradiol metabolism. Thus, FSH remains elevated for a time sufficient to allow follicles to undergo the physiological changes necessary to proceed to ovulation.
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The effects of two methods of inducing low progesterone concentrations on the shape of the plasma progesterone curve and on follicular characteristics in lactating cows were studied. A low ascending progesterone curve was elicited by three PGF2alpha injections on d 3 to 4 of the estrous cycle; a low constant curve by induction of corpus luteum regression on d 6 and insertion of two progesterone-containing intravaginal devices from d 6 to 15 of the cycle. Plasma progesterone concentration was highest in the untreated control group, intermediate in low ascending group, and lowest in the low constant group. On d 15, both control and low ascending groups had one large healthy and one large atretic follicle, suggesting a turnover of follicular waves; in the low constant group, the presence of only one very large healthy follicle indicated follicular persistence. Estradiol concentration in the follicular fluid and its production by granulosa cells were highest in the low constant, intermediate in the low ascending, and lowest in the control group. Androstenedione concentration in the follicular fluid and its production by theca cells were higher in the low constant than in the low ascending and control groups. The results indicate that the low ascending progesterone curve affected follicular development and steroidogenesis differently from the low constant curve. We suggest that the low ascending curve mimics the effects of naturally occurring low plasma progesterone concentrations better, and it might, therefore, be used as a model for studying the effects of low plasma progesterone on fertility.
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Heifers were assigned either low or high (HE) levels of energy intake and low or high concentrations of dietary crude protein. The effect of these diets on the plasma concentrations of insulin, insulin-like growth factor (IGF)-I, and urea on follicular growth and early embryo development is described. We propose that the observed dietary-induced changes in the ovarian IGF system increase bioavailability of intrafollicular IGF, thus increasing the sensitivity of follicles to FSH. These changes, in combination with increased peripheral concentrations of insulin and IGF-I in heifers offered the HE diet, contribute to the observed increase in growth rate of the dominant follicle. In contrast to follicular growth, increased nutrient supply decreased oocyte quality, due in part to increased plasma urea concentrations. Clearly a number of mechanisms are involved in mediating the effects of dietary energy and protein on ovarian function, and the formulation of diets designed to optimize cattle fertility must consider the divergent effects of nutrient supply on follicular growth and oocyte quality.
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The fertility of dairy cows decreases during the summer and remains low during the cooler autumn although the animals are no longer under heat stress. The aim of this study was to characterize a delayed effect of summer heat stress on oocyte quality in the autumn and to improve oocyte quality by enhanced removal of follicles damaged during the previous summer. Lactating cows (n = 16) were subjected to heat stress during the summer. In autumn, ovarian follicles (3-7 mm in diameter) were aspirated by an ultrasound-guided procedure during four consecutive oestrous cycles. Follicles were aspirated from control cows on day 4 and from treated cows on days 4, 7, 11 and 15 of each oestrous cycle. All cows received PGF(2alpha) and GnRH injections on days 19 and 21, respectively, and maintained cyclicity, as indicated by plasma progesterone concentrations. On day 4 of each cycle, the oocytes recovered were examined morphologically, matured and activated in vitro, and cultured for 8 days. In cycle 1 (early October) both groups showed low percentages of grade 1 oocytes, cleavage, four- and eight-cell embryos, morulae and parthenogenetic blastocysts. Subsequently, the number of grade 1 oocytes increased earlier (cycle 2) in treated than in control cows (cycle 3; P < 0.05). The cleavage rate in the control group remained relatively low throughout (32-58%), whereas in the treated group it increased from 40% (cycle 1) to 75% (cycles 3 and 4; P < 0.05). The number at each stage of embryo development increased slightly but remained low throughout in the control group, whereas in the treated group significant (P < 0.05) increases of all stages were observed in cycles 3 and 4. The results show a delayed effect of summer heat stress on oocyte quality and embryo development in the autumn. Enhanced removal of the impaired cohort of follicles led to earlier emergence of healthy follicles and high quality oocytes in the autumn.
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The objective of the present study was to evaluate changes in equine follicular fluid insulin-like growth factor binding protein (IGFBP) proteolytic activity as well as steroid, IGF, and IGFBP concentrations during follicular development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6 to 15 mm; n = 54), medium (16 to 25 mm; n = 23), or large (> 25 mm; n = 15), and follicular fluid was collected. Estradiol and androstenedione levels in follicular fluid were greater (P < 0.05), and IGFBP-3 concentrations tended to be greater (P < 0.10) in large than in small or medium follicles, whereas IGFBP-2, -4, and -5 levels were less (P < 0.05) in large than in small or medium follicles. Estradiol and androstenedione concentrations were negatively correlated (P < 0.01) with IGFBP-2, -4, and -5 but not IGFBP-3 concentrations. To evaluate proteolysis of IGFBP, follicular fluid was incubated with human 125I-labeled IGFBP-2, -3, and -5 and protein separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of 125I-lableled IGFBP-2 or -3, and the small amount of proteolysis of IGFBP-2 and -3 did not differ (P > 0.10) among follicle classes. However, more 125I-labeled IGFBP-5 was cleaved (P < 0.05) by follicular fluid from large follicles collected during the follicular phase than large follicles during the luteal phase, and small or medium follicles from follicular and luteal phase mares indicating that a protease to IGFBP-5 exists in estrogen-dominant equine follicles. This IGFBP-5 protease was inhibited by kallikrein/serine protease and metalloprotease inhibitors. We conclude that the tendency of estrogen-dominant follicles of mares to have greater levels of IGFBP-3 and lesser levels of IGFBP-2 does not appear to be due to differences in proteolysis, whereas changes in IGFBP-5 levels are likely due to changes in activity of a serine protease or metalloprotease. Changes in IGFBP may alter levels of bioavailable IGF that stimulate steroidogenesis and mitogenesis in developing mare follicles.
Article
In the first of three experiments, eight ovariectomised Greyface ewes primed with exogenous progesterone were used to provide quantitative data on the effects of two contrasting feeding levels (0.3 vs. 1.4 × maintenance) on plasma progesterone concentrations. Over the 9 day study period, mean (± SEM) daily progesterone concentrations were 4.3 ± 0.13 and 3.3 ± 0.17 μg l−1 for the low and high feeding regimens, respectively (P = 0.06), indicating that high feed intake suppressed circulating progesterone levels. The second experiment examined the effect in superovulated Finn-Dorset ewes of a diet supplying either 0.6 (Group L, n = 8) or 2.3 (Group H, n = 8) times their daily energy needs for maintenance, from 1 day before introduction of exogenous progesterone to the time of insemination, on plasma progesterone concentrations and the viability of ova recovered 4 days after insemination. Mean (± SEM) plasma progesterone concentrations were 4.5 ± 0.17 μg l−1 and 2.8 ± 0.16 μg l−1 for L and H ewes, respectively, during the 12 day priming period (P < 0.001). Eight hours after progesterone withdrawal, levels had fallen to 0.9 ± 0.06 μg l−1 and 0.8 ± 0.07 μg l−1, respectively, then rose to 17.8 ± 3.01 μg l−1 and 12.9 ± 2.50 μg l−1 (P > 0.10) at ovum collection. Intervals (mean ± SEM) to oestrous onset (14.5 ± 0.38 h) and the luteinising hormone (LH) surge (27.1 ± 0.98 h) were unaffected by feed intake. Mean (± SEM) ovulation rates (8.1 ± 1.57 vs. 7.8 ± 1.10) and numbers of ova recovered (5.0 ± 1.39 vs. 4.8 ± 1.11) were also similar for each group. However, the proportions of ova considered viable (over 32 cells) at recovery were 0.53 and 0.22 for L and H groups, respectively (P < 0.005). Following 72 h culture (Tissue Culture Medium-199 (M199) + 10% foetal calf serum (FCS)), 0.55 and 0.27, respectively, had developed to blastocysts (P < 0.025). Of ova assessed as viable at recovery, similar proportions (0.86 vs. 0.75) from L and H treatments developed to blastocysts, with corresponding nuclei counts (mean ± SEM) of 55 ± 5.2 and 55 ± 13.2. The third experiment used 12 superovulated Greyface ewes, each offered a different feed level within the range 0.6–2.5 × maintenance, to determine the nature of the relationship between feeding level, pre-ovulatory progesterone concentrations and ovum development at Day 2 following insemination and subsequently during 7 day co-culture (M199 + FCS). Increases in feeding level were accompanied by linear decreases in plasma progesterone (r2 = 0.79, P < 0.001), the interval to oestrous onset (r2 = 0.52, P < 0.01) and timing of the LH surge (r2 = 0.32, P < 0.06). Although undetectable at ovum collection, and somewhat equivocal after 4 day culture, high feeding levels prior to ovulation reduced the proportion of ova (0.16 vs. 0.58) developing to or beyond the expanding blastocyst stage after 7 day culture. Quantitative indices of cell division and protein synthesis confirmed this. In conclusion, excessive feeding during follicular recruitment and oocyte maturation in superovulated ewes imparts a legacy of embryonic loss and developmental retardation.
Article
The effect of insulin-like growth factors (IGFs) and insulin on the release of progesterone and oxytocin from bovine corpus luteum was investigated at early (days 5-7), mid- (days 8-12) and late (days 15-18) luteal phases of the oestrous cycle in an in vitro microdialysis system. The expression of specific receptors was evaluated in bovine corpora lutea of the respective luteal stages. A 30 min infusion of IGF-1, IGF-2 (1.3, 13 and 130 nmol l-1) or insulin (13, 130 and 1300 nmol l-1) caused a stimulation of the release of progesterone (P < 0.05). IGF-1 was most effective in releasing progesterone. Oxytocin release from corpora lutea was stimulated by insulin at all doses tested (13-1300 nmol l-1), whereas the IGFs were only effective at the highest dose (130 nmol l-1) applied. The high doses of IGFs (130 nmol l-1) and insulin (1300 nmol l-1) stimulated the release of progesterone and oxytocin throughout the luteal phase (P < 0.05). For all three peptides, greatest stimulation was seen during the late luteal phase (days 15-18 of the oestrous cycle) with the peak of progesterone release directly related to peptide infusion (P < 0.05). In addition, IGF-1 stimulated total release of progesterone (units in 4 h) after the beginning of the stimulation during this phase (P < 0.05). IGF-1 caused a gradual increase of progesterone even beyond the time of peptide perfusion, whereas IGF-2 and insulin stimulated progesterone release only during the peptide perfusion. Distinct receptors for IGF-1 and IGF-2 were present in corpora lutea membrane preparations at all stages investigated. Specific binding for insulin was also seen in all stages of the cycle without any cycle-dependent changes in the amount of binding. The displacement of labelled insulin by unlabelled IGF-1 and IGF-2 did not show the rank of order that has been described as typical for insulin receptors (i.e. insulin > IGF-1 > IGF-2), but comparable binding affinities were observed for the three unlabelled ligands. Specific binding of IGF-2 was markedly higher than that of IGF-1 or insulin throughout the cycle (1.9- and 4.9-fold higher compared with IGF-1 and insulin, respectively). Receptor specificity did not change during luteal development. Binding affinity and capacity of IGF-1 receptor was constant throughout the oestrous cycle. Specific IGF-2 binding increased and showed a positive co-operativity towards the end of the cycle. Specific binding of insulin was not significantly different in the three luteal stages examined.
Article
Both in animals and humans, before or after birth, angiogenesis appears to be closely coordinated in time and space with the formation of fat cell clusters. Monobutyrin, a novel fat-specific angiogenesis factor, may play a role in this process. The potential to acquire new fat cells appears to be permanent throughout life in both animals and humans, as revealed by in vitro experiments. Considerable evidence now supports the view that BAT and WAT are distinct organs; in addition, the existence of distinct BAT precursor cells is demonstrated by their unique ability to express the UCP gene. In bovine and ovine, the transformation of BAT into WAT is strongly suggested by the rapid disappearance after birth of UCP from the various BAT depots. Despite the initial cell heterogeneity of the stromal-vascular fraction, cultured stromal-vascular cells of adipose tissue are adipose precursor cells that show varying capacities for replication and differentiation, according to age and fat depot. Studies of adipose cell differentiation in vitro correspond to the sequence: adipoblast (unipotential cells)----commitment preadipose cell (preadipocyte)----terminal differentiation immature adipose cell----terminal differentiation mature adipose cell (adipocyte). Cell commitment is triggered by growth arrest and characterized by the expression of early markers (A2COL6/pOb24; clone 5; LPL), whereas only terminal differentiation of preadipocytes requires the presence of various hormones. Multiple signaling pathways have been characterized and shown to cooperate in the process of terminal differentiation. The concept that adipose cells behave as secretory cells is now emerging from in vitro data, since secretion of various proteins (LPL, adipsin, CETP) and important metabolites (fatty acids, monobutyrin, androgens, estrogens, prostaglandins) takes place both constitutively and upon hormonal stimulation. This suggests that adipose tissue participates more directly than previously thought in metabolic activities and energy balance.
Article
It will be interesting to see resolution of the seemingly paradoxical actions of follicular fluid extract on pituitary gonadotropin secretion - being selective in the suppression of tonic FSH levels, but effective in blocking the surge modes of both FSH and LH release by estrogen or GnRh action. Is inhibin activity derived from a single product of ovarian secretion? Is the hypothalamic release (pulse frequency) of GnRH altered by inhibin? Or is inhibin acting directly on the pituitary gonadotrophs, desensitizing them to GnRH- or estrogen-positive feedback? Perhaps these studies will clarify whether a single GnRH regulates both FSH and LH secretion. What role may inhibin have in polycystic ovarian disease or other endocrinopathies impairing the human HPO axis? Can we develop novel means to manipulate inhibin activity for fertility control or enhancement? Perhaps the answers will seed important insights into inhibin's role in male reproductive endocrinology as well. These are among the principal queries soon to be squarely addressed in the laboratory and the clinic. Such mysteries within the ovaries are likely to entertain us, clinical investigators and basic scientists alike, for the foreseeable future.
Article
Angus (n = 14) and Brahman (n = 14) cows were used to evaluate the effects of insulin administered concomitantly with FSH in a superovulation regimen. Cows were allotted to four pen replicates by treatment and breed, and received FSH (i.m.) twice a day for 5 consecutive days (first day of injections = day 0 of study) plus concomitant administration of either saline (control) or long-acting bovine insulin (0.25 iu kg-1 body mass; s.c.). Blood samples were collected at intervals of 6 h during the injection period and analysed for plasma insulin, glucose, insulin-like growth factor I (IGF-I) and IGF-I binding protein (IGFBP) activity. Cows were ovariectomized on day 5. The number and diameter of follicles were recorded. Follicular fluid was aspirated for determination of IGF-I, IGFBP activity, oestradiol and progesterone. Mean plasma concentration of glucose was lower in insulin-treated than in control cows averaged over days 1-5 (56 +/- 3 versus 82 +/- 3 mg dl-1; P < 0.01). Plasma concentration of IGF-I and IGFBP activity were not affected (P > 0.10) by treatment, but were higher in Brahman than in Angus cows (IGF-I: 41 +/- 6 versus 19 +/- 6 ng ml-1, P < 0.05; IGFBP activity: 17.5 +/- 0.4 versus 15.8 +/- 0.04% (10 microliters)-1; P < 0.03). Insulin treatment did not affect the number of small (1.0-3.9 mm), medium (4.0-7.9 mm) or large (> or = 8.0 mm) follicles. Brahman cows had a greater (P < 0.01) number of medium and total follicles (19.4 +/- 2.5 and 60.5 +/- 5.5, respectively) than did Angus cows (7.5 +/- 2.6 and 30.5 +/- 5.6, respectively). Diameter of large follicles was greater in insulin-treated than in control cows (11.4 +/- 0.2 versus 10.6 +/- 0.1 mm; P < 0.05). Follicular fluid IGF-I concentration in large follicles was higher in insulin-treated Brahman cows (60 +/- 2 ng ml-1) than in control Brahman cows (37 +/- 2 ng ml-1), but was lower in insulin-treated Angus cows (31 +/- 3 ng ml-1) than in control Angus cows (38 +/- 2 ng ml-1; treatment x breed interaction, P < 0.01). IGFBP activity in fluid from large follicles was not affected by insulin treatment in Brahman cows but was reduced (P < 0.05) by insulin treatment in Angus cows.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
Insulin and insulin-like growth factors (IGFs) have direct effects on cultured ovarian cells. These effects include stimulation of granulosa cell mitogenesis, granulosa and luteal cell progesterone production, and thecal cell androgen production and appear similar among species. However, species differences exist with regard to insulin and IGF-I effects on granulosa cell estradiol production. In addition to endocrine effects of insulin and IGFs, IGFs are produced by granulosa, thecal, and luteal cells, allowing for an intraovarian autocrine and paracrine system. Granulosa, thecal, and luteal cells contain receptors for insulin and IGFs, and these receptors appear to mediate the effects of insulin and IGFs. Adding to the complexity of the regulatory role of IGFs is the presence of IGF-binding proteins (IGFBPs) within the ovary. These IGFBPs are produced by granulosa, thecal, and luteal cells, and their production is hormonally regulated. Evidence for a coherent mechanism by which insulin, IGFs, and IGFBPs interact and regulate ovarian function in vivo has yet to be found.
Article
To evaluate the usefulness of morphology grading of the oocyte-corona-cumulus complex (OCCC) as a marker of oocyte nuclear maturity, fertilizability, embryo cleavage, and likelihood of pregnancy. Prospective cohort study. Academic fertility center. Eighty-three infertile couples undergoing IVF-ET/intracytoplasmic sperm injection treatment. All patients underwent a long stimulation protocol of GnRH agonist therapy followed by hMG administration and transvaginal oocyte recovery. All OCCCs, oocytes, and embryos were assessed. The relation among OCCC morphology and the nuclear maturity of denuded oocytes, the fertilization rate, and embryo development to the cleavage stage were analyzed. Of 909 OCCCs collected from 92 cycles, 2.5%, 4.2%, 79.9%, and 13.4% were prophase I, metaphase I, metaphase II, and degenerating, respectively. No statistically significant differences were found in the percentage of intact metaphase II oocytes, the fertilization rate, or the cleavage rate among complexes with different morphologic grades. The morphologic grade of the OCCCs of transferred embryos in the pregnant group was not different from that in the nonpregnant group. Most oocytes were in metaphase II at the time of retrieval after ovarian stimulation. However, no relation was observed between the OCCC morphologic grade and oocyte nuclear maturity, the fertilization rate, or embryo cleavage. These observations suggest that OCCC morphology grading is a poor marker of oocyte quality.
Article
A severely malnourished 87-yr-old man presented with hypoglycemia. Serum GH levels were elevated, and serum levels of insulin-like growth factor I (IGF-I), IGF-binding protein-3, and GH-binding protein were extremely reduced. The patient's GH was biologically active. Administration of GH for 4 consecutive days resulted in a slight increment in serum IGF-I levels, but no elevation of serum IGF-binding protein-3. The expression of GH receptor messenger ribonucleic acid in the liver was greatly reduced. An autopsy revealed a Rathke's cleft cyst confined to the sella turcica. Immunohistochemical studies for GH showed that there was nothing to suggest a tumor overproducing GH. In addition, TSH levels were elevated in the presence of normal thyroid hormone levels, and there was a cluster of cells showing strong immunohistochemical staining for the TSH beta-subunit in the pituitary. In this patient, the decreased expression of GH receptor messenger ribonucleic acid in the liver may have been responsible for the GH resistance, which was probably caused by malnutrition.
Article
We wished to compare cumulus oocyte complex (COC) recovery and follicle development after single and repeated ultrasound-guided transvaginal follicle aspiration (aspiration). Aspirations were performed in Holstein-Friesian heifers every once weekly (every 7 d; n = 12) or twice weekly (every 3 or 4 d; n = 6) starting on Days 3 or 4 of the estrous cycle (estrus = Day 0) and continuing for 4 wk.. During each session, all visible follicles >2 mm were aspirated using an 7.5 MHz transducer to guide an 18 ga × 60 cm single lumen needle and applying 50 mm Hg vacuum which generated 25 mL/min. The COC's harvested from each follicle were counted and classified into 4 categories. Post-aspiration follicle wave emergence was traced by daily ultrasound examinations.
Article
The effects of FSH-stimulated cumulus cells on the regulatory mechanisms of chromatin condensation and maturation-promoting factor (MPF) activation around the time of germinal vesicle breakdown (GVBD) in bovine oocytes were examined. Chromatin condensation occurred in oocytes arrested at the germinal vesicle (GV) stage by protein synthesis inhibitor, cycloheximide, but this condensation was blocked by FSH-stimulated cumulus cells. However, treatment with cyclic AMP (cAMP)-dependent protein kinase inhibitor, H-8, dramatically increased the proportion of oocytes possessing GVs with condensed bivalents. Under the condition of inhibited protein synthesis, the phosphorylation form of p34cdc2 kinase was not changed due to chromatin condensation, although the activity of histone H1 kinase was significantly increased compared with that of oocytes possessing GVs with filamentous bivalents. The cycloheximide-dependent GVBD block was overcome by okadaic acid (OA) in 48 and 13% of the oocytes in the absence and presence of FSH, respectively. An initial 6-h culture period critical for protein synthesis was necessary for OA to counteract the inhibitory effect exerted by cycloheximide on the induction of GVBD and activation of histone H1 kinase in the absence of FSH, whereas this first culture period was prolonged for 2 h in the presence of FSH. Furthermore, even in FSH-stimulated oocytes, H-8 facilitated an OA-counteracted overcome of the cycloheximide-dependent GVBD block after 2 h of initial culture for protein synthesis. From these results, it is concluded that cAMP-dependent protein kinase activity regulated by cumulus cells following FSH-stimulation requests plays a role in the complex mechanism of chromatin condensation and MPF activation leading to meiotic resumption in bovine oocytes.
Article
Novel regulatory proteins have been identified within oocytes that are crucially involved in folliculogenesis. One of the most exciting oocyte signaling molecules is a novel member of the transforming growth factor beta (TGF-beta) superfamily, growth differentiation factor 9 (GDF-9). Loss-of-function studies have established that GDF-9 is obligatory for proper folliculogenesis and fertility in female mice. The current challenges are to understand how oocyte morphogens regulate folliculogenesis and how their actions and interactions are integrated into the overall processes of physiology and pathophysiology. Who would have thought that oocyte morphogens would be so crucial for reproduction?
Article
In the primate ovary, androgens promote oocyte insulin-like growth factor (IGF) I expression as well as initiation of follicle development. In most other mammals, IGFs do not appear to be required for the entry of follicles into the pool of growing follicles and for their gonadotropin-independent development. In contrast, IGFs are involved in the increase in FSH-responsiveness of granulosa cells when follicles enter into the gonadotropin-dependent stages of follicular development (200 microm in mouse, 2 mm in sheep, 5 mm diameter in cattle). In the late stages of folliculogenesis, the decrease in IGFBPs participates in the increase in IGF bioavailability, leading to a further amplification of FSH action.
Article
Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.
Article
Insulin and glucose may be limiting factors for ovarian function in dairy cows genetically selected for high milk yield. The effects of nutrition on the intrafollicular content of insulin and glucose were investigated in Israeli Holstein dairy cattle fed a basic total mixed ration and producing 34-39kg of milk daily. In experiment 1, carried out in 11 oestrus-synchronised cows, little variation in insulin concentration was found in plasma sampled during the luteal phase, but high variation was found in plasma sampled during the follicular phase. Therefore, in order to prevent confounding the effects of diet and of phase in cycle in the following experiments, experimental diets were fed during the luteal phase of synchronised oestrus cycles. In experiment 2, designed as Latin-Square, six cows received sequentially diets containing 17.1 (control) or 19.7% of crude protein, using two sources of supplementary protein, i.e. soyabean meal (SBM) and corn gluten meal (CGM), differing in ruminal degradability and leucine content. When dry matter intake was used as covariant, plasma insulin on day 16 was 29.5 and 26.4% higher in cows fed diets containing SBM and CGM than in the control (P<0.05). In experiment 3, 17 cows were individually fed the basic diet and then switched to isoenergetic diets containing SBM (n=5), CGM (n=6) or corn grain (CG, n=6) given from day 10 to 16 of the synchronised oestrus cycle. On the eve of day 16, and in the morning of day 17, they were administered PGF(2alpha) and the content of 26 largest follicles was aspirated by using the transvaginal ovum pick-up technique. Follicles were sorted into two classes (preovulatory and subordinate) according to oestradiol concentration and the progesterone:oestradiol ratio in follicular fluid (FF). Higher concentrations of insulin (0.282 versus 0.127ng/ml, P<0.0001) and of glucose (0.614 versus 0.386g/l, P<0.002), were found in FF from preovulatory follicles. The insulin concentration in the FF of cows fed the CG diet was 26% higher than in their counterparts fed CGM (P<0.04), SBM being intermediate. Dietary effects did not reach significance in subordinate follicles. The finding that preovulatory follicular status is associated with increased intrafollicular insulin and glucose suggests that insulin is involved in follicular maturation. The nutritional effect on intrafollicular glucose and insulin may have practical implications to optimise feeding in dairy cows during phases of the oestrus cycle.
Article
The traditionally accepted theory has been that most of the biological effects of growth hormone (GH) are mediated by circulating (endocrine) insulin-like growth factor-I (IGF-I). This dogma was modified when it was discovered that most tissues express IGF-I that can act via an autocrine/paracrine fashion. In addition, both GH and IGF-I had independent effects on various target tissues. Using tissue-specific gene deletion of IGF-I in the liver, it has been shown that circulating IGF-I is predominantly liver-derived but is not essential for normal postnatal growth. Therefore, it is proposed that non-hepatic tissue-derived IGF-I may be sufficient for growth and development. Thus the original somatomedin hypothesis has undergone further modifications.
Article
Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-I (IGF-I) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.
Article
Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 +/- 1, 33 +/- 2, 53 +/- 1, and 84 +/- 2 h after the periovulatory peak in FSH concentrations. Follicles > or = 2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r > or = 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (> or = 48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.
Article
Follicle dynamics and oocyte viability in Holstein primiparous and multiparous cows and the relationships between fertility and the biochemical and physical properties of oocyte membranes with season were examined. The conception rates of primiparous (n = 70 885) and multiparous (n = 143 490) cows differed, peaking in the winter and decreasing in the summer. The number of follicles 3-8 mm in diameter per ovary was higher in winter (19.6) compared with summer (12.0). However, in winter the percentage of ovaries with fewer than ten follicles per ovary was 16%, in contrast to 50% in summer. After aspiration of follicles, 7.5 oocytes per ovary were found in winter and 5.0 oocytes per ovary in summer. Cleavage to the two- to four-cell stage after chemical activation was greater in winter than in summer; this was enhanced at the morula stage and embryo development to the blastocyst stage was significantly higher in winter than in summer. Determination of the lipid phase transition in oocyte membranes revealed a shift of 6 degrees C between summer and winter. Fatty acid composition of phospholipids from follicular fluid, granulosa cells and oocytes indicated that there was a higher percentage of saturated fatty acids during the summer and that the percentages of mono-unsaturated and polyunsaturated fatty acids were higher in oocytes and granulosa cells during the winter. Oocytes and granulosa cells had similar fatty acid compositions, in contrast to follicular fluid. These results may explain the differences in the ability of oocytes to develop to the blastocyst stage at different seasons. Thus, temperature changes may lead to changes in membrane properties, which, in turn, can influence oocyte function and fertility.
Article
The present study was designed to examine the influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Three experiments were conducted. In experiment 1, oocytes were classified by number of cumulus cell layers and morphology of the ooplasm as good, fair or poor. Oocytes were cultured for IVM, IVF and IVC in CR1aa medium. In experiment 2, good quality oocytes were cultured for maturation in: (1) CR1aa; (2) CR2aa; (3) TCM-199; (4) MEM or (5) RPMI-1640, and then fertilized using frozen thawed buffalo spermatozoa in CR1aa. The oocytes were cultured in the same medium used for maturation after fertilization. In experiment 3, oocytes were classified into three groups: group (1) was without gonadotropin and serve as a control; group (2) in which IVM medium was supplemented with 10microg/ml FSH and group (3) in which IVM medium was supplemented with 10IUml(-1) eCG. In all experiments, oocytes were kept at 38.5 degrees C under 5% CO(2) for IVM, IVF, IVC and examined for cleavage and embryo development rates on days 3 and 8, respectively. Good and fair quality oocytes produced a higher cleavage rate (P<0.01) than poor quality oocytes. Morula production rate was also higher (P<0.01) for good as compared with fair quality oocytes. Embryo development with poor quality oocytes was arrested at the two to sixteen cell stage. In experiment 2, the cleavage rate was higher (P<0.05) in CR1aa than CR2aa, and higher (P<0.01) than TCM-199, MEM and RPMI-1640. The numbers of morulae and blastocysts were higher (P<0.01) for oocytes cultured in CR1aa and CR2aa media than TCM-199 or MEM. In experiment 3, the addition of FSH or eCG to the maturation medium increased (P<0.01) cleavage and developmental rates of buffalo embryo compared with control medium. In conclusion, the IVM of good quality buffalo oocytes in CR1aa or CR2aa medium and the addition of FSH or eCG in maturation medium produced higher cleavage and developmental rates of IVF buffalo embryos.
Article
Fat supplementation in the diet influences reproductive performance of lactating ruminants. Changes in the fat supply alter fatty acid composition and this can affect physical properties of cell membranes. This study examined the effect of rumen bypass polyunsaturated fatty acid (PUFA) supplementation on oocyte quality, chilling sensitivity, and lipid phase transition in oocytes of the sheep. Ewes were fed a diet supplemented with calcium soaps of fish oil for 13 weeks. More follicles and oocytes were found in the ovaries of ewes supplemented with PUFA than in control ewes. The number of high-quality oocytes was higher in ewes fed PUFA than in control ewes (74.3 and 57.0%, P < 0.05, respectively). Evaluation of phospholipid fatty acid composition indicated that PUFA were present in small proportions in oocytes, and eicosapentaenoic acid and docosahexaenoic acid were absent. Supplementation with PUFA increased the proportion of long chain unsaturated fatty acid in the plasma and cumulus cells phospholipids by 7.4 and 12.7%, respectively (P < 0.05). Integrity of oocyte membranes following chilling (16°C, 15 min) was improved by PUFA supplementation increasing from 62.5 to 90.0% (P < 0.05). Due to changes in the oocyte's fatty acid profile, physical properties of the membrane were changed and the midpoint temperature of lipid transition reduced by 11°C. These results suggest that supplementation of rumen bypass PUFA to ruminant diets can change fatty acid composition of follicle components and influence parameters such as number and quality of oocytes and their chilling resistance. Mol. Reprod. Dev. 61: 271–278, 2002.
Article
The potential of the ovum pick-up technique, used over a long period, was evaluated in 6 Italian Mediterranean buffalo cows that had more than 500 d open. The cows were submitted to ovum pick-up twice weekly for 2 mo. An additional 2-mo cycle of ovum pick-up was performed in 3 of the buffalo. The ovum pick-up sampling did not affect the resumption of reproductive activity of these animals. In fact, all the buffalo conceived, on average, 47.5 +/- 27.5 d after the last ovum pick-up. An average of 5.48 follicles was punctured, and 2.71 oocytes were collected per session. However, only 53.5% of these oocytes were suitable for in vitro embryo production. The number of punctured follicles differed between individual cows. There were no differences in the number of collected oocytes or in the recovery rates. The number of punctured follicles, the number of collected oocytes and the recovery rate were similar in the first and second months; the quality of the oocytes was, however, better in the second than in the first month (P < 0.05). The increasing interval between 2 consecutive ovum pick-up sampling (intersession interval) caused an increase of the percentage of large follicles. Moreover, the increase of the intersession interval from 4 to 5 d decreased the quality of the collected oocytes (P < 0.05). The efficiency of in vitro production of embryos to expanded blastocysts was 16.7%.
Reproductive outcomes for dairy heifers treated with combinations of prostaglandin F2 alpha norgestomet, and gonadotropin-releasing hormone [2] Butler WR, Smith RD. Interrelationships between energy balance and postpartum reproductive function in dairy cattle
  • Js Stevenson
  • Jf Smith
  • Hawkins
Stevenson JS, Smith JF, Hawkins DE. Reproductive outcomes for dairy heifers treated with combinations of prostaglandin F2 alpha norgestomet, and gonadotropin-releasing hormone. J Dairy Sci 2000;83:2008–15. [2] Butler WR, Smith RD. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J Dairy Sci 1989;72:767–83.
SAS user’s guide, version 6 ed
  • Sas Institute
SAS Institute. SAS user's guide, version 6 ed. Cary, NC: SAS Institute Inc.; 1986.
Influence of oocyte quality, culture media and gonadotrophins on cleavage rate and development of in vitro fertilized buffalo embryos
  • Abdoon