Preovulatory follicular status affects the insulin and glucose content of the follicles in high yielding dairy cows

ArticleinAnimal Reproduction Science 64(3-4):181-97 · December 2000with 71 Reads
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Abstract
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.

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    not store these pages in any form except for your own personal use. All other usage or distribution is illegal under international copyright treaties. Permission to use any of these pages in any other way besides the before mentioned must be gained in writing from the publisher. This article is exclusively copyrighted in its entirety to OJVR. This article may be copied once but may not be, reproduced or re-transmitted without the express permission of the editors. This journal satisfies the refereeing requirements (DEST) for the Higher Education Research Data Collection (Australia). Linking: To link to this page or any pages linking to this page you must link directly to this page only here rather than put up your own page. ABSTRACT Sadeghi S, Moradi Kor N., Trace elements concentration of ovarian follicular fluid in relation to follicle size in dairy cows, Onl J Vet Res., 17(9):490-495, 2013. Ovarian follicular fluid concentration of trace elements in relation to follicular size in Holstein cattles is reported. Ovaries were recovered from 40 female adult cows (Holstein Friesian) 5–7 years of age with clinically normal reproductive tracts after slaughtering. Visible follicles on the surface of the ovaries were classified based on their diameter as small (3-5 mm), medium (6-9 mm) and large (10-20 mm). Follicular fluid samples were analyzed for iron, iodine, copper, manganese, zinc, cobalt, molybdenum and selenium. Results showed that concentration of trace elements were different between follicles sized categories. Follicular fluid concentrations of iodine and manganese between follicles sized categories were significant (p ≤ 0.05). The findings suggest that the levels of trace elements in the follicular fluid were related to follicular size in dairy cows.
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    In vitro production (IVP) of bovine embryos has been improved immensely throughout the last decades. Nevertheless, embryos generated in vitro still differ from their in vivo-produced counterparts. It is possible to achieve blastocyst rates of up to 70% if in vivo-matured oocytes are used. In contrast, if oocytes are matured in vitro, blastocyst rates are only half that of those matured in vivo. This rather limited success may be attributed to the heterogeneous population of oocytes which are normally retrieved from follicles of 3-8 mm rather than from preovulatory follicles. In contrast to the in vivo-ovulated oocyte, these oocytes lack development up to the preovulatory stage and are matured in vitro. Therefore, much effort has been devoted to the establishment of non-invasive and non-perturbing means for selecting the most competent oocytes, for example the extensiveness and compactness of the cumulus-corona investment and the granulation of the ooplasm. In vitro culture (IVC) conditions have been enhanced in the last few years, mainly by adjustment of media formulations, whereas the in vitro maturation (IVM) protocols stay invariable. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting matured oocyte. The scope of this review is to give an overview of the current situation of in vitro maturation of mammalian oocytes with emphasis on the bovine species. Special attention has been paid to the in vivo situation in the follicle and how a better understanding of these intrafollicular factors will aid to improve the in vitro maturation conditions.
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    The presence of corpus luteum may have a local effect on metabolite composition of follicular fluid (FF) and could indirectly influence follicular development and oocyte quality. The purpose of this study was to examine the influence of the corpus luteum on metabolite composition of follicular fluid (FF), harvested from different-sized follicles and the relationship between metabolite composition of FF to blood serum in dairy cows. Ovaries and blood samples were collected from 30 female adult Holstein Friesian cows, 4-7 years old, with clinically normal reproductive tracts. The animals were in the diestrus stage and selected post mortem. The ovaries collected were classified based on the presence and absence of corpus luteum (CL(+/-)). Visible follicles on the surface of the ovaries were classified into (i) small (3-5mm), (ii) medium (6-9mm) and (iii) large (10-20mm) based on their diameter. Follicular fluid was aspirated from follicles with different sizes in CL(+) and CL(-) ovaries. Blood and FF samples were analyzed for various biochemical constituents including glucose, cholesterol, triglyceride, total protein, albumin and globulin. The results showed that serum concentration of glucose, cholesterol and triglyceride was significantly different (p≤0.05) in FF from follicles of different size categories. Differences between various follicle size categories in CL(-) ovaries were only significant for concentrations of glucose, cholesterol and triglyceride. FF concentration of glucose and cholesterol in the same follicle size categories in CL(+) ovaries was significantly lower than that of CL(-) ovaries. These results indicate that levels of the biochemical metabolites in serum and FF differ significantly. In addition, FF concentrations of biochemical metabolites were related to follicular size and to the presence or absence of corpus luteum.
  • Article
    Full-text available
    Aim: Estimation of some biochemical constituents in follicular fluid of non-descript cows of Assam. Materials and Methods: Twenty five pairs of ovaries were collected from local slaughter house and the follicular fluid was aspirated from small (2-4 mm), medium (4-6 mm) and large (6-9 mm) follicles. Aspirated fluid samples were centrifuged at 4000 rpm for 20 min in a refrigerated centrifuge to remove granulosa cells and other cell debris. Supernatant was used for estimation of glucose, total protein, cholesterol, acid phosphatase and alkaline phosphatase. Data generated in the study were analyzed statiscally by SPSS (version 16.0). SPSS South Asia Pvt. Limited, Kacharakanahalli, Bangalore, 560043). Results: A non significant difference was recorded in case of total protein and cholesterol of follicular fluid of small, medium and large sized follicles of cow. However, the glucose level significantly (p
  • Article
    Full-text available
    The aim of this work was to study effect of the dry matter intake level on the concentrations of glucose, insulin, urea, estrogen and progesterone in the blood and the level of IGF-I in the follicular liquid of heifers. Twenty seven crossbred heifers were used, in three treatments: 1.2, 1.6 and 2.6% of the live weight of dry matter intake (DMI) daily. The sanguine concentrations of glucose and estrogen were higher in the animals with 1.6% of DMI and the insulin levels were higher in the animals with 2.6% of DMI. There was an increase in the sanguine levels of urea with the increase of the DMI. There was no effect of the DMI on the progesterone levels in the sanguine plasma collected from the jugular. The progesterone levels in caudal vena were higher in the animals with 2.6% of DMI. There was no effect of the DMI on the follicular levels of IGF-I.
  • Article
    Full-text available
    The experiment was carried out in two stages with the objective of determining the effect of parenteral supplementation of 50 mg Cu on the reproductive behaviour of both hypo- and normocupremic cyclic female cattle. In the first stage, 62 hypocupremic, cyclic animals were selected, 32 heifers (16 in the control group and 16 in the treatment group) and 30 cows (12 in the control group and 18 in the treatment group). In the second stage, 98 normocupremic, cyclic heifers were selected (49 in the control group and 49 in the treatment group). 50 mg of Cu were administered to the animals in the treatment groups parenterally (subcutaneously) every two months until three successive applications were completed, while the control animals were not given copper supplementation. The effects of the copper therapy on cupremia and the percentage of heat and gestations presented were determined. The relative risk of treated animals of presentating anoestrus and service repetition was evaluated by the formation of a 2 x 2 contingency table and the application of a chi(2) test. The levels of serum copper were compared using a t-test, while heat and gestations were compared using a proportion comparism test. The parenteral administration of 50 mg Cu in hypocupremic, cyclic heifers and cows led to significant increases of cupremia (P<0.001) and the percentages of heat presentations and gestations (P<0.01); in the normocupremic animals, there were significant increases in the values of serum copper (P<0.0001), heat presentations (P<0.01) and gestations (P<0.001), especially in animals with cupremia values below 14 mmol/L. The treatment reduced the relative risk of reproductive disorders. It is concluded that the parenteral administration of 50 mg Cu in female cattle with Cu blood serum level <14 mmol/L increased cupremia and the percentages of heat presentations and gestations, and reduced the relative risk of reproductive disorders.
  • Article
    Ovarian acyclicity is one of the most important causes of infertility in water buffalo. Recent studies have indicated alterations in the composition of follicular fluid during the condition. The aim of this study was to determine the changes in follicular fluid concentrations of estradiol, progesterone and insulin during ovarian acyclicity in water buffalo. Ovaries were collected from 50 acyclic and 95 cyclic (control) buffaloes and follicular fluid was aspirated from small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized follicles. Estradiol concentration was lower (P<0.0001) in acyclic (1.4 ± 0.09 ng/ml) than in cyclic (3.3 ± 0.18 ng/ml) buffaloes. Regardless of the ovarian cyclic status, there was an increase (P<0.01) in estradiol concentration with the increase in follicle size; the mean concentrations were 2.4 ± 0.16 ng/ml, 2.8 ± 0.29 ng/ml and 3.5 ± 0.41 ng/ml in small, medium and large follicles, respectively. A higher (P<0.001) progesterone concentration was recorded in acyclic (24.3 ± 2.61 ng/ml) compared to the cyclic (7.6 ± 0.79 ng/ml) group. Furthermore, acyclic buffaloes had a lower (P<0.05) concentration of insulin in the follicular fluid than that of cyclic buffaloes (15.2 ± 1.55 μIU/ml versus 25.9 ± 2.78 μIU/ml, respectively). In conclusion, acyclic buffaloes have lower concentrations of estradiol and insulin concurrent with higher concentrations of progesterone in the follicular fluid. These hormonal changes in the follicular microenvironment are possibly a manifestation of the disturbances in the normal follicular development leading to anovulation and anestrus in acyclic buffaloes.
  • Article
    High-producing dairy cows frequently suffer metabolic alterations that cause different diseases, which could decrease the reproductive efficiency of the herd. Among these reproductive disorders, cystic ovarian disease (COD) has been related to alterations in metabolites and hormonal factors such as insulin, adiponectin and leptin. The aim of this study was to determine the protein expression of adiponectin and some of its downstream targets in ovarian follicles of control cows and cows with clinical diagnosis of COD. We also analyzed some key metabolic sensors in plasma and follicular fluid from both groups. In follicular cysts, we detected higher protein expression of adiponectin receptor 2 (AdipoR2), 5' adenosine monophosphate-activated protein kinase (AMPK), carnitine palmitoyl transferase 1 (CPT1) and acyl-coenzyme A oxidase 1 (ACOX1) relative to control antral follicles (p < 0.05). This was related to higher plasma adiponectin concentration in cows with COD than in control cows (p < 0.05). On the other hand, insulin concentrations showed an opposite pattern (p < 0.05). Furthermore, we found alterations in local and systemic concentrations of several metabolites. In this regard, in follicular fluid of cystic cows, the concentrations of non-esterified fatty acids and beta-hydroxybutyrate were higher (p < 0.05), whereas the concentrations of glucose and triacylglycerol were lower than in follicular fluid from control cows (p < 0.05). Besides, in both follicular fluid and plasma of cows with COD, the concentration of cholesterol was higher than in control animals (p < 0.05). These results evidence a local altered scenario of some metabolic sensors in cystic follicles, which could generate an adverse microenvironment for the resumption of ovarian activity, possibly causing the persistence of follicles and the recurrence of COD.
  • Article
    Full-text available
    Developmental competence of bovine oocytes is known to decrease during the hot season. The mechanism underlying this disruption, however, is not entirely clear. The present study examined seasonal effects on the expression of genes involved in oocyte maturation, zygote formation and early embryonic development. Bovine oocytes were aspirated from ovaries collected from a local slaughterhouse during the summer (Jun-Sep) and winter (Dec-May). Oocytes were matured (22 h), in-vitro fertilized (18 h, 38.5°C, 5% CO2) and cultured (KSOM, 38.5°C, 5% CO2, 5% O2) for 8 days. In each season (× four replicates), samples (n = 20) of GV- and MII-oocytes and zygotes, and of preimplantation embryos at the 2-, 4- and 8-cell and blastocyst stages (n = 10, 5, 3 and 4, respectively) were collected and total RNA was isolated, using 500 µl Trizol reagent and 10 µl glycogen. cDNA was generated from each sample using M-MLV reverse transcriptase (for 1 h at 42°C) and qPCR was carried out with primers for GDF9, POU5F1 (previously known as OCT4) and C-MOS using 18S and GAPDH as reference genes. Unexpectedly, gene expression of 18S was stable throughout all developmental stages in both seasons, while that of GAPDH varied among and between seasons, with decreases in MII-oocytes and 8-cell-stage embryos during the summer. Thus, gene expression was referenced to the former gene. The average cleavage rate (2- to 4-cell stage, 44 h post fertilization) did not differ between seasons (80 ± 10.5 vs. 73 ± 9.2% for winter and summer, respectively) however, the proportion of 2-cell stage in the summer was twofold higher from that of 4-cell. The percentage of embryos that developed to the blastocyst stage was higher in the winter than in the summer (23 ± 2.3 vs. 9 ± 3.5 %, respectively; P<0.05). In GV oocytes and putative zygotes, expression of all the examined genes was stable and did not differ between seasons. On the other hand at the MII stage, the expression of all genes decreased in the summer, most markedly that of C-MOS and POU5F1. In 2-cell-stage embryos, expression of all the examined genes was unstable among and between seasons. At the 4-cell stage, opposite patterns were noted between seasons, with decreased expression of GDF9 in the winter and of POU5F1 in the summer. Similarly, the expression of POU5F1 in 8-cell-stage embryos and blastocysts was lower in the summer than in the winter. Taken together, results indicated seasonal variations in gene expression in oocytes and their resultant embryos, which might explain, at least in part, the mechanism underlying the disruption of oocyte developmental competence during the hot season.
  • Article
    Full-text available
    Abstract The present study was carried out to investigate the possible etiology and pathology of cystic ovarian follicles by determining the expression levels of mRNA for LH-r gene in follicular cells of dominant follicles in comparison with cystic ovarian follicles in Iraqi cows. This study were performed in two steps, firstly aspiration of follicular fluids from dominant follicles and cystic follicles and stored in (-20 ℃) until estradiol and progesterone assay, secondary section of the follicular wall in to two hemispheres, and stored at -70 ℃ to -80 ℃ to molecular study. The macroscopic examination of the ovaries revealed that the numbers of dominant follicles are [1] samples while cystic follicles were in [2] samples. There was a significant difference (P<0.05) in the diameter of cystic ovarian follicles (37.56 ± 0.64 mm) compared with dominant follicles (19.93 ± 0.32 mm). Results of hormonal assay showed higher estradiol-17β (865.96±10.64 ng/ml) and progesterone (84.8±1.35 ng/ml) concentrations in follicular fluids of cystic ovarian follicles, which were significantly higher (P<0.05) in comparison with those of dominant follicles which were (314.39±2.55 ng/ml) and (50.25±1.57 ng/ml) respectively. Molecular study, to evaluate the relative quantification of LH-r gene in dominant and cystic follicular cells, has been done by extraction of the total RNA and assay its concentration from these cells, synthesis data of the complementary DNA (cDNA), that done by reverse transcription PCR (q-RT-PCR) technique, of target gene and compared of the gene expression in dominant and cystic follicular cells, the our results referred to down regulation of LH-r gene expression in follicular cells of the cystic ovarian follicles, that may be assistance to understand the etiology and pathology of this case (disease), thus the up-regulation of the this gene in cells of dominant follicles may explain the important role of the LH in ovulation mechanism and increasing follicular ovulation chance. Keywords Gene Expression; Cows; Ovarian Follicles; LH-r Gene
  • Thesis
    Full-text available
    The study was carried out at Maryout Research Station located 34 Km west of Alexandria that belongs to the Desert Research Center (DRC), Ministry of Agriculture and Land reclamation, and at the Department of Animal Production, Faculty of Agriculture, Alexandria university. The aim of the study was to improve the reproductive performance of she-camels under semi-arid conditions by using different regimens of progestins on controlling and synchronizing the ovarian activity and AI program based on real-time ultrasonography of ovarian structures The efficiency of hormonal synchronization of estrus and induction of ovulation in addition to the changes in some blood biochemical constituents during the course of synchronization of ovulation and early days of pregnancy were determined. Eighteen mature one-humped she-camels, aging between 7 to 10 years, with an average body weight of 450 ± 0.2 kg were used in this study from November 2012 to March 2013. Animals were randomly divided into 3 groups of 6 animals each, the first group served as control and received no treatment, while the second group received (0.5 gm/h/day) melengestrol acetate (MGA) orally for 10 consecutive days and finally the third group was fitted with controlled internal drug release (CIDR, 1.38 gm) device for 10 consecutive days. On day nine of progesterone treatment (CIDR, MGA), each animal was injected i.m. with 500 μg PGF2α and on day ten, upon CIDR withdrawal and MGA treatment was stopped, each animal was injected i.m. with 3000 IU PMSG. Immediately before insemination, each female received 5000 IU i.m. injection of hCG to induce ovulation. Monitoring the ovarian activity was done 3 times after 4 days from CIDR removal or MGA withdrawal from feed to determine the appropriate time for insemination according to the size of the dominant follicle. In addition, the uterine contents were scanned on days 18, 30, 60 and 90 from AI for early pregnancy diagnosis. Blood samples were collected from all animals before and after supplementation of melengestrol acetate (MGA) and CIDR in on days (day 0 is the day CIDR-in) and days 10 is the day of CIDR-out and day 15, 17, 20 before AI). In addition, blood samples were collected from she-camels on days 18, 30, 60, 90 after AI. 133 The obtained results can be summarized as follows: 1. Hormonal treatment had no effect on either total number of follicles or size of the dominant follicles, however days of synchronization had significant (P < 0.01) effect on the diameter of the dominant follicles in she-camels. The diameter of the dominant follicle was largest in MGA and CIDR groups on day 7 after CIDR removal and MGA withdrawal (1.1 and 1.2 cm) respectively, this was equivalent to day 17 for control group (1.1 cm),while the lowest diameter was recorded day 10 in all groups. 2. The overall mean of serum progesterone concentration of the CIDR group (4.30 ng/ml) was significantly greater (P < 0.01) than those of control and MGA groups (0.47 and 0.80 ng/ml, respectively). Also, serum progesterone showed significant increase during days of synchronization (P<0.01) in CIDR group being (11.9 ng/ml) on day two, then the concentration declined to (3.58 ng/ml) on day 10 (day of CIDR removal), and decreased to a basal level of (0.73 ng/ml) on day 20, while in the control group P4 concentration was (0.35 and 0.93 ng/ml) for the MGA group. 3. The overall means of serum estradiol 17-β concentrations were not significantly different among MGA and CIDR groups (44.56 and 47.31 pg/ml), respectively compared with the control group (54.19 pg/ml), during the synchronization period. Nevertheless, the treated groups that received the progestin treatments had lower estradiol 17-β concentrations on days 4, 6, 8 and 10 compared to the control. 4. Numerical increases were observed in single ovulation and ovulation rate (%) in MGA group 83.33% and 66.67% compared to CIDR group (66.66% and 33.33%) respectively. However the CIDR group tended to have more multiple-ovulation (33.33%) than the MGA group (16.67%). Also the MGA and CIDR treatments had slight different conception rates (66.67%) and (50%), respectively. 5. The results revealed no-significant effect of treatment on all the studied hematological parameters [hemoglobin (Hb), packed cell volume (PCV), red blood cell counts (RBCs), white blood cells (WBCs), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume (MCV)] in she-camels during synchronization period. However, all hematological parameters were affected (P < 0.01) by days of synchronization except Hb concentration and WBCs counts. 134 6. Serum total protein, albumin, cholesterol, high density lipoprotein and aspartate aminotransferase concentrations were not significantly affected in MGA or CIDR groups as compared to the control group, while, days effect was significantly observed (P < 0.01) on serum concentrations of albumin and aspartate amino- transferase enzyme activity. 7. Numerical increase in serum urea nitrogen was noted in the CIDR group (20.12 mg/dl) compared with the control and MGA groups (18.52, 18.11 mg/dl) respectively. There was a marked effect of days (P<0.01) on serum urea concentrations, where the least serum urea concentration (17.58 mg/dl) was recorded at day 6 compared to other days. 8. Serum glucose concentration was not significantly affected in the MGA or CIDR groups compared to the control. However, in the MGA and CIDR groups serum glucose increased to its highest (P <0.01) on days 17 and 20 compared to the control and to other previous days. 9. The level of serum alanine aminotransferase concentrations were greater (P < 0.05) in the CIDR (5.80 IU/L) and MGA (5.72 IU/L) groups than in the control group (5.13 IU/L) as well as days. 10. The results revealed no significant effect of treatment on serum mineral concentrations of calcium (Ca), phosphorus (P) and sodium (Na) in she-camels during synchronization period. However, There was a marked effect of days (P<0.05) on serum phosphorus where the highest value was recorded on day 20 in the MGA and CIDR groups compared to control. However, serum potassium (K) concentrations were significantly higher (P < 0.01) in the CIDR group (3.84 mEq/l) compared with control and MGA (3.42 and 3.46 mEq/l) groups respectively. Days effect (P < 0.05) was recorded on overall mean serum K concentrations, with day 20 recorded highest overall serum k concentrations (4.34 mEq/L) compared to previous days. 11. The results also clarified that overall means of serum progesterone concentrations were significantly higher (P < 0.01) in pregnant she-camels (10.72 ng/ml) compared with the non-pregnant ones (0.52 ng/ml). Serum progesterone concentration increased in pregnant animals increased from (5.98 ng/ml) on day 18 following insemination, and reached the highest level on day 60 of pregnancy (18.05 ng/ml) then declined on day 90 (12.00 ng/ml). Also, there were significant interaction 135 (P<0.05) in progesterone among days of pregnancy concentrations. Serum progesterone concentration for non-pregnant she-camels remained at a basel level of (0.66, 0.34, 0.57 and 0.52 ng/ml) on days 18, 30, 60 and 90. 12. Numerical increase was observed in estradiol 17-β concentrations in non-pregnant animals (40.39 pg/ml) as compared with pregnant she-camel ones (27.53 pg/ml). 13. Serum total protein, albumin and cholesterol were not significantly affected by pregnant status, or days of pregnancy. 14. The results showed that overall mean serum urea concentration was greater (P < 0.05) in the pregnant she-camels (23.18 mg/dl) compared with the non-pregnant animals (19.79 mg/dl). Also days of pregnancy showed a significant (P < 0.01) effect on serum urea nitrogen concentrations, and increased from (16.2 mg/dl) on day 18 to (33.81 mg/dl) on 90d of pregnancy. 15. Data also indicated that overall means of serum glucose concentrations were not different between pregnant and non-pregnant she-camels, while days had a significant (P < 0.01) effect. Days 18 and 30 had elevated serum glucose concentrations compared to days 60 and 90 after insemination in both pregnant and non-pregnant animals. 16. The results showed that overall mean serum of high density lipoprotein concentrations was greater (P < 0.05) in the pregnant (21.17 mg/dl) compared with the non-pregnant she-camels (13.41 mg/dl). 17. Serum aspartate aminotransferase concentrations were greater (P < 0.05) in pregnant (25.75 IU/L) than the non-pregnant she-camels (17.52 IU/L), while serum alanine aminotransferase concentrations were not significantly affected by pregnancy (12.77 and 11.13 IU/L) respectively. 18. There were no significant effects of pregnancy status on serum mineral concentrations [phosphorus (P), sodium (Na) and Potassium (K)] in she-camels during 90 days after insemination. However serum calcium concentration was greater (P < 0.05) in the pregnant she-camels (10.06 mg/dl) compared with the the non-pregnant (7.85 mg/dl). However, all mineral concentrations were not affected by days of pregnancy except P concentration.
  • Article
    Acid-base balance is one of the most vigorously regulated variables of the body, including genital organs. Subacute ruminal acidosis is a common disturbance in dairy cows that disturbs several biochemical indices in the blood, cerebrospinal fluid, and urine. The possible negative effects of metabolic acidosis on the follicular fluid (FF) composition and, subsequently, on oocyte quality, are not fully elucidated. This study aimed to evaluate the changes in acid-base balance (ABB) in FF and blood during acute metabolic acidosis in dairy heifers. Ten Holstein heifers were stimulated with FSH in eight decreasing doses at 12-hour intervals (D0–D3). Acidosis was induced by oral administration of sucrose at 9 g/kg of body weight, dissolved in 10 L of warm tap water, at D3. Samples were collected from each cow at 0, 8, 12, 16, 24, 32, 40, and 48 hours after treatment. Samples of FF, obtained by transvaginal follicular aspiration, and peripheral blood were examined for ABB parameters: pH, pCO2, pO2, HCO3⁻, and base excess (BE). A significant decrease in pH, HCO3⁻, and BE values in the blood, as well as FF, occurred after sucrose treatment. The lowest pH values occurred in blood at 16 hours, and in FF at 24 hours, after treatment (7.30 ± 0.05 and 7.33 ± 0.05, respectively). The lowest HCO3⁻ values in blood (18.75 ± 3.2 mmol/L) and FF (18.07 ± 2.84 mmol/L) occurred 24 hours after treatment, as did the lowest BE values (−6.61 ± 3.7 mmol/L and −7.53 ± 3.89 mmol/L, in blood and FF, respectively). Significant correlations for HCO3⁻ (r = 0.928), BE (r = 0.946), pH (r = 0.889), and pCO2 (r = 0.522) existed between blood and FF samples. The results demonstrated that metabolic acute acidosis substantially influences the characteristics of both serum and FF.
  • Conference Paper
    Full-text available
    Folliculogenesis serves as maturation of the ovarian follicle, a densely packed shell of somatic cells that contains an immature oocyte. During Folliculogenesis, follicular fluids containing growth factors, hormones and nutrients pave the way for oocyte growth and development. The present study was aimed to compare glucose, urea, cholesterol and triglyceride levels of follicular fluids in different sized follicles and serum of Arab sheep. The blood and ovaries samples of 30 slaughtered sheep were taken to be studied. Follicular fluid was taken in three set of small (less than 2mm), medium (2 to 4 mm) and large (up to 4mm) sizes. Concentrations for glucose, urea, cholesterol and triglyceride levels of ovary fluids in different sized follicles and serum were measured. Results showed that serums glucose and urea was more than three follicular classes significantly; however, glucose concentration among three classes did not significantly. As follicle got larger, urea and triglyceride levels were reduced. Serums cholesterol concentration was significantly more than small and medium sized follicles but there was no significant difference between large follicle and serum among others. Generally, results showed that ovaries follicles size associate with changes in serums glucose and cholesterol concentration of blood. As follicle got larger, urea and triglyceride levels were reduced. Identification of essential biochemical substances for follicle growth and development is promising to improve developing oocyte quality and finally reproduction.
  • Article
    Full-text available
    In the present study, ovarian follicular fluid concentrations of trace elements and biochemical metabolites in relation to follicular size were investigated in dairy cows. Ovaries were recovered from 40 female adult Holstein Friesian cows 5–7 years of age with clinically normal reproductive tracts after slaughtering. The stage of the cycle in the cows slaughtered was diestrus determined post mortem. Visible follicles on the surface of the ovaries were classified, based on their diameter, into (i) small (3-5 mm), (ii) medium (6-9 mm) and (iii) large (10-20 mm) categories. Follicular fluid samples were analyzed for elements (iron, iodine, copper, manganese, zinc, cobalt, molybdenum and selenium) and biochemical metabolites (glucose, cholesterol, triglyceride, total protein, albumin, globulin, urea and creatinine). Results showed that concentrations of trace elements were different between follicles sized categories. Differences in follicular fluid concentrations of iodine and manganese between follicles sized categories were significant (p ≤ 0.05). In addition, differences in follicular fluid concentration of glucose and cholesterol between follicles sized categories (Small, Medium and large follicles) were significant (p ≤ 0.05). These results of the present study suggest that the levels of trace elements and biochemical metabolites composition in the follicular fluid were related to follicular size in dairy cows.
  • Article
    Full-text available
    The aim of this study was to evaluate a new device for the ultrasound-guided transvaginal aspiration of follicular fluid for acid-base balance analysis (ABB set) in comparison with the original modified commercial OPU set. In the ABB set, an aspiration syringe was placed in the front part of the new tool's handle, next to the transducer, so as to enable direct collection of the sample into the syringe. To obtain a sufficient amount of testable fluid, reservoirs of urine (rubber balloons) were used for later aspiration under laboratory conditions in Experiment 1. Fifteen triads of samples (each triad with two punctures) were collected. While the first sample of each triad was taken using the ABB set (ABB sample), two samples were taken by one puncture using the original modified commercial OPU set: aerobic phase of sampling (AE sample) with air present in the tubing at the start of sampling and the subsequent anaerobic phase of sampling (AN sample). Values determined in the second sample from the triad (AE) varied from the values in both ABB and AN samples (pH 7.685 vs. 7.704 vs. 7.692, pCO2 11.13 vs. 10.3 vs. 10.85, pO2 6.87 vs. 8.67 vs. 7.02). In Experiment 2, ultrasound-guided transvaginal aspirations were carried out in 13 cows bearing ovarian cysts with diameters of at least 3 cm, using plastic aspiration syringes (Experiment 2P) and in 12 cows using glass aspiration syringes (Experiment 2G). The sequence of samples was the same as in Experiment 1. We found a significantly higher pH in AE in comparison to AN (7.357 vs. 7.348), lower pCO2 (6.85) and higher O2 (14.12) in samples of AE in comparison to samples of ABB and AN (pCO2 7.36, 7.30; O2 9.95, 10.63 respectively) in cystic fluid in Experiment 2P. We found a significantly higher pH (7.4), lower pCO2 (5.98) and a higher pO2 (12.35) in AE samples in comparison to ABB and AN samples of cystic fluid (pH 7.386, 7.385; pCO2 6.39, 6.35 and O2 10.56, 10.65, respectively) from Experiment 2G. We conclude that the acid-base balance assay was affected by air, present in the tubing during aerobic sampling in comparison to anaerobic and ABB set sampling. These pre-analytical changes can be prevented by the use of the ABB set because the results obtained with the ABB set were not different from that of the AN samples. We also confirmed pre-analytical changes in acid-base balance parameters in the cystic fluid after it had been stored in plastic aspiration syringes. Our new ABB set equipped with a glass aspiration syringe is suitable for sampling follicular fluid for both acid-base balance and gas analysis.
  • Article
    Polychlorinated biphenyls (PCBs), DDT and its metabolite (DDE) belong to estrogen-like endocrine disruptors. However, though their activity is approximately 1000-fold lower than the activity of estradiol (E2), this steroid's high concentration in follicular fluid and incubation media does not inhibit the influence of these xenobiotics. It was hypothesized that these xenobiotics might affect Steroidogenic Factor-1 (SF-1) and impair ovary function. To test this hypothesis, granulosa cells were obtained from ovarian follicles >1 or <1cm in diameter, which were treated with PCB-77, PCB-153, DDT or DDE (each at 10ng/ml), alone or jointly with an SF-1 antagonist (F0160). Treatment with the SF-1 antagonist inhibited (P<0.05) the secretion of P4 from cells of both sizes of follicles, as induced (P<0.05) by an SF-1 activator (HxP), DDE or PCB-153. All xenobiotics and HxP stimulated (P<0.05) the synthesis and secretion of oxytocin (OT). However, the effect on mRNA expression for NP-I/OT, which is OT precursor, was inhibited (P<0.05) by F0160 in all cultures treated with PCB-77, except for granulosa cells derived from follicles <1cm. Moreover, F0160 inhibited the effect on OT secretion of HxP, as well as all xenobiotics except for PCB-77 and DDE, in granulosa cells derived from follicles <1cm. Xenobiotic treatment did not affect (P>0.05) the expression for SF-1 mRNA. It is suggested that the SF-1 receptor may be involved in the adverse effects of xenobiotics on P4 secretion as well as the synthesis and secretion of OT.
  • Article
    Insulin signaling regulates various aspects of physiology, such as glucose homeostasis and aging, and is a key determinant of female reproduction in metazoans. That insulin signaling is crucial for female reproductive health is clear from clinical data linking hyperinsulinemic and hypoinsulinemic condition with certain types of ovarian dysfunction, such as altered steroidogenesis, polycystic ovary syndrome, and infertility. Thus, understanding the signaling mechanisms that underlie the control of insulin-mediated ovarian development is important for the accurate diagnosis of and intervention for female infertility. Studies of invertebrate and vertebrate model systems have revealed the molecular determinants that transduce insulin signaling as well as which biological processes are regulated by the insulin-signaling pathway. The molecular determinants of the insulin-signaling pathway, from the insulin receptor to its downstream signaling components, are structurally and functionally conserved across evolution, from worms to mammals - yet, physiological differences in signaling still exist. Insulin signaling acts cooperatively with gonadotropins in mammals and lower vertebrates to mediate various aspects of ovarian development, mainly owing to evolution of the endocrine system in vertebrates. In contrast, insulin signaling in Drosophila and Caenorhabditis elegans directly regulates oocyte growth and maturation. In this review, we compare and contrast insulin-mediated regulation of ovarian functions in mammals, lower vertebrates, C. elegans, and Drosophila, and highlight conserved signaling pathways and regulatory mechanisms in general while illustrating insulin's unique role in specific reproductive processes. This article is protected by copyright. All rights reserved.
  • Article
    The negative energy balance (NEB) during the early postpartum period in high yielding dairy cows has clearly been linked to diminished reproductive performance. As for follicular growth and development, nearly half of all modern dairy cows suffer from an ovarian dysfunction during the first weeks after calving. Several hormones and metabolites may act as metabolic cues for the NEB, thereby affecting follicular development at both the hypothalamic and the ovarian levels. The main metabolic cue for the hypothalamus seems to be metabolic fuel availability, i.e. glucose, and hormones like insulin and leptin may have a direct permissive effect on gonadotrophin secretion or an indirect effect by affecting fuel partitioning and availability. At the ovarian level, NEB can affect follicle growth by the reduced insulin, insulin-like growth factor I and leptin concentrations. Metabolites like non-esterified fatty acids may influence follicle growth at both the hypothalamic and the ovarian levels.
  • Article
    Full-text available
    Diets can alter the concentrations of circulating hormones such as insulin and IGF-I. Such responsive hormones are related directly to nutritional status and moreover, directly or indirectly, associated with reproductive function and fertility. Metabolic hormones are involved in follicular development, number and size of ovarian structures, circulating concentrations of steroid hormones, duration of estrus, steroidogenesis, ovulation and embryonic development. However, circulating metabolic hormones in excess, resulting from high dry matter/energy intake can also contribute to the reduction of oocyte and embryo quality. Although changes in dietary intake affect ovarian function in Bos taurus and Bos indicus cattle, it seems that overfeeding influences more profoundly oocytes/embryos from heifers and cows of Bos taurus than of Bos indicus breeds. There is also a distinct effect of nutrition on in vitro vs. in vivo embryo production, in which metabolic hormones seem to affect more the later stages of follicle development. Thus, this paper presents and discusses the results of some relevant studies on the role of feed intake and its association with metabolic hormones in bovine reproduction.
  • Article
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    Copyright: © 2014 Izquierdo AC. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editorial During the last two decades, advances in genetic improvement, the area of nutrition and the improvement in the handling of animals, have made production averaged specialized cow's milk production is increasing; however, such an increase in milk production, has had a negative impact on reproductive performance of these animals, such as low conception rates, which do not correspond to those that were achieved 20 to 30 years ago; so that before they could achieve conception rates 60%, at present and in the best case, only be achieved 40 to 50%. From all this, we can say that today, you can take the 15 to 16% of cows with estrus repetitions problems, which means achieving at least 45% of conceptions. At present, the problem of repeat breeders, is considered as the second reproductive problems after problems postpartum anestrus, mainly in units bovine production tropics, which is considered as a problem of low fertility, sometimes, is below 30% of pregnancies [1-3]. From the practical point of view, the problems of low fertility, is present when the following symptoms are observed: -Return to estrus 19 to 23 days after the third service or artificial insemination, without apparent cause. -Display of normal service intervals. -No abnormal genital discharge. -Without the presence of abnormalities in the genital organs, detectable by rectal palpation [2,4]. It is known that fertility problems in animal production units (UPA), are multifactorial disorders among them in oogenesis, degenerated oocytes, disorders of ovulation, fertilization failure, inflammation of the ovaries, disorders of the oviducts, the uterus changes as metritis and endometritis, early embryonic mortality, among others [5-7]. On the other hand, the low fertility of the animals in UPA, is directly related to metabolic disorders, resulting in the high producing cows, usually, may have negative energy balance during the first 70-80 days postpartum.
  • Article
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    Data derived principally from peripheral tissues (fat, muscle and liver) show that insulin signals via diverse interconnecting, intracellular pathways and that some of the major intersecting points (known as critical nodes) are the IRSs (insulin receptor substrates), PI3K (phosphatidylinositol 3-kinase)/Akt and MAPK. Most of these insulin pathways are probably also active in the ovary and their ability to interact with each other and also with follicle stimulating hormone (FSH) and luteinizing hormone (LH) signalling pathways enables insulin to exert direct modulating influences on ovarian function. This paper reviews the intracellular actions of insulin and the uptake of glucose by ovarian tissues (granulosa, theca and oocyte) during the oestrous/menstrual cycle of some rodent, primate and ruminant species. Insulin signals through diverse pathways and these are discussed with specific reference to follicular cell types (granulosa, theca and oocyte). The signalling pathways for FSH in granulosa cells and LH in granulosa and theca cells are summarized. The roles of glucose and of insulin-mediated uptake of glucose in folliculogenesis are discussed. It is suggested that glucose in addition to its well established role of providing energy for cellular function may also have insulin-mediated signalling functions in ovarian cells, involving AMPK and/or hexosamine. Potential interactions of insulin signalling with FSH or LH signalling at critical nodes are identified and the available evidence for such interactions in ovarian cells is discussed. Finally the action of the insulin sensitizing drugs, metformin and the thiazolidinedione, rosiglitazone on follicular cells is reviewed
  • Article
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    La enfermedad quística ovárica bovina es una importante enfermedad que afecta la fertilidad del ganado bovino lechero. Diversos procesos ováricos tales como la proliferación celular y la esteroidogénesis se encuentran regulados por numerosas hormonas entre las que se encuentra la insulina. La respuesta a esta hormona se desencadena luego de la unión a su receptor específico. El objetivo de la presente revisión es analizar la función de intermediarios claves en la vía de señalización intracelular, que actúan en los primeros estadios involucrados en la respuesta a insulina. Se analizan el receptor de insulina (IR), el sustrato -1 del IR (IRS1) y la enzima fosfatidilinositol 3-quinasa (PI3K) denotando modificaciones ocurridas en folículos quísticos que podrían influenciar negativamente en la funcionalidad de ovarios y contribuir a la persistencia folicular.
  • Article
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    Background Insulin has been used as a stimulatory factor for in vitro cell culture since many years. Even for routine in vitro embryo production (IVP), insulin is added to the media during different steps. There is a strong difference in concentrations used in vitro compared to what is measured in vivo in follicular fluid or serum. We performed a pilot study on insulin stability to explain possible reasons for that variation. ResultsWe measured insulin concentrations before and after bovine oocyte maturation in an experiment by using a quantitative ELISA (Mercodia bovine insulin ELISA immunoassay) and found that concentrations were stable up to 22 h of incubation. We compared our results with eleven in vivo studies measuring insulin in either serum or follicular fluid and nine IVP-protocols using insulin. In all studies, in vitro concentrations were much higher compared with those found physiologically in vivo. Limited knowledge is available concerning the different activity and stability of insulin in vitro versus in vivo. Conclusions The concentrations of insulin used in vitro are quite high in comparison to physiological concentrations found in serum or follicular fluid. One explanation may be a different stability or activity of insulin in vitro even if we could measure stable concentrations of insulin in our pilot study. More precise dose–effect studies have to be performed to draw clear conclusions about the consequences of the use of such high doses as they might have negative consequences for the developing embryo. Insulin has direct effects on the regulation of the metabolism and could even influence the epigenetic programming of the metabolism with unknown consequences for the offspring later in life.
  • Article
    Introduction Separation of chromosomes in meiosis is a well-guarded process such that errors in chromosome segregation are rare events. For instance, only 1 in every 100000 divisions in yeast is associated with non-disjunction. Aneuploidy in germ cells of mammals like the mouse is generally much higher, in the range 0.5–1% [1]. Furthermore, there is a gender-specific difference in susceptibility to meiotic errors during germ cell formation in mammals, particularly in humans. On average, only 1–4% of sperm in healthy men have numerical chromosomal aberrations, while on average about 20% of all human meiosis II oocytes are aneuploid [1–4]. The correlation between the incidence of the birth of a trisomic child with Down's syndrome and maternal age was first recognized in 1933 by Penrose [5], confirmed by chromosomal analysis of spontaneous abortions and live births and, since the introduction of assisted reproduction, by evidence from polar bodies, oocytes, and embryos (e.g., [3, 6–8]). However, the cause(s) of the extraordinary susceptibility of aging oocytes to meiotic errors was obscure until recently. In typical mitosis there is division of sister chromatids derived from replication of each chromosome, and each pair therefore carries the same alleles along their arms (Figure28.1A). In contrast, in meiosis I the two originally paternally and maternally derived homologs, each containing two sister chromatids, separate during first meiotic division (termed reductional division, Figure28.1B, Ciii). They are normally physically attached to each other by at least one chiasma from recombination between sister chromatids of parental homologs (indicated by X in Figure28.1B, Ci, Ciii). Chiasmata are held in place by cohesion between sister chromatid arms and centromeres (Figure28.1Ci–Ciii) placed on chromatids before S-phase (Figure28.1Ci), which is maintained until anaphase I (Figure28.1Ciii, B) (reviewed in [9]). The paternal and maternal alleles along chromatid arms of recombined chromosomes switch left and right of a chiasma or exchange (indicated by different coloring in Figure 28.1B, Ci, E–J). Hence, the distribution of polymorphisms can be used to trace the origin and recombinational history of a chromosome in a zygote or child.
  • Article
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    The aim of this study was to evaluate the applicability of ovum pick-up equipment for follicular fluid collection from various follicular structures (experiment 1) and for recovery of follicular fluid for acid-base balance analysis (experiment 2). An ultrasound scanner equipped with a 5-MHz convex transducer was used for transvaginal ultrasound-guided follicular aspiration. A 17-gauge, 60-cm aspiration needle was connected with a shortened aspiration line. The fluid was aspirated manually into a 2 ml plastic syringe at a speed of approximately 0.2 ml/s. The success of aspiration was higher in ovarian cysts (100%) and single follicles larger than 13 mm (76.7%) compared to single follicles smaller than 12 mm (20%, p < 0.001). The success of aspiration of multiple follicles on day 4 (diameter of 7-9 mm) was higher (90.9%) compared to follicles on day 2 (diameter of 4-6 mm) (66.7%, p < 0.05) in experiment 1. The fluid from ovarian cysts > 25 mm in diameter was aspirated in a two-step procedure (samples 1 and 2) for the determination of pH, HCO(3), BE, pCO(2) and pO(2) (experiment 2). The indicators were compared between samples 1 and 2. Higher pO(2) as well as pH and lower pCO(2) in sample 1 compared to sample 2 showed insufficient anaerobic conditions during the first phase of the puncture in experiment 2. Our study brings for the first time the finding that the ovum pick-up equipment used in our experiments is suitable for the collection of follicular fluid only from larger follicular structures. The sampling of follicular fluid for acid-base balance assays requires the development of a special new device to prevent samples from coming into contact with air during aspiration.
  • Article
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    La nutrición tiene una influencia en el comportamiento reproductivo de la hembra bovina, pero el mecanismo por el cual se ve afectado dicho desempeño es poco conocido. Aunque las investigaciones se han centrado en la influencia de la nutrición sobre el eje hipotálamo-hipófisis, estudios más recientes han probado la hipótesis que las hormonas metabólicas, como señales nutricionales, ejercen un efecto directo en el ovario. En el ganado, el tratamiento con somatotropina bovina recombinante (rGH) aumenta la población de pequeños folículos ováricos; esto está asociado con el aumento en las concentraciones circulantes de insulina y con el factor de crecimiento insulínico I (IGF-I). Otros estudios, tanto in vitro como in vivo, demuestran la importancia de la acción conjunta del IGF-I o la insulina con la FSH y la LH. También, se han obtenido evidencias de que al alimentar novillas supliendo el 200% de las necesidades de mantenimiento durante un corto periodo, se aumentan las concentraciones circulantes de insulina y la población de pequeños folículos ováricos. Teniendo en cuenta los últimos hallazgos, los trabajos se han encaminado a resolver algunos problemas prácticos en la reproducción del ganado de leche. En primer lugar, tanto el tratamiento previo con rGH como el aumento de la ingesta alimentaria mejoran la viabilidad embrionaria. En segundo lugar, al formular dietas destinadas a aumentar la concentración de insulina durante la lactancia temprana, se nota un pequeño aumento del consumo voluntario y que estos animales llegan más prontamente a la primera ovulación posparto, lo cual, además, muestra un aumento de la tasa de concepción al primer servicio.
  • Article
    Metabolic changes in blood serum may be reflected in the biochemical composition of follicular fluid and can be indirectly influenced oocyte quality. The purpose of this study was to investigation metabolite composition variations of follicular fluid and blood serum in Iranian dromedary camels during the peak breeding season (October-march). Following slaughter, blood samples were collected from 50 female camels and follicular fluid aspirated from small (5-9 mm) and large (10-20 mm) follicles were analyzed for various metabolite concentrations, using the commercial kits.
  • Article
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    This study is performed to evaluate the effect of insulin in the porcine parthenogenetic embryo development. In porcine embryo culture, insulin is helpful factor in the process of embryo development. To identify this, insulin is used in pig embryos development. Therefore, this study was performed to investigate the effect of insulin on early embryonic development in pigs. For that, insulin positive or negative (0, 10 ug/mL) was supplemented in the porcine IVM media and then compared two groups divided by the cytoplasm of the black groups and white ring groups based on the distribution of lipid material of the cell cytoplasm in microscope. In maturation rates of porcine oocytes, significant higher black group rates were shown in the insulin positive groups compared with other groups (56.0±2.1 vs 46.2±0.3). In the embryo culture, black groups were showed the significant higher cleavage rates (82.1±0.8, 78.3±0.1 vs 63.2±0.3, 63.4±0.0), and blastocyst formation rates (15.5±3.6, 16.6±0.4 vs 11.7±1.3, 7.4±0.2) regardless of whether the addition of insulin. Also, black groups were showed higher cell number of blastocyst (33.2±2.5, 35.5±2.6 vs 31.2±2.1, 31.3±2.2). In conclusion, supplement of insulin producing black group in vitro maturation, it was effective in vitro maturation and embryonic development of pig embryos. The capability of porcine oocytes to in vitro mature, fertilize, and develop has been proved by several researchers, and successful methods for in vitro maturation and fertilization of porcine oocytes and culture of porcine embryos have been developed. But, even if pig oocytes develop to blastocyst stages by fertilization in vitro developmental potential is higher than in vivo (Day 1993). In the first experiment, determining of first polar body extraction oocytes were selected for in vitro maturation of parthenogenesis.
  • Article
    The formation of the meiotic spindle is a critical process to assure accurate chromosome segregation and subsequent embryo development. Coordinated formation and organization of microtubules, centrosomes, and chromosomes is important for meiotic spindle formation at the oocyte's center after germinal vesicle breakdown (GVBD), for the formation of the MI (meiosis I) spindle to segregate homologous chromosomes, and for the formation of the MII (meiosis II) spindle to segregate chromatids, resulting in oocyte haploidy. The human oocyte is particularly susceptible to errors in chromosome segregation which may be related to defective centrosome and microtubule organization and to defective chromosome attachment to kinetochore microtubules and loss of molecular surveillance factors. The present chapter is focused on (1) formation of central, MI and MII spindle, with focus on microtubules and centrosomes; (2) chromosome dynamics and segregation during MI and MII, with focus on molecular aspects and surveillance mechanisms; and (3) spindle abnormalities, environmental influences, and possible treatments to restore spindle integrity with implications for assisted reproductive technologies (ART). The formation of the meiotic spindle is a critical step during oocyte maturation and begins when the germinal vesicle breaks down (GVBD) as a result of stimulation by luteinizing hormone (LH). Spindle formation in most mammalian oocytes takes place at the oocyte's center and involves significant restructuring of the cytoskeleton that will impact subsequent cellular and molecular functions that are also important for later development [1]. Coordinated formation and organization of microtubules, centrosomes, and chromosomes begins directly after GVBD with remodeling of these major spindle components in the oocyte's center to form the meiotic spindle.
  • Article
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    The positive relationship between nutritional state and ovulation rate in sheep may involve the action of specific nutrients on gonadotrophin release. LH and FSH secretion is controlled in part by hypothalamic GnRH, which is in turn influenced by central adrenergic and serotonergic neuronal systems. In this experiment the branched chain amino acids (BCAAs) leucine, isoleucine and valine were examined for effects on LH and FSH secretion. A mixture of the three amino acids was infused into ewes for 5 days immediately before luteolysis, a time when nutritional effects on ovulation rate occur. The infusion significantly increased ovulation rate without any associated increase in FSH or LH. However, the infusion did increase plasma insulin concentrations and this effect, together with the high levels of blood urea observed, suggests that these amino acids had increased the supply of energy substrates to the follicles. An increase in insulin-mediated glucose uptake by follicles could be the stimulus responsible for the increase in ovulation rate. The ability of the animal to utilize BCAAs for energy metabolism may be an important component of the ovulation responses to nutrition.
  • Legislation on animal welfare (defending animal rights), Paragraph 14 Kenesset Law Pub Effects of energy balance on hormones, ovarian activity and recovered oocytes in lactating Holstein cows using transvaginal follicular aspiration
    • Jerusalem
    • S Israel
    • Landau
    Israel Council on Animal Care Guidelines, 1994. Legislation on animal welfare (defending animal rights), Paragraph 14. Kenesset Law Pub., Jerusalem, Israel (in Hebrew). r196 S. Landau et al./Animal Reproduction Science 64 (2000) 181–197 Kendrick, K.W., Bailey, T.L., Garst, A.S., Pryor, A.W., Ahmadzadeh, A., Akers, R.M., Eyestone, W.E., Pearson, R.E., Gwazdauskas, F.C., 1999. Effects of energy balance on hormones, ovarian activity and recovered oocytes in lactating Holstein cows using transvaginal follicular aspiration. J. Dairy Sci. 82, 1731–1740
  • Article
    To examine endocrine and biochemical differences between dominant and subordinate follicles and how the dominant follicle affects the hypothalamic-pituitary-ovarian axis in Holstein cows, the ovary bearing the dominant follicle was unilaterally removed on Day 5 (n = 8), 8 (n = 8), or 12 (n = 8) of synchronized estrous cycles. Follicular development was followed daily by ultrasonography from the day of detected estrus (Day 0) until 5 days after ovariectomy. Aromatase activity and steroid concentrations in first-wave dominant and subordinate follicles were measured. Intact dominant and subordinate follicles were cultured in 4 ml Minimum Essential Medium supplemented with 100 microCi 3H-leucine to evaluate de novo protein synthesis. Five days after unilateral ovariectomy, cows were resynchronized and the experiment was repeated. Follicular growth was characterized by the development of single large dominant follicles, which was associated with suppression of other follicles. Concentrations of estradiol-17 beta (E2) in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles (438.9 +/- 45.5 ng/ml; 875.4 +/- 68.2 pg E2/follicle) compared to subordinate follicles (40.6 +/- 69.4 ng/ml; 99.4 +/- 104.2 pg E2/follicle). Aromatase activity in first-wave dominant follicles was higher at Days 5 (1147.1 +/- 118.1 pg E2/follicle) and 8 (1028.2 +/- 118.1 pg E2/follicle) compared to Day 12 (450.7 +/- 118.1 pg E2/follicle). Concentrations of E2 and androstenedione in first-wave dominant follicles were higher at Day 5 (983.2 +/- 78.2 and 89.5 +/- 15.7 ng/ml) compared to Days 8 (225.1 +/- 78.6 and 5.9 +/- 14.8 ng/ml) and 12 (108.5 +/- 78.6 and 13.0 +/- 14.8 ng/ml). Concentrations of progesterone in subordinate follicles increased linearly between Days 5 and 12 of the estrous cycle. Plasma concentrations of FSH increased from 17.9 +/- 1.4 to 32.5 +/- 1.4 ng/ml between 0 and 32 h following unilateral removal of the ovary with the first-wave dominant follicle. Increases in plasma FSH were associated with increased numbers of class 1 (3-4 mm) follicles in cows that were ovariectomized at Day 5 or 8 of the cycle. Unilateral ovariectomy had no effects on plasma concentrations of LH when a CL was present on the remaining ovary. First-wave dominant follicles incorporated more 3H-leucine into macromolecules and secreted high (90,000-120,000) and low (20,000-23,000) molecular weight proteins that were not as evident for subordinate follicles at Days 8 and 12.(ABSTRACT TRUNCATED AT 400 WORDS)
  • Article
    In this study we examined, in two experiments, patterns of follicular development and dominance under conditions of heat stress. Estrous cycles were programmed to include two follicular waves (wave 1 and 2). On Day 1 of the estrous cycle (Day 0 = estrus), cows were assigned randomly to cooled (C; n = 6) or heat-stressed (H; n = 6) groups. In experiment 1, on Day 12 prostaglandin (PG) F2 alpha was injected and a controlled intravaginal drug release device (1.9 g progesterone) was inserted (this was removed on Day 17). In experiment 2, PGF 2 alpha was injected on Day 14. Ovarian structures were examined daily by ultrasonography, and blood samples were collected at each scanning. Cycle lengths were 20 and 17 days in experiments 1 and 2, respectively. Mean maximal body temperatures were higher (p < 0.01) in H (40.3 degrees C) than in C (38.8 degrees C) cows. In experiment 1, the rate of increase in number of large follicles (> or = 10 mm) was greater in H than in C cows (p < 0.01), resulting in 53% more large follicles in H cows during wave 1; this was associated with a lower (p < 0.05) number of medium-sized (6-9 mm) follicles between Days 7 and 10 of the cycle. Heat stress hastened (p < 0.02) the decrease in size of the first-wave dominant follicle and hastened (p < 0.01) the emergence of the second dominant (preovulatory) follicle by 2 days.(ABSTRACT TRUNCATED AT 250 WORDS)
  • Article
    The objective was to determine the effects of selection for milk yield in Holsteins on blood plasma concentrations of somatotropin, prolactin, insulin, and thyroxine. Two genetic groups, one selected since 1964 for milk yield and one an unselected control, were used for this purpose and averaged 9878 and 7402 kg milk, respectively. All cows initiating normal lactations between August 1983 and August 1984 (29 selection and 23 control) were blood sampled at 42 and 161 d postpartum. On these days, three blood samples were taken at 3.5-h intervals from each cow via jugular venipuncture for radioimmunoassay. Data were analyzed as split-plots with repeated measures. Whole plot effects included genetic group, season of sampling, and parity. Subplot effects included stage of lactation and sample number within day. Somatotropin concentration increased, insulin concentration decreased, and prolactin and thyroxine concentrations remained unchanged with selection for milk yield. Thyroxine and insulin concentrations tended to decrease with age. Somatotropin concentrations decreased from peak to midlactation, whereas insulin and thyroxine concentrations increased.
  • Article
    The effect of ruminally degradable starch on the reproductive performance of sheep was studied in 50 Booroola x Assaf crossbred ewes, of which 32 were carriers of the FeCB (Booroola) allele and 18 were not carriers. The sheep were group-fed, according to body weight (BW), diets composed of pea straw, soybean concentrate and corn grain offered in two meals per day. Dietary differences in ruminally undegradable starch (RUS) were obtained during the 29 day treatment period by feeding grain as whole corn (WC, 1.4 g RUS day−1 kg−1 BW) or extruded (EC, 0.4 g RUS day−1 kg−1 BW ). Oestrus was synchronized by inserting progestagen pessaries at Day 8, and withdrawing pessaries and injecting 400 IU of pregnant mare serum gondaotrophin at Day 20 after initiation of dietary treatments (Day 0). Ovulation rate (OR) was estimated by laparoscopy. The FecB allele was associated with a significant increase in OR (2.88 vs. 1.40, P⪕ 0.0001) and prolificacy (1.85 vs. 1.32 lamb born per ewe lambing, P⪕0.09). Within FecB allele carriers, all WC-fed ewes had multiple ovulations, compared with 61% of EC-fed ewes (P⪕0.005). OR values were 3.29±0.27 and 2.46±0.31 corpora lutea (CL) per ewe ovulating and prolificacy was 2.13±0.22 and 1.57±0.23 lamb per ewe lambing in WC- and EC-fed ewes, respectively (P⪕ 0.09). In non-carrier ewes, dietary treatments did not affect OR (1.44 and 1.36 CL per ewe ovulating in WC- and EC-fed ewes) or prolificacy (1.20 and 1.44 lamb per ewe lambing, in the same order). In a second experiment, aimed to assess the effect of dietary RUS on circulating insulin levels, blood was sampled before and at hourly intervals for 10 h after a meal from ten ewes fed rations with WC or EC. Insulin levels were higher in EC than in WC-fed ewes before and 3 h after a meal (P⪕ 0.05). EC feeding may be associated with higher peripheral insulin concentration but does not enhance OR. Data are used to show that: (1) provision of about 100 g RUS day−1 exerts a positive effect on the OR and prolificacy of FecB carriers; (2) dietary treatments may affect OR in sheep of different ovulation potential differently; (3) no positive relationship was found between circulating plasma insulin concentration and OR.
  • Article
    Supplementing mature rams with lupin seed (Lupinus angustifolius, a highly digestible source of energy and protein) increases gonadotrophin secretion within 5-10 days. When sheep receive a post-ruminal supplement of protein and energy equivalent to that in the lupin supplement, LH secretion is increased but not to the same extent as with lupin seed itself. This suggests that lupin seed contains a specific mixture of nutrients or an unknown component that exerts effects on the brain centres that control gonadotrophin secretion. This hypothesis was tested by comparing the responses to isonitrogenous and isoenergetic supplements of 3 legumes: lupins, cowpeas, and soybeans. Rams were fed the supplements for 10 days and blood was sampled every 20 min for 24 h on days -1, 5, and 10 relative to the start of supplementation. The plasma was assayed for LH, FSH, and the metabolic hormones insulin and insulin-like growth factor 1 (IGF-1). Both the cowpea and the lupin supplements increased LH pulse frequency and the mean concentrations of insulin and IGF-1 on days 5 and 10 compared with day -1 (P < 0.05). The soybean supplement did not affect LH pulse frequency or the concentrations of insulin or IGF-1. Only the cowpea supplement increased the mean concentration of FSH. The nutritional stimulation of the reproductive centres of the brain appears not to be specific to lupins, because the diet supplemented with cowpeas evoked similar responses. The lack of response to soybean seed suggests that the effects of diet on secretion of metabolic and gonadotrophic hormones are not due simply to the total energy and protein content of the diets, but to subtle differences in their chemical constituents.
  • Article
    In situ evaluation of the duodenal amino acid (AA) profile was attempted in a 4 × 4 Latin square study using four lactating Holstein cows fitted with ruminal and duodenal cannulas. Dietary supplemental crude protein (CP) sources, making up 40% of the dietary CP, were: soybean meal, cottonseed meal, corn gluten meal or urea. Duodenal flow of organic matter (OM) and CP was determined in vivo by means of constant infusion of ytterbium and chromium, and predicted in situ from rumen degradation. Purines were used as microbial markers. In situ calculation of the AA profile, expressed as g AA in 100 g of total AAs, was based on the composition of AA in feed and in isolated bacteria as well as on in situ rumen degradation of OM and CP. The AA profile in the duodenum when assessed in vivo was affected by the nature of the supplemental protein; in situ assessment of the AA profile resulted in comparable profiles. The data are interpreted to suggest that the in situ method enables prediction of the profile of AA (g AA/100 g total AA) flowing to the duodenum, and that the undegraded portion of supplemented protein affects that profile.
  • Article
    To obtain information on the physiological mechanisms controlling the partition of dietary energy between body weight and milk production in lactating cattle, the concentrations of hormones (prolactin, growth hormone, insulin and thyroxine) and metabolites (glucose, non-esterified fatty acids, β-hydroxybutyric acid and l-lactic acid) in plasma obtained from eight high-yielding and seven low-yielding cows, matched for diet and stage of lactation, were compared. Blood samples were taken via a jugular catheter, throughout four 48 h periods at various times during lactation (days 40, 80, 120 and 180) and also when the animals were dry. The milk yield rose for 7 weeks after parturition, reaching peak values of 24·8 and 10·1 kg/day in the high- and low-yielding groups respectively. The body weights of the low-yielding animals rose steadily, whereas cows in the high-yielding group lost weight up to week 14 of lactation and thereafter gradually gained weight. No differences were found between the groups in the digestibility of the diets, although analysis of the rumen fluid indicated small differences in the proportions of volatile fatty acids. Milk analysis suggested that the protein content of milk obtained from low-yielding cows was greater than that of milk from high-yielding animals. Throughout lactation, the concentrations of growth hormone (P < 0·001), non-esterified fatty acids (P < 0·01) and β-hydroxybutyric acid (P < 0·05) were higher in the high-yielding than in the low-yielding group, whereas the concentration of insulin was higher (P < 0·01) in the low-yielding animals. The level of thyroxine was higher (P < 0·05) in the low-yielding group when the animals were dry. A comparison of peak lactation (days 40 and 80) with the dry period demonstrated that the concentration of prolactin was higher in both groups high-yielding, P < 0·01; low-yielding, P < 0·001) when the animals were dry and the concentration of thyroxine in the low-yielding cows was higher (P < 0·01) when the animals were dry. Significant reductions in the concentrations of growth hormone (P < 0·001) and non-esterified fatty acids (P < 0·001) were accompanied by an increase in the concentration of glucose (P < 0·01) when the high-yielding animals were dry.
  • 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
    Full-text available
    Glucose metabolism was studied in ewes fed 800 g chopped alfalfa hay (H) or 400 g alfalfa hay and 400 g corn grain given in whole (HWC), ground (HGC) or extruded (HEC) form. Daily intake of metabolisable energy and crude protein were: 5.8 MJ, 109 g; 9.0 MJ, 84 g; 9.5 MJ, 84 g and 8.5 MJ, 88 g in H, HWC, HGC and HEC, respectively. In situ ruminal degradability ranked whole, ground, and extruded corn in ascending order. Ruminal pH and concentration of acetic acid were lower and of propionic acid higher (P less than 0.05) in HEC than in HGC and HWC groups. Plasma level of glucose (P less than 0.10), insulin (P less than 0.05), and the ratio of insulin to non-esterified fatty acids (NEFA) (P less than 0.01) were higher in HEC than in other groups. Glucose irreversible loss (GILR) and entry rate (GER), recycling (GRec) and reentry (GRee) were determined by double isotope dilution procedure. GER, but not GILR, was higher in HWC than in H and HGC (6.98 mg/min/kg BW0.75 vs 3.97 and 4.24 mg/min/kg BW0.75, respectively; P less than 0.05) and than in HEC (4.84 mg/min/kg BW0.75; P less than 0.10). GRec and GRee were higher in HWC than in the other treatments. Grinding or extruding the grain increased ruminal degradability and decreased glucose entry rate.
  • Article
    Thirty-four multiparous, lactating, cyclic beef cows which calved in moderate body condition were used to determine effects of restricted nutrition on corpus luteum (CL) development and endocrine status. At 78 d postpartum, six cows were assigned to a control (CON) diet (26.0 Mcal ME), fed to increase bodyweight (BW) and body condition score (BCS), and the remaining 28 cows were fed to lose BW and BCS on a restricted (RES) diet (14.0 Mcal ME). Following a 40-d adjustment period on respective diets, estrous cycles were synchronized and cows bled daily for determination of progesterone (P4), luteinizing hormone (LH) and insulin (INS) beginning at the synchronized estrus. Ultrasonography was used to determine the ovulatory follicle and CL development. Control cows were maintained for one estrous cycle and were ovariectomized on day 11 of their second cycle. Ten cows on restricted diet (RES-C) continued to form a functional CL (P4 > 1.5 ng/ml at day 10 of an estrous cycle) through as many as 5 cycles, after which observations were discontinued. Fourteen cows on restricted diet (RES-A) were ovariectomized on day 11 of a cycle when a CL was identified by ultrasonography, but was subfunctional (P4 < 1.5 ng/ml on day 10 of that cycle). Four additional RES-A cows which had subfunctional CL were not ovariectomized but were bled for an additional 25 d. At ovariectomy, CL and ovarian weights were collected. Luteal tissue was prepared for evaluation of P4 synthesis, LH responsiveness in vitro, and for determination of P4 content and total LH receptors. Bodyweight and BCS increased in CON cows; whereas, RES cows lost BW and BCS (P < .05). In the cycle prior to ovariectomy, serum P4 and LH were not different in 18 RES-A cows which developed subfunctional CL in comparison to CON cows. Four RES-A cows not ovariectomized but bled for an additional 25 d neither exhibited estrus, ovulated, nor had P4 concentrations greater than .3 ng/ml. Serum INS was lower in RES-A cows during the cycle prior to ovariectomy than in CON cows (P < .05). During the 11-d period prior to ovariectomy, mean serum P4 and INS were lower in RES-A cows than in CON cows (P < .05); however, serum LH was not different. Furthermore, CL and ovarian weights, P4 content of CL, secretion of P4 by luteal tissue in response to LH in vitro and LH receptor number were not different between CON and RES-A cows. In conclusion, nutritional anestrus may be preceded by the formation of a CL with lower steroidogenic output in vivo. However, luteal tissue, collected from RES-A cows, did not appear to be subfunctional during in vitro incubation when substrate availability and gonadotropin support were equal between diets.
  • Article
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    To examine endocrine and biochemical differences between dominant and subordinate follicles and how the dominant follicle affects the hypothalamic-pituitary-ovarian axis in Holstein cows, the ovary bearing the dominant follicle was unilaterally removed on Day 5 (n = 8), 8 (n = 8), or 12 (n = 8) of synchronized estrous cycles. Follicular development was followed daily by ultrasonography from the day of detected estrus (Day 0) until 5 days after ovariectomy. Aromatase activity and steroid concentrations in first-wave dominant and subordinate follicles were measured. Intact dominant and subordinate follicles were cultured in 4 ml Minimum Essential Medium supplemented with 100 microCi 3H-leucine to evaluate de novo protein synthesis. Five days after unilateral ovariectomy, cows were resynchronized and the experiment was repeated. Follicular growth was characterized by the development of single large dominant follicles, which was associated with suppression of other follicles. Concentrations of estradiol-17 beta (E2) in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles (438.9 +/- 45.5 ng/ml; 875.4 +/- 68.2 pg E2/follicle) compared to subordinate follicles (40.6 +/- 69.4 ng/ml; 99.4 +/- 104.2 pg E2/follicle). Aromatase activity in first-wave dominant follicles was higher at Days 5 (1147.1 +/- 118.1 pg E2/follicle) and 8 (1028.2 +/- 118.1 pg E2/follicle) compared to Day 12 (450.7 +/- 118.1 pg E2/follicle). Concentrations of E2 and androstenedione in first-wave dominant follicles were higher at Day 5 (983.2 +/- 78.2 and 89.5 +/- 15.7 ng/ml) compared to Days 8 (225.1 +/- 78.6 and 5.9 +/- 14.8 ng/ml) and 12 (108.5 +/- 78.6 and 13.0 +/- 14.8 ng/ml). Concentrations of progesterone in subordinate follicles increased linearly between Days 5 and 12 of the estrous cycle. Plasma concentrations of FSH increased from 17.9 +/- 1.4 to 32.5 +/- 1.4 ng/ml between 0 and 32 h following unilateral removal of the ovary with the first-wave dominant follicle. Increases in plasma FSH were associated with increased numbers of class 1 (3-4 mm) follicles in cows that were ovariectomized at Day 5 or 8 of the cycle. Unilateral ovariectomy had no effects on plasma concentrations of LH when a CL was present on the remaining ovary. First-wave dominant follicles incorporated more 3H-leucine into macromolecules and secreted high (90,000-120,000) and low (20,000-23,000) molecular weight proteins that were not as evident for subordinate follicles at Days 8 and 12.(ABSTRACT TRUNCATED AT 400 WORDS)
  • Article
    To determine whether systemic and/or intraovarian concentrations of insulin-like growth factor-I (IGF-I) are affected by short-term fasting, 24 heifers were blocked by weight and, within block, were assigned to one of three treatments: fasted for 0 h (controls; n = 8), fasted for 24 h (n = 8), or fasted for 48 h (n = 8). Blood plasma was collected every 8 h from -64 h to 0 h before ovariectomy (OVEX). OVEX was performed per vagina under local anesthesia during the follicular phase of an estrous cycle (36-42 h after synchronization with prostaglandin-F2 alpha). Follicular fluid (FFL) and granulosa cells were collected individually from follicles greater than or equal to 6 mm (large), and FFL was pooled from follicles 1.0-5.9 mm (small) in diameter. Fasting did not affect (p greater than 0.20) the number (mean +/- SE) of small (52 +/- 7) or large (1.5 +/- 0.4) follicles per heifer, specific binding of 125I-hCG to granulosa cells of follicles greater than or equal to 8 mm in diameter, or concentrations of progesterone in FFL of small follicles. At OVEX, body weight was less (p less than 0.01) for 24 h- and 48 h-fasted heifers (412 +/- 7 kg and 399 +/- 7 kg, respectively) than for 0 h-fasted heifers (442 +/- 7 kg). At OVEX, plasma concentrations of IGF-I were lower (p less than 0.05) in the 48 h-fasted group (105 +/- 8 ng/ml) than in the 0 h-fasted group (140 +/- 8 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)
  • Article
    The effects of intravenous infusion of 17 amino acids, each at a dose of 3 mmol/kg over 30 min, on the secretion of insulin, glucagon, and growth hormone (GH) were studied in 6 castrated male sheep. Insulin-like growth factor I (IGF-I) secretion was also studied using eight of the amino acids. Plasma alpha-amino nitrogen reached a peak at 30 min followed by a gradual decrease thereafter. The greatest increase was obtained using aspartic acid and the smallest with methionine, responses to the remaining amino acids lying between these two. Leucine was the most effective amino acid in stimulating insulin secretion but did not produce any increase in glucagon and GH secretion. Alanine, glycine, and serine induced a greater enhancement of both glucagon and insulin secretion than other amino acids. No amino acid was able to specifically stimulate glucagon secretion without also increasing insulin or GH secretion. With regard to insulin and glucagon secretion, amino acids could be divided into groups according to their R groups. Neutral straight-chain amino acids stimulated both insulin and glucagon secretion, with a greater secretory response to shorter C-chain amino acids. Branched-chain amino acids tended to enhance insulin and suppress glucagon secretion. Acidic amino acids caused an increase in GH secretion. Aspartic acid caused the strongest stimulation of GH secretion, exceeding that induced by arginine. No changes in plasma IGF-I were brought about by any of the amino acids tested.
  • Article
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    The net release of insulin, glucagon and somatostatin by the portal-drained viscera (PDV) and their net uptake by the liver in response to 3-d abomasal infusions of casein were measured in seven multicatheterized beef steers. The steers were fed 4.3 kg DM/d of a high-concentrate diet in 12 equal meals (13.1 Mcal ME/d and 95 g N/d). In two separate experiments, the abomasal infusion of 300 g casein/d (300C) or 150 g casein/d (150C) was compared to a water infusion. Plasma flow was measured by indicator dilution and net flux by venoarterial concentration difference x plasma flow. Arterial plasma concentrations of insulin were increased (P less than .02) by either 300C or 150C. The 300C increased (P less than .03) PDV insulin release but did not affect hepatic uptake, resulting in an increased (P less than .03) total splanchnic (TSP) insulin flux. The 300C increased (P less than .05) plasma concentrations of glucagon as the result of decreased (P less than .06) hepatic extraction ratio and not as the result of increased portal release. The portal and hepatic flux of somatostatin measured as somatostatin-like immunoreactivity (SLI) were highly variable and not affected by casein infusions. Arterial plasma concentrations of somatomedin-C were not responsive to abomasal casein infusions. The abomasal infusion of 300C resulted in increased plasma concentrations of insulin via increased PDV release and increased plasma glucagon via decreased hepatic extraction ratio.
  • Article
    The ruminant corpus luteum synthesizes and secretes oxytocin, but little is known of the regulation of these processes in the ovary. In the present work we describe a method for the preparation of cells from the early bovine corpus luteum (1-5 days postovulation) and their maintenance in serum-free culture. The release of oxytocin and progesterone from these cells was increased by the addition of insulin or insulin-like growth factor I (IGF-I), but not by IGF-II. Hormone release (measured between 60 and 84 h of culture) was increased approximately 5-fold (oxytocin) and 2.5-fold (progesterone) by maximally effective concentrations of IGF-I (EC50, 0.27 nM) and insulin (EC50, 1.94 nM). Sustained exposure (0-84 h) to prostaglandins (PGs) caused a dose-dependent reduction in oxytocin release in the presence of IGF-I (PGF2 alpha EC50, 31 nM; rank order of potency, PGF2 alpha greater than PGE2 greater than PGE1), but did not markedly reduce progesterone release. The inhibitory effect of PG on oxytocin production was mimicked by sustained exposure to a protein kinase-C activator (phorbol 12,13-dibutyrate), supporting the proposed role for this enzyme as a mediator of PG action. These data provide the first demonstration that oxytocin release from early bovine corpus luteal cell cultures can be regulated by insulin, IGF-I, and PGs. Since granulosa and/or luteal cells produce and respond to IGF-I and PGF2 alpha, our data indicate functional interaction of these compounds in the regulation of luteal cell activity.
  • Article
    To assess the mechanisms of insulin resistance in the ruminant, severe and adult-onset obesity was produced in Dorset ewes by overfeeding a high-energy ration over a 1- to 2-yr period. Body weights increased to 100 kg compared with 50 kg in lean control sheep; significant hyperinsulinemia (40 +/- 4 vs 10 +/- 1 microU/ml) also developed as did a moderate hyperglycemia (62 +/- 2 vs. 52 +/- 1 mg/100 ml). Tissue sensitivity and responsiveness to insulin were then determined in both obese and lean sheep by the euglycemic glucose-clamp technique. Insulin was infused at eight different rates from 0.2 to 50 mU.kg-1.min-1 and [6-3H]-glucose was infused for measurement of glucose kinetics. The mean dose-response curves for glucose utilization and clearance rates were displaced to the right in obese compared with lean sheep. As a result, the half-maximally effective insulin concentrations usually were elevated two- to fourfold, indicating decreased insulin sensitivity in obese sheep, and this is consistent with decreased insulin receptors in peripheral tissues. On the basis of fat-free body weight, the maximal glucose responses, however, were not significantly different between obese and lean sheep, indicating that postreceptor defects do not exist in muscle tissue. Furthermore, lean ruminants are more resistant to insulin than are humans, but this resistance seems only because of the sheep's decreased responsiveness to insulin and thus only because of postreceptor insulin effects in peripheral tissues.
  • Article
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    Twenty-two nonlactating Hereford cows exhibiting normal estrous cycles were fed either maintenance (M) or restricted (R) diets until most of the R cows became anestrus; R cows then were fed 160% of the M diet until estrous cycles resumed. Concentrations of progesterone, glucose, insulin and nonesterified fatty acids (NEFA) were determined in weekly blood samples. Blood also was collected frequently, before and after i.v. infusion of 300 ml of a 40% glucose solution, to evaluate responses in blood concentrations of glucose and insulin when cows were exhibiting normal estrous cycles, when R cows were initiating anestrus, during anestrus, and at the reinitiation of estrous cycles. Losses in BW and body condition score in R cows were associated with reduced (P less than .01) concentrations of glucose and insulin and greater (P less than .01) concentrations of NEFA in blood plasma compared with those of M cows. During normal estrous cycles, disappearance of infused glucose from plasma and concentrations of insulin in serum were similar for R and M cows. Glucose disappearance from plasma was retarded and serum concentrations of insulin remained increased for a longer time after glucose infusion in R at the start of anestrus compared with M cows (P less than .01). Similarly, during anestrus, the rate of glucose disappearance was slower for R cows (P less than .01). During refeeding of R cows, disappearance of infused glucose was similar for R and M cows. In conclusion, reduced concentrations of glucose and insulin and increased concentrations of NEFA in blood were associated with nutritional anestrus and the glucoregulatory effects of insulin were compromised during nutritional anestrus.
  • Article
    Plasma insulin and growth hormone concentrations have been measured by radio· immunoassay in fasting sheep and in sheep fed either a restricted amount of a concentrate diet or virtually ad libitum a variety of dried forage diets providing a wide range of energy and protein intakes.
  • Article
    A rapid and convenient commercial radioimmunoassay kit, developed for quantifying hormones in specimens from human beings, was validated for use in measuring insulin in serum of dogs, cattle, and horses. The procedure uses polypropylene assay tubes treated with rabbit anti-porcine insulin serum and porcine [125I]iodoinsulin. Specificity was proven by demonstrating that standard solutions of porcine insulin and serial dilutions of canine, bovine, and equine sera and pancreatic extracts inhibited binding of [125I]iodoinsulin to the antibody in a parallel manner. Gel-filtration chromatography of pancreatic extracts yielded a major peak of immunoreactive material that eluted identically with [125I]iodoinsulin. Immunoreactivity was not associated with fractions that contain larger and smaller molecular weight peptides (eg, proinsulin and C-peptide, respectively). Biological specificity of the assay was shown by demonstrating increased insulin in serum after injection of glucose into heifers and glucagon into dogs and horses. Purified insulin and insulin in pancreatic extracts could be quantitatively recovered from serum, thereby demonstrating accuracy of the assay. Interassay precision of 5 control specimens run in 20 consecutive assays ranged from 6.7% to 20.1% (coefficient of variation) and intra-assay precision of 6 control specimens each assayed 10 times ranged from 4.4% to 10.7% (coefficient of variation). Sensitivity of the assay was 3.2 microIU/ml. This radioimmunoassay for insulin is ideal for veterinary research and diagnosis, because a single set of reagents and procedures can be used for at least 3 species.
  • Article
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    The effect of nutrition on gonadotrophin secretion may be exerted through a central metabolic signal that reflects nutritional status. We have previously found that glucose and insulin concentrations are elevated in the cerebrospinal fluid (CSF) of rams in which the secretion of gonadotrophins has been stimulated by a nutritional supplement of lupin grain (Lupinus angustifolius). In the present study, we tested the hypothesis that insulin and/or glucose is a metabolic modulator of GnRH secretion and mediates the effects of nutrition on gonadotrophin secretion. Six mature rams were fed a diet that maintained live weight and then given a series of infusions, each for 12 h/day for 4 days, in a cross-over design. The treatments were: artificial CSF (aCSF), glucose (50 mumol/h) in aCSF, insulin (0.6 ng/h) in aCSF, and glucose (50 mumol/h) plus insulin (0.6 ng/h) in aCSF; all infused at a rate of 5 microliters/min. At the same time as the infusion treatments, two other groups of four rams without cerebral cannulae were fed either the maintenance diet or the same diet supplemented with 750 g lupin grain per head per day for 4 days, again in a cross-over design. Rams fed the lupin supplement showed an increase in both LH pulse frequency and mean FSH on day 4 (P < 0.05). Infusion of aCSF or glucose did not affect gonadotrophin secretion. Rams infused with insulin or insulin plus glucose showed an increase (P < 0.05) in LH pulse frequency but no increase in FSH concentrations on day 4 of infusion.(ABSTRACT TRUNCATED AT 250 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
    The effects of high and low dietary dietary intake on reproduction in female cattle and sheep will be considered at the level of the pituitary gland, ovary and uterus. In sheep, increased dietary intake for a relatively short time will increase ovulation rate, by increasing gonadotropin secretion. Dietary intake can affect steroids such as progesterone and also intra-follicular concentrations of some growth factors such as IGF-1 and IGF-2. The effects of altered energy intake on gonadotropins and steroids in cattle are not as repeatable as those in sheep but follicular growth rates can be altered. High nutrition has a negative effect on oocyte quality, with animals on ad-libitum high energy diets particularly at risk. Overfeeding can decrease embryo quality in both sheep and cattle and it appears that this results from changes primarily at the level of the follicle or oocyte. Restricted nutrition for a short time will enhance pregnancy rates in cattle; most of this benefit appears to occur if food is restricted before insemination. Thus feeding levels before mating are particularly important to subsequent reproductive success. High dietary crude protein may decrease pregnancy rate in lactating cows. In ewes and heifers supplementation with urea failed to have any effect on pregnancy rates when good quality embryos were transferred to recipient animals exposed to high dietary crude protein. In donor ewes there were adverse effects on early embryo development following urea treatment, suggesting that the mechanism affecting the reproductive process was primarily operating at the level of the oocyte. Collectively, these data identify the overall deleterious effects of high dietary intake and excess crude protein on fertility and highlight the importance of dietary intake before ovulation on the likelihood of establishing a viable pregnancy.
  • Article
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    In this study we examined, in two experiments, patterns of follicular development and dominance under conditions of heat stress. Estrous cycles were programmed to include two follicular waves (wave 1 and 2). On Day 1 of the estrous cycle (Day 0 = estrus), cows were assigned randomly to cooled (C; n = 6) or heat-stressed (H; n = 6) groups. In experiment 1, on Day 12 prostaglandin (PG) F2 alpha was injected and a controlled intravaginal drug release device (1.9 g progesterone) was inserted (this was removed on Day 17). In experiment 2, PGF 2 alpha was injected on Day 14. Ovarian structures were examined daily by ultrasonography, and blood samples were collected at each scanning. Cycle lengths were 20 and 17 days in experiments 1 and 2, respectively. Mean maximal body temperatures were higher (p < 0.01) in H (40.3 degrees C) than in C (38.8 degrees C) cows. In experiment 1, the rate of increase in number of large follicles (> or = 10 mm) was greater in H than in C cows (p < 0.01), resulting in 53% more large follicles in H cows during wave 1; this was associated with a lower (p < 0.05) number of medium-sized (6-9 mm) follicles between Days 7 and 10 of the cycle. Heat stress hastened (p < 0.02) the decrease in size of the first-wave dominant follicle and hastened (p < 0.01) the emergence of the second dominant (preovulatory) follicle by 2 days.(ABSTRACT TRUNCATED AT 250 WORDS)
  • Article
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    Effects of postpartum energy restriction, parity and time after parturition on energy status (measured by glucose, insulin, non-esterified fatty acids (NEFAs) and beta-hydroxybutyrate), LH secretion and follicular growth were investigated in ten primiparous and nine multiparous suckled cows. Females were allocated by parity, body mass and body condition score at calving to diets supplying either 100% (CE, n = 10) or 70% (LE, n = 9) of energy requirements until day 70 postpartum. Metabolic parameters were measured every week from calving to day 70 postpartum. Blood samples were collected at intervals of 15 min for 10 h on day 30 and day 50 after parturition for LH measurement. Ovaries were examined between days 20 and 30 and days 40 and 50 postpartum by ultrasonography. Energy supply affected mean plasma concentrations of glucose (CE: 0.64 +/- 0.01 g l-1 versus LE: 0.61 +/- 0.01 g l-1; P < 0.05) and NEFA (CE: 168 +/- 17 mu eq l-1 versus LE: 309 +/- 18 mu eq l-1; P < 0.01) but by day 70 postpartum, glucose and NEFA concentrations were not significantly different between the two groups. LH pulse amplitude and frequency were not affected by energy supply (P > 0.10). However, at day 30 postpartum, LH pulse frequency was negatively correlated with plasma concentration of NEFA (r = -0.61; P < 0.01). Cows fed diets supplying 100% of energy requirements had more large follicles than did cows fed low energy diets (CE: 0.82 +/- 0.05 versus LE: 0.31 +/- 0.05; P < 0.05). The size of the largest follicle was greater in CE cows than in LE cows (CE: 10.2 +/- 0.1 mm versus LE: 8.7 +/- 0.2 mm; P < 0.05). Between 40 and 50 days postpartum, the size of the largest follicle was negatively correlated with NEFA concentration (r = -0.5; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
  • Article
    The effects of the form in which amino acids are presented to the abomasum on the milk production of dairy cows receiving a basal diet of grass silage and a barley-based supplement were examined in two experiments. Effects of abomasal infusions of sodium caseinate were compared with the effects of corresponding levels of either an enzymic hydrolysate of casein (Expt 1) or a corresponding mixture of free amino acids (FAA; Expt 2). In Expt 1, although the yield of protein in milk increased progressively with each level of infusion, the yields of protein were greater for the caseinate than for the hydrolysate. Again, in Expt 2, for milk protein yield, sodium caseinate was superior to FAA at the lower level of infusion. In both experiments, the hydrolysate and FAA treatments were associated with higher concentrations of fat in the milk. There were indications of differences in the pattern of secretion of glucagon between the caseinate and FAA treatments. It is concluded that the differences between treatments relate either to the kinetics of absorption of amino acid residues or to the action of bioactive peptides released during digestion of casein.
  • Article
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    The effect of environmental factors on first service conception rate, days from calving to first service, weeks from calving to conception, days from first to second service, and number of services per conception and the relationship of production with reproductive performance were estimated using 1.42 million records. The production trait was 305-d mature equivalent milk production. Season and age affected fertility: reproductive performance was lowest for lactations initiated during warm months (May to July) and decreased with parity. The association of production and fertility was antagonistic. High producing cows had 12 d more from calving to first service, .15 lower first service conception rate, .32 more services per conception, and 4.8 wk more from calving to conception than low producing cows in the same herd. The negative effect of production on fertility was reduced by superior herd management associated with high production. The difference in reproductive performance of cows in high and low producing herds represented only about 35% of the difference in the performance of high and low producing cows within herds for all fertility traits. The negative effect of production on fertility for interval between first and second service was totally compensated for by better management.
  • Article
    Correlations between reproductive traits and measures of milk yield indicate that higher yield is associated phenotypically and genetically with reduced reproductive performance in lactating cows. Numerous recent studies have reported that reproductive performance is compromised, primarily through delayed ovarian activity and reduced conception rates, by the demands of high milk yield. However, daily managerial decisions to obtain efficient reproductive performance have considerable impact. Management can offset depression in fertility, because high yielding herds often achieve the fewest days open. Selection for milk yield has increased blood concentrations of somatotropin and prolactin, stimulators of lactation, and decreased insulin, a hormone that is antagonistic to lactation and may be important for normal follicular development. These changes in hormone concentrations promote higher milk yield but may be potentially detrimental to other physiological functions, such as reproduction, if the management is not adequate to meet the metabolic demands of lactation. Timing and magnitude of negative energy balance apparently interact to determine the extent to which negative energy balance alters hypothalamic secretion of GnRH and its effect on gonadotropin secretion and, therefore, ovarian secretion of progesterone, which affects expression of estrus and support of the uterus during early pregnancy.
  • Article
    Sheep fed ad lib. on a good-quality pelleted diet (g/kg; hay 500, barley 300, molasses, fish meal and minerals) were infused via the hepatic portal vein with mixtures of the sodium salts of volatile fatty acids, acetate and propionate, and a variety of equivalent osmotic loads. Propionate infused at rates between 0.6 and 2.5 mmol/min consistently reduced food intake in a linear, dose-related manner. Propionate infusions resulted in consistent dose-related increases in peripheral venous plasma glucose concentration but variable changes in insulin concentration. Infusion of osmotically balanced mixtures of propionate, acetate, mannitol or saline (9 g NaCl/l) indicated that at constant osmotic loading propionate caused a greater reduction in intake than other infusions. Acetate infusions only depressed food intake when administered as a 1 M solution. Lower concentrations had little effect, similar to that of equivalent osmotic loads of mannitol or saline. It is concluded that portal propionate flow has a potential role in the control of food intake in ruminants which is independent of osmotic effects or changes in plasma insulin concentration.
  • Article
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    The objectives of the present studies were to determine the effect of insulin, insulin-like growth factor I (IGF-I), testosterone, and FSH on proliferation, progesterone production, and(or) estradiol production of bovine granulosa cells. In addition, existence of IGF-I mRNA in granulosa cells and in vitro IGF-I production by granulosa cells were assessed. Cells from small (1 to 5 mm) and large (> or = 8 mm) follicles were collected from cattle and cultured for either 3 or 4 d. When cells from small follicles were cultured, insulin (.1 to 10 micrograms/mL) and IGF-I (100 to 400 ng/mL) increased (P < .05) cell numbers compared with controls. Insulin alone or IGF-I alone increased (P < .05) progesterone production per cell by severalfold on d 4. In cells from both sizes of follicles, insulin (1 microgram/mL), in the presence of FSH, increased estradiol production per cell. In contrast, IGF-I (100 ng/mL) inhibited estradiol production by cells from small follicles and stimulated estradiol production by cells from large follicles. Insulin-like growth factor II (100 ng/mL) and insulin at higher doses (> or = 5 micrograms/mL) had no effect on estradiol production by cells from small and large follicles. Granulosa cells contained four IGF-I mRNA transcripts and produced IGF-I in vitro. These results support the hypothesis that insulin and IGF-I may have direct local effects on bovine ovarian function, and that these effects are influenced by dose and size of follicle.
  • Article
    Twenty-four multiparous Holstein cows were divided into six blocks of 4 cows based on their previous 305-d mature equivalent milk yield and were used in a 2 x 3 factorial experiment for the first 84 DIM. Objectives were to determine the effects of forage and RUP supplementation of DM and nutrient intakes and digestibilities, milk yield and composition, BW change, and plasma concentrations of insulin, triiodothyronine, thyroxine, cortisol, NEFA, urea N, and protein. One cow from each block was assigned to wheat or sorghum silage plus one of three concentrates. Protein treatments were 1) all supplemental CP from soybean meal; 2) 33.3% of supplemental CP from heat soybean meal, fish meal, and corn gluten meal; and 3) protein treatment 2 plus an additional 4.6% CP from soybean meal. Intakes of DM, CP, NDF, ADF, NEL, and RUP were higher in cows fed sorghum silage; RUP tended to increase DMI. Milk and 3.5% FCM yields were higher for cows fed sorghum silage than for those fed wheat silage (42.3 vs. 40.0 kg/d and 41.2 vs. 38.7 kg/d, respectively). Milk protein, lactose, and SNF were increased by RUP. Insulin was increased, cortisol was decreased, and thyroid hormones were unaffected by RUP. Concentrations of NEFA were highest at wk 4. For early lactation cows fed diets containing sorghum or wheat silage, RUP is a necessity.
  • Article
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    The objectives of this study were to relate concentrations of plasma (PUN) and milk (MUN) urea nitrogen to pregnancy rate in dairy cows and compare various methods of analysis and preparation of milk for measuring MUN. In two experiments, blood or milk samples were collected on the day of AI from Holstein cows (n = 160 and n = 155, respectively). Three methods of MUN analysis were compared. Two laboratory chemical procedures yielded similar results, whereas a quick dipstick method overestimated chemical analyses. Before and after milking strip samples had MUN concentrations equivalent to those in composite milk. Concentrations of PUN or MUN greater than 19 mg/dL were associated with decreased (P < .02) pregnancy rates (18 and 21 percentage point reduction in the two experiments). In two subset groups of cows (n = 51 and n = 23, respectively), plasma progesterone or MUN concentrations were monitored during the 5-d period after AI. Plasma progesterone concentrations increased similarly during the period for cows divided into low vs high PUN but were greater in pregnant than in nonpregnant cows on d 4 and 5 (P < .04). The MUN concentrations showed low within-cow variation (CV = 8%) but were lower in pregnant cows and had a decreasing trend over time compared with nonpregnant cows (P < .05). Based on this study, plasma and milk will yield similar results for monitoring urea nitrogen in dairy cows; PUN and MUN concentrations > 19 mg/dL were associated with approximately a 20 percentage point decrease in pregnancy rate after AI in lactating dairy cattle.
  • Article
    The objectives of the present study were to determine the interaction among basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, and insulin-like growth factor-I (IGF-I) on cell numbers, steroidogenesis, and IGF-I binding sites in cultured thecal cells. Cells from large (> or = 8 mm) follicles were collected from cattle and cultured for 4 days. Treatment with 1-30 ng/ml of bFGF for 2 days increased (p < 0.05) thecal cell numbers but inhibited (p < 0.05) androstenedione and progesterone production in the presence of insulin alone or insulin plus LH. Treatment with 1-30 ng/ml of bFGF for 2 days also inhibited (p < 0.05) the number of specific 125I-IGF-I binding sites in thecal cells, with maximal inhibition being detected at 1 ng/ml; coculture with 100 ng/ml LH did not influence this effect. In the absence of bFGF and the presence of LH, 30 ng/ml of IGF-I increased thecal cell numbers and production of androstenedione and progesterone. However, cotreatment with 10 ng/ml bFGF inhibited thecal cell response to IGF-I. Cotreatment consisting of 3-100 ng/ml EGF with insulin increased (p < 0.05) thecal cell numbers but decreased (p < 0.05) androstenedione and progesterone production and the number of specific 125I-IGF-I binding sites in thecal cells, with maximal inhibition observed at 3 ng/ml of EGF. Also, cotreatment consisting of 3 and 10 ng/ml of EGF with IGF-I plus LH inhibited (p < 0.05) cell numbers and production of androstenedione and progesterone by thecal cells. These results suggest that bFGF, EGF, insulin, and IGF-I may interact to play a significant role in basal and LH-modulated thecal cell steroidogenesis and mitogenesis during follicular development in cattle.
  • Article
    The influence of source of protein during the luteal phase before a synchronized oestrus on the dynamics of follicular development, observed daily by ultrasonography, was assessed in ewes that were beginning the sexual season. Iso-nitrogenous amounts of soybean meal (SBM) or of a corn-gluten meal-ground-corn grain mixture (CGM-GC), or an iso-energetic amount of ground-corn grain (GC), were fed from four days before to four days after treatment with prostaglandin F2 alpha (PGF2 alpha). Feeding with SBM was associated with a higher frequency of short luteal phases (P < 0.02). Dynamics of follicular population were studied in ewes that ovulated after normal cycles. More follicles > or = 2 mm in diameter were observed on the ovaries of ewes fed SBM four days before PGF2 alpha treatment (P < 0.02), but the highest number was seen in ewes fed CGM-GC at the time of injection of PGF2 alpha (P < 0.08). Ewes fed SBM had larger follicles at last detection and ovulated earlier after PGF2 alpha treatment than their counterparts fed other diets (P < 0.001). Ovulatory follicles developed over a greater range of days in ewes with twin ovulations compared with ewes with single ovulations (P < 0.08). Serum concentrations of insulin were increased after four days of feeding with CGM-GC (P < 0.01), but not with SBM or GC, and reached a peak at the time of oestrus. In summary: (1) the source of dietary protein during the late-luteal phase affected follicular maturation after PGF2 alpha treatment; (2) insulin and glucose may be involved in this response and may play a role in ovarian follicular activity; and (3) twin ovulations appeared to result from both reduced atresia and increased recruitment of follicles.
  • Article
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    The objectives of this study were to characterize early postpartum (PP) follicular development in dairy cows and to evaluate the influence of energy balance (EB) and the level of dietary fat on dominant follicle development and function. Forty-five multiparous Holstein cows were fed either low (LF, 3.3%), moderate (MF, 5.2%), or high (HF, 7.1%) total dietary fat beginning at parturition, and daily EB was determined for all cows. Follicular development was monitored by ultrasonography, and blood plasma was analyzed for metabolites, metabolic hormones, progesterone, estradiol, and FSH. After an increase (p < 0.01) in mean plasma FSH during Days 1-5, all cows experienced a wave of follicular development during the second week PP regardless of diet or EB. Feeding the MF (p < 0.05) or HF (p < 0.06) diet resulted in a greater number of class IV (> 15 mm) follicles on Day 14 PP as compared to the LF diet. Cows fed the MF diet had higher (p < 0.05) peak plasma estradiol during the first follicular wave and a shorter (p < 0.05) interval to first ovulation than both LF and HF cows. Follicular development prior to first ovulation was characterized by either ovulation of the first dominant follicle (OV, n = 19), one or more waves of nonovulatory dominant follicles (NOV, n = 18), or the formation of a follicular cyst (CYST, n = 8). Ovulation failure during the first follicular wave PP was accompanied by lower (p < 0.001) peak plasma estradiol (0.96 +/- 0.36 vs. 5.0 +/- 0.35 pg/ml), a smaller (p < 0.01) maximum follicle diameter (16.0 +/- 0.9 vs. 20.0 +/- 1.0 mm), lower (p < 0.09) levels of plasma insulin-like growth factor-I (IGF-I; 73.5 +/- 10.1 vs. 102.9 +/- 9.9 ng/ml), and a longer (p < 0.02) interval to the EB nadir (14.6 +/- 1.9 vs. 6.4 +/- 1.9 days). The number of days to first ovulation was positively correlated with days to the EB nadir (r = 0.55), with dominant follicles that emerged after the EB nadir exhibiting enhanced (p < 0.01) production of estradiol and greater (p < 0.01) ovulatory success. The development of large (> 10 mm) dominant follicles was not a limiting factor in PP reproductive recovery, and moderate dietary fat shortened the interval to first ovulation. Follicular competence early PP was associated with higher plasma IGF-I and a shorter interval to the EB nadir.
  • Article
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    The working hypothesis was that dietary fats differing in fatty acid composition would differentially influence ovarian follicular growth. Cows (n = 27) were fed isoenergetic, isonitrogenous, and isofibrous diets containing no added fat (control; CT, n = 7) or diets supplemented with fats containing primarily saturated (SAT, n = 7), polyunsaturated (PU, n = 7), or highly polyunsaturated (HPU, n = 6) fatty acids. Coincident changes in serum lipid metabolites, insulin, and GH and the concentration of IGF-I in large and medium-sized follicles also were examined. Body weights and body condition scores remained similar for all groups throughout the study. Polyunsaturated fat increased (diet x day, P = .06) the number of medium-sized follicles on d 5 through 9 of a synchronized estrous cycle within 3 wk of onset of feeding and maximized (P < .001) this to a fourfold difference at ovariectomy after 7 wk. Fats with predominantly SAT and HPU tended (P < .10) to produce these effects after 7 wk. All fat-supplemented diets increased serum concentrations of total cholesterol (P < .05), GH (P < .05), and follicular fluid IGF-I in large follicles (P < .065) compared to CT but differentially influenced serum concentrations of insulin. Polyunsaturated fat stimulated a marked increase (P < .001) in serum insulin relative to controls within 3 wk, whereas SAT and HPU increased (P < .05) serum insulin only after 6 to 7 wk. We conclude that consumption of PU fatty acids stimulates a greater rate of ovarian follicular growth in cattle compared to CT, AT, and HPU. Future research should investigate the potential role of insulin in mediating PU effects on follicular growth.
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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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    To examine hormonal regulation of genes pertinent to luteal steroidogenesis, bovine theca and granulosa cells derived from preovulatory follicles were cultured with various combinations of forskolin and insulin. On Day 8 of culture, progesterone production was measured, and mRNA levels of steroidogenic factor-1 (SF-1), cytochrome P450 side-chain cleavage enzyme (P450scc), and steroidogenic acute regulatory protein (StAR) were determined by means of semiquantitative reverse transcription-polymerase chain reaction. Notably, the combination of forskolin plus insulin stimulated progesterone production in luteinized theca cells. This was probably a result of a synergistic interaction between forskolin and insulin, observed on both StAR and P450scc mRNA levels. However, in luteinized granulosa cells (LGC), forskolin and insulin each independently were able to up-regulate the levels of P450scc and StAR mRNA levels, respectively. Moreover, insulin alone was sufficient to maintain the high steady-state levels of StAR mRNA in LGC. Both insulin and insulin-like growth factor I enhanced StAR gene expression in LGC. SF-1 was constitutively expressed in bovine luteal cells; its amounts did not vary between the two luteal cell types or with hormonal treatments. In summary, this study demonstrates a distinct, cell-type specific regulation of StAR and P450scc mRNA in the two bovine luteal cell types.
  • Article
    I. Introduction II. Insulin and Insulin Receptor A. Structures of insulin and insulin receptor B. Presence of insulin and insulin receptor in the ovary C. Insulin action and the ovary D. Summary III. IGFs and Their Receptors A. IGF peptides and receptors B. Expression of IGFs and IGF receptors in the ovary C. Role of IGFs in ovulatory function and steroidogenesis D. Summary IV. IGF-Binding Proteins (IGFBPs) and Proteases A. Structural relationships among IGFBPs B. IGFBP expression in the ovary C. IGFBP proteases in the ovary D. IGFBP actions in the ovary E. Role of IGFBPs in follicular development and atresia F. Summary V. Polycystic Ovary Syndrome (PCOS) A. Clinical features B. Theories of pathogenesis C. Insulin resistance in PCOS D. Alterations of IGFs and IGFBPs in PCOS E. Summary VI. The Insulin-Related Ovarian Regulatory System: Implications for Therapy A. Treatment of PCOS B. Therapeutic use of IGF-I and IGF-II C. Use of GH in ovulation induction VII. Summary and Conclusions
  • Article
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    The effects of energy balance on hormonal secretion patterns and the structure of recovered oocytes were evaluated in 20 lactating Holstein cows during two trial periods. Cows were randomly assigned to one of two dietary treatments formulated so that dry matter consumption was 3.6% of body weight (high energy; 1.78 Mcal/kg) or 3.2% of body weight (low energy; 1.52 Mcal/kg). Ovum recovery procedures were conducted twice weekly between d 30 and 100 of lactation. Follicle size and number were recorded. Follicular fluid aspirated from the largest follicle and serum samples were collected for hormone assay. Milk yield averaged 41.6 +/- 0.3 kg/d (mean +/- SE) for high energy fed cows and 32.8 +/- 0.3 kg/d for low energy fed cows. Oocyte numbers increased linearly from d 30 to 100 postpartum. Cows fed high energy diets produced more good (+) oocytes than did cows fed low energy diets.
  • Article
    A preliminary investigation was performed to examine whether insulin resistance is a factor in the pathogenesis of cystic ovarian disease (COD) in high-yielding dairy cows. In total 30 cows, of which 15 were diagnosed as suffering from COD based on the anamnesis and clinical examination, and the other 15 served as matched controls, were subjected to an intravenous glucose tolerance test (IVGTT). The aim of the study was to investigate whether insulin activity was altered in COD cows. Differences in glucose clearance between the COD cows and their controls were analyzed comparing the fractional turnover rate (k), the glucose half-time (T1/2), and the area under the curve (AUC) 60 and 120 min after infusion. Differences in insulin response were analyzed comparing the insulin increment, the insulin peak concentration, and the AUC 60 and 120 min after glucose infusion. Although insulin resistance, attended by a secondary hyperinsulinemia, is stated to directly contribute to the ovarian abnormalities that characterize the polycystic ovary syndrome (PCOS) in human medicine, this was not observed in COD cows. On the contrary, COD cows appeared to have a low insulin response following an intravenous glucose load as compared with their matched controls. This was illustrated by significantly lower insulin increments (P = 0.04) and lower insulin peak concentrations (P = 0.04). As COD cows had a significantly lower insulin response to a standard glucose load, it was concluded that insulin could be a factor in the pathogenesis of COD in dairy cows.
  • Article
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    Ovarian function of nutritionally induced anoestrus cows was evaluated in vivo (Expt 1) and in vitro (Expt 2). In Expt 1, 32 nutritionally induced anoestrous beef cows were divided into four treatment groups receiving: (1) saline infusions at one pulse every 4 h for 13 days (control); (2) 2 micrograms GnRH at one pulse every 4 h (2 micrograms infused in 1.8 ml saline over 5 min) for 13 days (GnRH-4); (3) 2 micrograms GnRH at one pulse every 1 h for 13 days (GnRH-1); and (4) continuous infusion of 2 micrograms GnRH (a total of 2 micrograms in 34 ml h-1) for 13 days (GnRH-C). On the last day of treatment, cows were killed, ovaries were removed and follicular fluid samples (n = 149) were collected. The percentage of cows with luteal activity on day 13 was significantly different (P < 0.01) among treatments (0, 25, 75 and 25% for control, GnRH-4, GnRH-1 and GnRH-C cows, respectively). Owing to the large percentage of ovulatory cows in the GnRH-1 group (n = 6), anovulatory cows (n = 2) were removed from this treatment group for statistical analysis, as were cows with luteal tissue from the GnRH-4 (n = 2) and GnRH-C (n = 2) groups. The numbers of small (1.0-4.9 mm) and medium plus large (> or = 5 mm) follicles were not affected (P > 0.10) by treatment. However, GnRH-4 cows (n = 6) had greater (P < 0.05) concentrations of oestradiol in follicular fluid than did control (n = 8) but not GnRH-1 (n = 6) or GnRH-C (n = 6) cows. Concentrations of insulin-like growth factor I were greater (P < 0.05) in the follicular fluid of GnRH-1 cows than in all other treatment groups. Concentrations of androstenedione and progesterone in follicular fluid were not affected (P > 0.10) by treatment or follicle size. The binding activity of insulin-like growth factor binding proteins was not affected by GnRH treatment. However, the binding activity of insulin-like growth factor binding protein 2, 29-32 kDa and 22 kDa insulin-like growth factor binding proteins were greater (P < 0.05) in small versus medium plus large follicles. In Expt 2, granulosa cells were collected from nutritionally anoestrous cows to determine whether ovarian cells from anoestrous cows have the capacity to respond to insulin-like growth factor I or insulin in vitro. Both insulin-like growth factor I (20 and 200 ng ml-1) and insulin (10, 100 and 1000 ng ml-1) increased (P < 0.05) granulosa cell proliferation and progesterone production. In conclusion, pulsatile infusion of 2 micrograms GnRH (every 1 or 4 h) for 13 days into nutritionally induced anoestrous cows results in increased intrafollicular oestradiol and insulin-like growth factor I concentrations and can stimulate ovulation without markedly affecting concentrations of androstenedione or progesterone, or the binding activity of insulin-like growth factor binding proteins, in follicular fluid. In addition, granulosa cells from nutritionally induced anoestrous cows have the capacity to respond to insulin-like growth factor I and insulin in vitro, indicating that the decrease in trophic factors observed with restricted feeding does not reduce the response of the ovary to insulin-like growth factor I and insulin.
  • Article
    Ovarian follicular growth and development is an integrated process encompassing both extraovarian signals, such as gonadotrophins and metabolic hormones, and intraovarian factors. Follicular development has been classified into gonadotrophin-independent and -dependent phases. In the latter, FSH provides the primary drive for follicular recruitment and LH is required for continued development of follicles to the preovulatory stage. A transient increase in circulating FSH precedes the recruitment of a group of follicles, and these recruited follicles are characterized by expression of mRNAs encoding P450scc and P450arom in granulosal cells. As follicles mature, there is a transfer of dependency from FSH to LH, which may be part of the mechanism(s) involved in selection of follicles for continued growth. Indeed, changes in the pattern of expression of mRNA for gonadotrophin receptors and steroid enzymes within follicular cells appear to be closely linked to changes in peripheral concentrations of gonadotrophins. The mechanism of selection of dominant follicles still requires clarification, but seems to be linked to the timing of mRNA expression encoding LHr and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in granulosal cells. Additional intraovarian systems, including the ovarian IGF and activin/inhibin systems, also exert a role. For example, it appears that the development of follicular dominance in cows is associated with the FSH-dependent inhibition of the expression of mRNA encoding insulin-like growth factor binding protein 2 (IGFBP-2) in granulosal cells. In conclusion, the integration of these endocrine signals and intraovarian factors within follicles determines whether follicles continue to develop and become dominant or are diverted into apoptotic pathways leading to atresia.
  • Article
    Short-term effects of nutrition on conception rate (CR), ovulation rate (OR), ova and embryo losses (OEL) during the first 50 days following insemination and total reproductive wastage after ovulation (TRW), were investigated in primiparous lactating Sarda ewes after oestrous synchronisation and cervical [corrected] artificial insemination (AI). Eighty ewes grazing a green high-quality pasture were offered one of three iso-energetic supplements from day 14 before to day 2 after AI: whole maize grain (M); soyabean meal (S); maize gluten meal (G); or served as controls (C). Supplements G and S were iso-nitrogenous but provided different amounts of rumen undegradable digestible protein. The intake of herbage and digestible dry matter, measured by the n560 mg/l was associated with lower CR. Ranking by ovulation groups of CR was single<double=triple; and of TRW was double<triple<single. In conclusion, OR in primiparous lactating Sarda ewes grazing on a good quality pasture is high, and CR after AI is reduced by ova as well as embryo loss, especially following single ovulations. Short-term supplementation with a soyabean-based concentrate may help alleviate these losses.
  • Article
    Meiotic arrest refers to the nuclear stage of the oocytes within the follicles. Meiotic resumption occurs when oocytes are isolated from their follicular environment and placed in a simple maturation medium. The sine qua none condition for meiotic resumption is that the oocytes must be competent to resume meiosis. It has been shown that competent oocytes must reach a minimum diameter before been able to resume meiosis. In the bovine, competent oocytes measure at least 110 μm in diameter (24, 39).It appears that the inhibitory factors of meiotic resumption might only be necessary after the oocyte has acquired its competence. However, once the oocytes become competent, they need these factors to maintain meiotic arrest. It is generally recognized that follicular cells produce inhibitors necessary to maintain the oocyte in meiotic arrest. The removal of the oocyte from its follicular environment deprives the oocyte of inhibitory factors. Oocytes then resume meiosis (63). This article will first review the different chemical modulators to emphasize the importance of protein synthesis and the role of different kinases and phosphatases. Then it will review the follicular aspects involved in the control of meiotic arrest of oocytes competent to resume meiosis.
  • Article
    A study was conducted to modify the routinely used ovum pick up (OPU) devices to permit use of disposable needles and to simplify the technique and to make it more economical and practical to use. Long nondisposable needles are commonly used in transvaginal OPU despite several disadvantages. A new OPU device was developed using 19-g disposable needles to eliminate these disadvantages and to make the technique more successful. The disposable needle was connected to silicone tubing by means of a stainless steel connector. The system was inserted into a stainless steel tube, creating a rigid structure within which to move the needle back and forth. A blunt needle can be changed simply by replacing it with a new one, even while the device is in the vagina of a cow. The needle guidance system is incorporated into a new OPU device together with the transducer of an ultrasonographic scanner with an unilateral orientated scanning field, making it possible to utilize the needle length to its maximum. This combination permits easy manipulation of the ovaries, easy positioning of follicles on the puncture line, and enables the use of shorter needles which directly enter the scanned area without loss of useful needle length. As a preliminary result we obtained an overall oocyte recovery rate of 42%. Although this is promising, additional puncture sessions are needed to establish more consistent recovery rates. When OPU is used routinely, application of short disposable needles is more practical and economical.
  • Article
    The efficacy of transvaginal ultrasound-guided puncturing of ovarian follicles for collecting immature oocytes in cattle was studied. Three experiments were conducted to examine the effects of puncturing on follicle recruitment and on the number of oocytes collected. Puncture sessions were executed twice weekly at regular intervals of 3 and 4 d respectively. The oocytes were matured, fertilized and allowed to develop in vitro and the number of transferable embryos was recorded. The health of the cows was checked daily. In Experiment 1, dairy cows (n=10) were punctured over a period of 5 mo, and the collected oocytes were fertilized with the semen of 1 bull. In Experiment 2, oocytes were collected from one 12 year old high pedigree dairy cow and an one month pregnant cow were punctured. The oocytes of the old cow were fertilized with semen of 8 different bulls. In Experiment 3, beef cows (n=6) were punctured over a 2 mo period and the semen of 2 different bulls of the same breed was used to fertilize the oocytes from 3 of these cows. In Experiment 1, 14.5 +/- 0.4 (mean +/- SEM) follicles were punctured per session, and 8.0 +/- 0.3 (mean +/- SEM) oocytes were recovered. A mean of 16% of the oocytes developed into transferable embryos with a pregnancy rate of 40%. The results did not differ between the months of the experiments, indicating that the transvaginal puncturing method can be used successfully over a 5 mo period. No detrimental effects were observed after clinical and post mortem examinations, nor did breed, age or reproductive status appear to affect the results. However, large differences were observed between individual cows and between cow/bull combinations.