Subclinical,chronic intramammary infection lowers steroid concentrations and gene expression in bovine preovulatory follicles

Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.     

Insulin‐Like Growth Factor‐1 Regulates the Expression of Luteinizing Hormone Receptor and Steroid Production in Bovine Granulosa Cells

Luteinizing hormone LH plays important roles in follicular maturation and ovulation. The effects of LH are mediated by LH receptor (LHR) in the ovary. However, the factors that regulate the expression of LHR in bovine granulosa cells (GCs) are not well known. Insulin‐like growth factor‐1 (IGF‐1) is known to play a key role in the acquisition and maintenance of functional dominance. To better understand the roles of LHR expression and IGF‐1, we conducted three experiments to determine (i) mRNA expression of LHR in the GCs of developing follicles, (ii) the effects of IGF‐1 on LHR mRNA expression in cultured GCs and (iii) the effects of IGF‐1 on estradiol (E2), progesterone (P4) and androstenedione (A4) production by non‐luteinized GCs. In experiment 1, small follicles (<6 mm Ø) expressed lower levels of LHR than mid‐sized follicles (6–8 mm Ø) and large follicles (≥9 mm Ø) expressed the highest levels of LHR mRNA (p < 0.05). In experiment 2, IGF‐1 (1 and 100 ng/ml) increased (p < 0.05) the expression of LHR mRNA in GCs from small and large follicles. In experiment 3, IGF‐1 (0.1–100 ng/ml) increased A4 and E2 in GCs from both small and large follicles but increased P4 only in large follicles. IGF‐1 in combination with LH (0.1 and 1 ng/ml) increased P4 and A4 in large follicles, and increased E2 and A4 in GCs of small follicles. These findings strongly support the concept that IGF‐1 upregulates LHR mRNA expression as well as A4 and E2 production in GCs and that IGF‐1 is required for determining which follicle becomes dominant and acquires ovulatory capacity.     

Expression and localization of adiponectin and its receptors in ovarian follicles during different stages of development and the modulatory effect of adiponectin on steroid production in water buffalo

Adiponectin is an adipocyte‐derived hormone regulating energy metabolism, insulin sensitivity and recently found to regulate reproduction. The current study was carried out to investigate gene and protein expression, immunolocalization of adiponectin and its receptors AdipoR1 and AdipoR2 in ovarian follicles of different developmental stages in water buffalo (Bubalus bubalis) and to investigate the effect of adiponectin on steroid production in cultured bubaline granulosa cells. qPCR, western blotting and immunohistochemistry were applied to demonstrate mRNA expression, protein expression and immunolocalization, respectively. The results indicate that adiponectin, AdipoR1 and AdipoR2 were present in granulosa cells (GC) and theca interna (TI) of ovarian follicles and the expression of adiponectin, AdipoR1, AdipoR2 in GC and AdipoR1 and AdipoR2 in TI increased with increase in follicle size (p < .05). Expression of adiponectin was high in small and medium size follicles in TI. The adiponectin and its receptors were immunolocalized in the cytoplasm of GC and TI cells. Further, in the in‐vitro study, GCs were cultured and treated with recombinant adiponectin each at 0, 1 and 10 µg/ml alone or with follicle stimulating hormone (FSH) at 30 ng/ml) or Insulin‐like growth factor I (IGF‐I) at 10 ng/ml for 48 hr after obtaining 75%–80%s confluency. Adiponectin at 10 µg/ml increased IGF‐I‐induced estradiol (E₂) and progesterone (P₄) secretion and FSH‐induced E₂ secretion from GC and also increased the abundance of factors involved in E₂ and P₄ production (cytochrome P45019A1 [CYP19A1] and 3‐beta‐hydroxysteroid dehydrogenase [3β‐HSD]). In conclusion, this study provides novel evidence for the presence of adiponectin and its receptors in ovarian follicles and modulatory role of adiponectin on steroid production in buffalo.     

Increased abundance of aromatase and follicle stimulating hormone receptor mRNA and decreased insulin-like growth factor-2 receptor mRNA in small ovarian follicles of cattle selected for twin births

Cattle genetically selected for twin ovulations and births (Twinner) exhibit increased ovarian follicular development, increased ovulation rate, and greater blood and follicular fluid IGF-1 concentrations compared with contemporary cattle not selected for twins (Control). Experimental objectives were to 1) assess relationships among aromatase (CYP19A1), IGF-1 (IGF1), IGF-2 receptor (IGF2R), and FSH receptor (FSHR) mRNA expression in small (≤5 mm) antral follicles and 2) determine their association with increased numbers of developing follicles in ovaries of Twinner females. Ovaries were collected from mature, cyclic (d 3 to 6) Twinner (n = 11), and Control (n = 12) cows at slaughter and pieces of cortical tissue were fixed and embedded in paraffin. Expression of mRNA was evaluated by in situ hybridization using (35)S-UTP-labeled antisense and sense probes for CYP19A1, FSHR, IGF1, and IGF2R mRNA. Silver grain density was quantified within the granulosa and theca cells of individual follicles (2 to 7 follicles/cow) by Bioquant image analysis. Follicles of Twinners tended to be smaller in diameter than Controls (1.9 ± 0.1 vs. 2.3 ± 0.1 mm; P = 0.08), but thickness of granulosa layer did not differ (P > 0.1) by genotype. Relative abundance of CYP19A1 (P < 0.01) and FSHR (P < 0.05) mRNA was greater in granulosa cells of Twinners vs. Controls, respectively, whereas IGF2R mRNA expression was less in both granulosa (P < 0.01) and theca (P < 0.05) cells in follicles of Twinners vs. Controls, respectively. Abundance of CYP19A1 mRNA in granulosa cells was correlated negatively with IGF2R mRNA expression in both granulosa (r = -0.33; P < 0.01) and theca (r = -0.21; P = 0.05) cells. Expression of IGF1 mRNA was primarily in granulosa cells, including cumulus cells, and its expression did not differ between Twinners vs. Controls (P > 0.10). Detected increases in CYP19A1 and FSHR, but not IGF1, mRNA expression along with decreases in IGF2R mRNA expression in individual follicles of Twinners support the hypothesis that increased follicular development and steroidogenesis in Twinner females result from increased extra-ovarian IGF-1 production. Furthermore, a reduction in follicular IGF2R mRNA expression accompanied by a reduction in receptor numbers would increase availability of free IGF-2 and its stimulation of follicular development in Twinners.     

Effects of low-dose follicle-stimulating hormone administration on follicular dynamics and preovulatory follicle characteristics in dairy cows during the summer

The well-documented phenomenon of reduced conception rate in dairy cows during the hot season involves impaired functioning of the ovarian follicles and their enclosed oocytes. Three experiments were performed to examine the administration of low doses of follicle-stimulating hormone (FSH) to induce turnover of follicles that are damaged upon summer thermal stress and to examine whether this FSH administration has beneficial effects on preovulatory follicles. In experiment 1, synchronized heifers were treated with 100 mg of Folltropin-V (n = 7) or 4.4 mg of Ovagen (n = 6) on day 3 of the estrous cycle. Treatment with both FSH sources resulted in greater (P < 0.05) numbers of follicles than in control animals (n = 12) on day 6 of the estrous cycle, indicating that low doses of FSH can increase the number of emerging follicles in a follicular wave. In experiment 2, milking cows were assigned to a control group (n = 4) or treated with 2.2 mg (FSH-2.2; n = 6) or 4.4 mg (FSH-4.4; n = 5) Ovagen. Follicle-stimulating hormone was administrated on day 3 or 4 and day 10 or 11 of the estrous cycle, coinciding with emergence of the first and second follicular waves, respectively. The number of follicles emerging during the first wave tended to be higher (P < 0.1) in FSH-4.4-treated cows than in controls. The second-wave dominant follicles emerged 2 d later in the treated cows and were smaller in diameter (P < 0.05) than controls, 2 d before aspiration. Despite being younger, the preovulatory follicles of FSH-4.4 cows expressed a steroidogenic capacity that was similar to controls with a tendency toward greater insulin concentrations (P < 0.09). In experiment 3, milking cows were assigned to a control group (n = 6) or treated with 4.4 mg Ovagen (FSH-4.4; n = 6). Follicle-stimulating hormone was administrated on day 3 and day 12 or 13 of the estrous cycle. The number of emerging follicles was higher (P < 0.05) in the treated vs control cows. However, the features of the preovulatory follicle developed in the subsequent cycle did not differ between groups. In summary, low doses of FSH can efficiently induce follicular turnover accompanied by a modest effect on the preovulatory follicle of the treated cycle. It appears that the administration of low doses of FSH, precisely timed to synchronize with the emergence of follicular waves, might have a beneficial effect on the preovulatory follicle and its enclosed oocyte.     

Co-culture model reveals the characteristics of theca cells and the effect of granulosa cells on theca cells at different stages of follicular development

Theca cells (TCs) play an important role in follicular development, which cannot be separated from granulosa cells (GCs). However, compared with mammals, the TCs and the effects of GCs on TCs at different follicular development stages (FDSs) have specific characteristics in avian species, but none of them have been clearly defined. In this study, we established an in vitro co-culture (with GC at the corresponding stage) model of goose TCs at different FDSs (pre-hierarchical, hierarchical and F1) by using a transwell system. The properties of TCs in co-culture at the three FDSs, including cell morphology, activity and intracellular lipid content, as well as the expression of key genes involved in de novo lipogenesis, steroidogenesis, proliferation and apoptosis, were examined and defined. We further compared the mono-culture and co-culture groups. After co-culture, the activity of TCs showed significant (p < .01) increases in all stages; moreover, in pre-hierarchical TCs, the expression levels of FAS, SREBP, 3β-HSD and CCND1 were promoted, and PPARγ, CYP19, BCL2 and CAS3 were inhibited (p < .05); in the hierarchical TCs, the expression levels of PPARγ, FAS, CYP19, CCND1 and BCL2 were promoted, and SREBP, STAR, 3β-HSD and CAS3 were inhibited (p < .05), whereas in the F1 TCs, the expression levels of PPARγ, FAS, 3β-HSD, CYP19 and CCND1 were promoted, and STAR and CAS3 were inhibited (p < .05). These results suggested that GCs at the three FDSs have dynamic and complex influences on the physiological characteristics of TCs, and the influences on TCs at the three FDSs were varied.     

Serum hormones in response to estradiol and (OR) progesterone in ovariectomized cows after thyroidectomy

To evaluate the effect of gonadal steroid treatment and thyroidectomy on concentrations of gonadotropins and thyroid-stimulating hormone in the bovine, nonlactating Holstein cows were either thyroidectomized and ovariectomized (THYOVEX; n=6) or ovariectomized only (OVEX; n=4), and subsequently treated with no gonadal steroids (control), estradiol-17β (E₂), progesterone (P₄), or P₄+E₂ in a 2 × 4 factorial experiment. Averaged across steroid treatments, baseline concentrations of luteinizing hormone (LH; P < .05) and follicle-stimulating hormone (FSH; P <.10) were higher in THYOVEX cows than in OVEX cows. Pulse frequencies and amplitudes of LH and FSH did not differ between THYOVEX and OVEX cows. Secretion of TSH was pulsatile and all concentrations and pulsatile characteristics of TSH were increased (P < .05) in THYOVEX compared to OVEX cows. Treatment with E₂ and P₄ decreased (P < .05) baseline concentrations and magnitude of LH pulses, whereas P₄+E₂ increased (P < .01) pulse frequency of LH and FSH. Amplitude of LH and FSH pulses were not affected by treatment with either steroid. Treatment with P₄+E₂ decreased (P < .05) baseline concentrations of TSH, whereas pulse frequency, and magnitude and amplitude of TSH pulses were not altered by treatment with steroids. Mean concentrations of LH and FSH were similar during 48 hr after termination of E₂ and P₄+E₂ treatments, but concentrations of TSH were higher (P = .06) after P₄+E₂ than after E₂. Secretion of TSH showed a diurnal variation, with the lowest concentrations in the morning and highest in the afternoon. These results indicate that thyroidectomy influenced secretion of gonadotropins in OVEX cows.     

Evaluation of numbers of microscopic and macroscopic follicles in cattle selected for twinning

We hypothesized that the number of microscopic follicles present in the ovaries of cattle selected for twin births (Twinner) would be greater than in the ovaries of contemporary Controls. Ovaries were collected from seven Control and seven Twinner cows at slaughter. The number of Small (1 to 3.9 mm), Medium (4 to 7.9), and Large (> 8 mm) surface follicles was counted and one ovary was fixed for histological evaluation. Fifty to sixty consecutive 6-microm slices were taken from a piece of cortical tissue, approximately 1 cm x 1 cm in area, located between the surface follicles. Microscopic follicles were classified as primordial (oocyte surrounded by a single layer of squamous pregranulosa cells), primary (oocyte surrounded by a single layer of one or more cuboidal granulosa cells), secondary (oocyte surrounded by two or more layers of granulosa cells), or tertiary (oocyte surrounded by multiple layers of granulosa cells with initiation of antrum formation to < or = 1 mm in diameter). The total number of follicles was counted in 200 fields (2 mm x 2 mm) per ovary. A field containing no follicles was classified as empty. There were significantly more secondary follicles in Twinner compared with Control ovaries (12.9 vs 6.3; P < .05). Twinners also tended to have more small surface follicles (35.4 vs 49.0; P < 0.1). We conclude that ovaries of Control and Twinner cows do not differ in the number of primordial follicles or in the number of follicles activated into the growing pool; however, Twinner cows are able to maintain more growing follicles at the secondary and subsequent stages of development.     

Effects of the phytoestrogen,genistein, and protein tyrosine kinase inhibitor–dependent mechanisms on steroidogenesis and estrogen receptor expression in porcine granulosa cells of medium follicles

The use of soy-based products in pig diets had raised concerns regarding the reproductive toxicity of genistein, the predominant isoflavone in soybeans. Genistein was reported to exhibit weak estrogenic activity but its mechanism of action is not fully recognized. The aim of the study was to examine the in vitro effects of genistein on (1) progesterone (P₄) and estradiol (E₂) secretion by porcine granulosa cells harvested from medium follicles, (2) the viability of cultured granulosa cells, and (3) the mRNA and protein expression of estrogen receptors α and β (ERα and ERβ) in these cells. In addition, to verify the role of protein tyrosine kinase (PTK)–dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. We found that genistein inhibited (P < 0.05) basal P₄ secretion by granulosa cells harvested from medium follicles of pigs. In contrast, lavendustin C did not affect basal P₄ secretion by the cells. Moreover, genistein increased (P < 0.05) basal granulosal secretion of E₂. In contrast, lavendustin C did not alter basal E₂ secretion by porcine granulosa cells. In addition, we demonstrated that genistein increased mRNA and protein expression of ERβ (P < 0.05) in the examined cells. The expression of ERα mRNA was not affected by genistein and ERα protein was not detected in the cultured granulosa cells of pigs. In summary, the genistein action on follicular steroidogenesis in pigs involved changes in the granulosal expression of ERβ. However, the genistein action on P₄ and E₂ production by granulosa cells harvested from medium follicles did not seem to be associated with PTK.     

Overfeeding and underfeeding have detrimental effects on oocyte quality measured by in vitro fertilization and early embryonic development in sheep

To determine effects of maternal diet on in vitro fertilization (IVF) and early embryonic development, ewes (n = 48) were divided into control, overfed (ad libitum feeding), and underfed (60% of control) nutritional planes for 8 wk before oocyte collection. Follicular development was induced by twice-daily injections of FSH on days 13 and 14 of the estrous cycle, and ovaries and blood samples were collected on day 15 of the estrous cycle. During the 8-wk experiment, for control ewes BW and BCS did not change, but for overfed ewes mean (± SEM) BW and BCS increased (11.8 ± 1.1 kg and 2.0 ± 0.1, respectively) and for underfed ewes decreased (14.2 ± 0.9 kg and 0.7 ± 0.1, respectively). The number of follicles was determined; oocytes were collected and subjected to in vitro maturation and fertilization. After IVF, developing embryos were evaluated throughout the 8-d culture period. The proportion of cleaved oocytes after IVF and developing morula and blastocyst were less (P < 0.0001) in overfed and underfed ewes than in control ewes. However, number of visible follicles, total number of oocytes, number of healthy oocytes, and percentage of healthy oocytes were similar for control, overfed, and underfed ewes. Serum insulin concentration was greater (P < 0.05) in overfed ewes than in underfed ewes, estradiol 17-β (E₂) concentration was greater (P < 0.05) in underfed ewes than in overfed ewes, but triiodothyronine (T₃) and thyroxine (T₄) concentrations were similar in all treatment groups. These data show that inadequate feeding has a negative effect on oocyte quality which results in lower oocyte cleavage after IVF and morula and blastocyst formation; overfeeding increased serum insulin and underfeeding increased serum E₂ but not T₃ or T₄. These data emphasize the importance of diet for reproductive and metabolic functions. Furthermore, the mechanisms through which enhanced or decreased energy in diet affect oocyte quality and serum insulin and E₂ concentrations remain to be elucidated.     

Effects of growth hormone treatment on the expression of somatotropic axis genes in the skeletal muscle of lactating Holstein cows

This study focused on the expression of somatotropic axis genes in the skeletal muscle of dairy cattle. A slow-release recombinant bovine growth hormone (GH) (rbGH) formulation was administered to 5 cows, and saline solution (control) was administered to another 5 cows every 2 wk for a total of 10 wk, starting from the peak of lactation. Tissue and blood samples were collected on days 2 and 14 after each rbGH injection. As target genes insulin-like growth factor (IGF)-1, IGF-2, IGFBPs (1, 2, 3, 4, 5, 6), acute labile subunit (ALS), IGF-1 receptor (IGF-1R), GH receptor (GHR), and the known GHR 5′-UTR variants were selected as target genes, and their relative expression was measured using real-time polymerase chain reaction. In GH-treated cows, an increase in expression was observed for GHR 5′-UTR variant 1I on day 14 (P < 0.05), whereas a significant down-regulation of GHR (P < 0.05) was found after comparing values of treated cows between day 2 and day 14. However, only IGF binding proteins (BP)-5 was found to be appreciably up-regulated in GH-treated cows (P < 0.001), which may indicate the importance of this gene in the overall molecular response to GH administration. Our study indicated that GH treatment did not affect the expression of most somatotropic axis genes, despite the marked increase in GH and IGF-1 in blood (P < 0.001). Nor did it have a large impact on the proportion of GHR 5′-UTR variants in the skeletal muscle of lactating cows. Finally, although we observed a significant variation in the expression of some genes, it would appear that the differences between GH-treated cows and controls were not great enough to be considered as reliable indirect indicators of GH treatment in dairy cattle.     

Effects of body condition and supplementation on ovarian function,growth factors and response to estrus synchronization in postpartum beef cows in Tamaulipas,Mexico

ABSTRACT

Eighteen beef cows with their calves were assigned to 3 groups considering body conditions scores at calving (BCSC): T1 (high BCSC, maintained throughout the study); T2 (low BCSC, increased during the study); and T3 (low BCSC, maintained throughout). An estrus synchronization protocol was implemented, with fixed time artificial insemination on day 70. Follicular or luteal growth, and pregnancy rate were similar between groups. Of all cows, five showed multiple ovulations (2–6); increasing number of ovulations reduced preovulatory follicle diameter and individual luteal volume (P?<?0.05), but total luteal volume was greater in cows with 2 or 3 ovulations on day 14 after insemination (P?<?0.05). Estradiol and progesterone levels were associated to follicular and luteal growth, respectively. No difference was found in IGF1 or GDF9 levels between groups or associated to fertility. No association was found between BCS, IGF1 or GDF9 levels and the reproductive process.     

Fine mapping the number of corpora lutea quantitative trait loci on SSC3: Analysis of the porcine follicle‐stimulating hormone receptor gene

In females, follicle‐stimulating hormone (FSH) targets a FSH receptor (FSHR) expressed only on granulose cells, inducing maturation of the ovarian follicles. We hypothesized that genetic variants in the FSHR gene influence litter size by affecting the number of corpora lutea. We fine‐mapped a region of Sus Scrofa chromosome 3 that contains quantitative trait loci for corpora lutea. Polymorphisms were detected in the exons and 5′ flanking region of the porcine FSHR gene, a positional candidate for the statistically most significant of the quantitative trait loci. Finally, 248 F₂ animals from a Duroc and Meishan cross were genotyped for three FSHR SNPs at positions 74, 532 and 1166, and these were correlated with the phenotypes of litter size and corpus luteum number. Three haplotypes were identified: M1 (G/G/C), M2 (C/A/T) and D (C/A/C). In the F₂ population, the M1 haplotype was associated with a greater number of corpora lutea (P < 0.01) and also seemed to be associated with increased litter size, although the association was not significant (P = 0.2571). Some polymorphisms resulting in amino acid substitutions in these genes were excluded from the polymorphisms possibly responsible for the number of corpora lutea.     

Ovarian Follicular Dynamics in Buffalo Cows (Bubalus bubalis)

Follicular growth in Egyptian buffalo cows was monitored using genital tracts from 200 buffalo cows collected immediately after slaughter. According to the morphological appearance of the corpus luteum (CL), the corresponding oestrous cycle was divided into four stages: A (days 1–4), B (days 5–10), C (days 11–17) and D (days 18–21). Within these stages the follicular population on the ovaries was evaluated and the dominant follicle (DF) determined in all recovered ovaries. The functional status of the DF and the largest sub‐dominant follicles was examined by histological examination in 31 cases, and Radio Immunoassay (RIA) analyses for estradiol‐17β (E₂) and progesterone (P₄) was performed in the follicular fluid in 23 of the DF. The results showed that DFs changed their endocrine character within the stages of the oestrous cycle. The DFs between days 5 and 10 were functionally active (E₂‐dominant; non‐atretic) in most of the cases. Between days 11 and day 17 half of the DFs became functionally inactive (P₄‐dominant; atretic). At days 18–21 all of the DF became functionally active and non‐atretic. In the specimens that carried two large follicles one of them was regularly atretic and P₄‐dominant whereas the other was non‐atretic and E₂‐dominant. Between days 18 and 21 all ovaries examined showed at least one large follicle. These findings suggest that in most of the cases follicular dynamics occurs in two wave‐like patterns in the Egyptian buffalo cows.     

Utilization of substrates for testosterone and estradiol-17β production by small follicles of the chicken ovary

A study was conducted to characterize steroidogenesis in small ovarian follicles (1–10 mm in diameter) of the hen. The aims of our study were: 1) to determine basal estradiol-17β (E₂) production by different sizes of small follicles; 2) to determine the ability of intact small follicles to utilize exogenous substrates for testosterone (T) and E₂ production; and 3) to investigate the preferred steroidogenic pathway in small follicles. Small follicles which had not entered the hierarchy were isolated from ovaries obtained 2 hr after oviposition and divided into three groups: small white follicles (SWF; 1–2 mm in diameter), large white follicles (LWF; 2–4 mm in diameter), and small yellow follicles (SYF; 5–10 mm in diameter). Yolk and granulosa cells were removed from LWFs and SYFs and the remaining theca layer was called a follicle shell. Intact follicles or follicle shells (4/4 ml/tube) were incubated in avian Ringer's buffer supplemented with 10 mM HEPES and 0.1% BSA at 37°C for 3 hr with various treatments. Testosterone and E₂ were measured in the medium. The SYFs and their corresponding follicle shells produced the greatest amount of E₂ when E₂ production was expressed per follicle. Addition of 2 mM 8-Br-cAMP to the incubation medium stimulated E₂ production by all sizes of follicles and follicle shells. However, follicle shells produced lower basal- and 8-Br-cAMP-stimulated E₂ secretion compared to corresponding intact follicles. There was no significant difference in E₂ production in response to various concentrations of 25-hydroxycholesterol (25-OH-CHOL; 0–100 μM) by intact follicles and follicle shells. On the contrary, intact follicles and follicle shells produced T and E₂ in a dose-dependent manner in response to increasing concentrations (0–100 μM) of pregnenolone (P₅). Intact follicles also used progesterone (P₄) and dehydroepiandrosterone (DHEA) as substrates for T and E₂ production. DHEA was the preferred substrate for steroid production compared to P₄. In summary, we found that: 1) steroidogenesis in small follicles is regulated by a cAMP-dependent protein kinase A second messenger system; 2) adequate amounts of endogenous cholesterol are available for steroidogenesis; and 3) both Δ⁵ and Δ⁴ pathways are functional in small follicles and the Δ⁵ pathway may be the preferred steroidogenic pathway. pathway.     

Effects of different cyclooxygenase inhibitors on prostaglandin E2 production,steroidogenesis and ovulation of bovine preovulatory follicles

Ovulation is an inflammation-like process, and cyclooxygenase-2 (COX-2)-dependent production of prostaglandin E₂ (PGE₂) is its key mediator. Balanced regulation of inflammatory processes in high-yielding dairy cows may be essential for physiological ovulation and fertility. This study aimed to elucidate the mechanisms underlying ovulation failure and cyst development after disturbing intrafollicular inflammatory cascades. Therefore, nonselective (indomethacin and flunixin-meglumine), COX-2 selective (meloxicam), and highly COX-2 selective (NS-398) inhibitors were injected into preovulatory follicles 16 h after administration of GnRH, and ovulation was monitored via ultrasound examination. Additionally, follicular fluid was collected after injection of indomethacin, meloxicam, and NS-398. Moreover, primary granulosa cell cultures from preovulatory follicles were prepared and treated with indomethacin, meloxicam, and NS-398. The concentrations of 17β-estradiol, progesterone, and prostaglandin E₂ (PGE₂) in the follicular fluid and cell supernatant were estimated. Indomethacin and flunixin-meglumine blocked ovulation, even at low doses, and led to ovarian cyst development. The selective and highly selective COX-2 inhibitors meloxicam and NS-398 were not effective in blocking ovulation. However, indomethacin, meloxicam, and NS-398 significantly and comparably reduced PGE₂ concentration in vivo and in vitro (P < 0.05) but had no effect on estradiol or progesterone production. This may contradict the generally accepted hypothesis that PGE₂ is a key mediator of ovulation and progesterone production. Our results suggest a connection between ovarian disorders and inflammatory actions in early postpartum cows.     

Effects of feed supplementation on growth,blood parameters and reproductive performance in Sanga and Friesian-Sanga cows grazing natural pasture

This study determined the effects of feed supplementation during the postpartum period on the weight gain, milk yield, blood profiles and reproductive performance of Sanga and Friesian-Sanga cows grazing on natural pasture. 20 Sanga and 20 Friesian-Sanga cows were randomly allocated either to serve as a control on grazing only or to be supplemented with 2.5 kg of concentrate a day for 10 weeks during the dry season. Each week, all cows were weighed and scored for body condition. Partial milk yield of cows was determined daily. Plasma concentrations of blood metabolites were assessed fortnightly from weeks 1 to 10 postpartum. Resumption of postpartum ovarian activity was determined by measuring progesterone concentration in the plasma from weeks 1 to 10. Supplemented cows had a better body condition score (6.2 versus 5.8; P?<?0.05) and higher partial milk yield (1.94 versus 1.55 L/day; P?<?0.01) than non-supplemented cows. Sanga cows had a better body condition score (6.2 versus 5.8; P?<?0.05) but lower milk yield (1.58 versus 1.92 L/day; P?<?0.01) than the Friesian-Sanga crossbreds. Total protein (P?<?0.05) and albumin (P?<?0.01) concentrations were higher in the supplemented than in the non-supplemented cows. Sanga cows recorded higher globulin (P?<?0.05) and total cholesterol (P?<?0.01) but lower albumin (P?<?0.01) concentrations than Friesian-Sanga crossbred cows. Feed supplementation did not affect (P?<?0.05) the interval from calving to resumption of ovarian activity, and the days to resumption of ovarian activity in the Sanga and Friesian-Sanga cows were also similar (P?>?0.05). The results demonstrate the beneficial effects of feed supplementation in terms of improved body condition and metabolic status and increased milk yield.     

Effect of peripheral concentrations of progesterone on follicular growth and fertility in ewes

The effects of progesterone (P₄) on follicular growth and fertility in ewes were examined. In Experiment 1, 22 ewes received either one or three packets of P₄ (5 g/packed) or an empty packet subcutaneously (sc) from Days 5 to 15 of the estrous cycle (estrus = Day 0). On Day 6, P₄-treated ewes received 12.5 mg of prostaglandin F₂α. Follicles ⩾3 mm in diameter were observed via transrectal ultrasonography daily from Day 4 through estrus, corpora lutea (CL) were observed 5 to 7 d after estrus. Ewes with low (LOW; ⩽1 ng/ml; n = 5), intermediate (MED; > 1 and <2 ng/ml; n = 10), or normal (NOR; ⩾2 ng/ml; n = 7) P₄ in jugular plasma on Days 7 through 15 differed in follicular development. The largest follicle at estrus was larger in ewes with LOW vs. MED and NOR P₄ (7.8 ± 0.3 vs. 6.9 ± 0.2 mm; P < 0.05). Treatments differed in proportions of multiple-ovulating ewes, in which the oldest ovulatory follicle was first observed before Day 10 (LOW: 3 of 3, MED: 6 of 10, NOR: 0 of 5, respectively; P < 0.05). Estradiol was higher early in the treatment period in LOW ewes than in MED and NOR ewes (day × treatment; P < 0.05). In Experiment 2, ewes received 5 mg of P₄ in corn oil (low progesterone [LP]; n = 51) or 2 ml of corn oil (CON; n = 49) sc every 12 hr on Days 6 through 14 of the estrous cycle before mating. LP ewes received 15 mg of prostaglandin F₂α on Day 6. Mean serum P₄ on Days 7 through 15 was 0.6 ± 0.1 ng/ml in LP and 1.9 ± 0.1 ng/ml in CON ewes. Eleven LP and 12 CON ewes were scanned daily from Day 4 through mating, and in all ewes (n = 93), CL were counted 10 d after mating and embryos were counted at 25, 40, and 60 d of gestation. In multiple-ovulating ewes, day of cycle of appearance was earlier for the oldest (Day 6.1 ± 0.8 vs. 10.4 ± 0.8) but not second oldest (Day 11.7 ± 1.0 vs. 12.2 ± 0.9) ovulatory follicles in LP compared with CON ewes. The conception rate was lower in LP (72%) than in CON ewes (98%; P < 0.01). However, numbers of CL 10 d after mating, and in pregnant ewes, numbers of embryos 25 d after mating and lambs born, did not differ with treatment. In summary, low P₄ increased the size of the largest follicles and the age of the oldest ovulatory follicles. Embryos resulting from the ovulation of older and younger follicles in the same ewe did not differ in their ability to survive.     

Effect of daidzein on production performance and serum antioxidative function in late lactation cows under heat stress

Forty Chinese Holstein cows were randomly divided into 4 groups according to birth, milk yield, and close lactation. The cows in 4 groups were fed with basic diet, basic diet plus 200, 300, 400 mg/day daidzein, respectively; and the experimental period was 60 days. The milk yield in all cows was monitored everyday and the quality of milk was examined regularly. Blood was collected on days 0, 10, 30, 60 of experiment to isolate serum, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), malondialdehyde (MDA) content, and total antioxidant capacity (TAC) in the serum were measured. Results showed that the milk yield and the percentage of milk fat were significantly higher after feeding daidzein (P<0.01), and the percentage of milk protein was significantly elevated on day 60 in three daidzein groups (P<0.05 or P<0.01), especially in 400 mg/day daidzein group (P<0.01). Compared with the control group (basic diet), the TAC and activities of SOD, GSH-Px and CAT were markedly elevated in three daidzein groups (P<0.05 or P<0.01), while the MDA content was significantly reduced in three daidzein groups (P<0.05 or P<0.01). All data suggest that daidzein has positive effects on increasing the production performance and antioxidative activity in late lactation cows, and enhancing the body adaptability and resistance to heat stress.     

Follicular characteristics and intrafollicular concentrations of nitric oxide and ascorbic acid during ovarian acyclicity in water buffalo (Bubalus bubalis)

The objective of this study was to examine the follicular characteristics and intrafollicular concentrations of nitric oxide and ascorbic acid during ovarian acyclicity in buffaloes. Ovaries were collected from 56 acyclic and 95 cyclic buffaloes at slaughter, surface follicle number was counted and follicles were classified into small (5.0–6.9 mm), medium (7.0–9.9 mm), and large (≥10.0 mm) size categories based on their diameter. Follicular fluid was aspirated and assayed for nitric oxide, ascorbic acid, estradiol, and progesterone. Acyclic buffaloes had a higher (P < 0.05) number of medium-sized follicles and a lower (P < 0.001) number of large follicles than the cyclic ones. In acyclic animals, the number of large follicles was lower (P < 0.01) than in medium size category which in turn was lower (P < 0.001) than the number of small follicles. In contrast, the number of medium and large follicles was not different (P > 0.05) in the cyclic control. However, the number of small-sized follicles was higher (P < 0.001) compared to the other two categories. The incidence of large-sized follicles was lower (P < 0.05) in acyclic buffalo population compared to the cyclic control. Evaluation of estrogenic status demonstrated that all the follicles of acyclic buffaloes are estrogen-inactive (E ₂/P ₄ ratio < 1). Small- and medium-sized follicles of acyclic buffaloes had higher concentrations of nitric oxide (P < 0.05 and P < 0.001, respectively) and lower concentrations of ascorbic acid (P < 0.05 and P < 0.01, respectively) than the corresponding size estrogen-active follicles of their cyclic counterparts. In conclusion, this study indicates that follicular development continues during acyclicity in buffaloes. Although follicles in some acyclic buffaloes attain a size corresponding to morphological dominance, they are unable to achieve functional dominance, perhaps due to an altered balance of intrafollicular nitric oxide and ascorbic acid and, as a result, these follicles instead of progressing to ovulation undergo atresia.