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A. Singh and R. Puthiyandy

Summary. Milk samples were collected on Days 20, 24, 28 and 40 after insemination. The percentage of fat and the progesterone concentration in the milk were correlated (P < 0·01). Progesterone concentration in the milk of pregnant buffaloes (24·83 ± 3·85 ng/ml) was significantly higher than that in non-pregnant animals (2·89 ± 1·25 ng/ml) on Day 20 and the difference between the two increased with time after insemination. The detection of non-pregnant animals was 100% successful at all times but the diagnosis was correct for 66, 68, 81 and 83% of animals tested on Days 20, 24, 28 and 40 respectively and predicted as pregnant.

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GUY H. KIRACOFE, A. R. SINGH and N. D. NGHIEM

Summary.

Rabbit antiserum to sheep lh inhibited uterine growth in adult rats following uterine trauma on Day 4 of pseudopregnancy. The weights of the horns subjected to trauma were similar to those obtained in rats ovariectomized at the time of trauma and given only progesterone, and in rats with the adenohypophysis transplanted beneath the kidney capsule on the day of ovulation. Antiserum to lh given to rats with an autotransplanted pituitary did not alter decidual growth. Oestrone restored maximal uterine growth in rats receiving lh antiserum and in rats with an autotransplanted pituitary. Simultaneous injections of fsh and lh resulted in maximal decidual cell response in pituitary autotransplanted rats, while either, given alone, had only slight stimulatory effects. It is suggested that lh antiserum inhibits the oestrogen production necessary for maximal uterine growth. Luteinizing hormone does not appear to be necessary, unless in minute quantity, for production of progesterone in amounts large enough to support maximal uterine growth after trauma.

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J. Singh, R. A. Pierson and G. P. Adams

Heifers were studied to determine whether computer-assisted quantitative echotexture analysis of ultrasound images reflect functional and endocrine characteristics of dominant and subordinate follicles at specific stages of development. Heifers were examined using transrectal ultrasonography each day until ovariectomy on day 3 (n = 8) and day 6 (n = 9) of wave 1, day 1 of wave 2 (n = 7), or after onset of pro-oestrus ≥ 17 days after ovulation (n = 8) to obtain growing, early-static, late-static and regressing dominant follicles of wave 1, subordinate follicles, preselection follicles and preovulatory dominant follicles. Ultrasound images of the follicles were obtained in vitro and analysed using custom-developed computer algorithms. Mean pixel (picture element) values (grey-scale: black = 0, white = 255) for the follicle wall and stroma increased (P < 0.05) progressively from the growing to the regressing phases of the dominant follicle of wave 1. The antrum and wall of subordinate follicles had higher (P < 0.05) mean pixel values than that of the corresponding dominant follicles. Pixel heterogeneity (a measure of variation of grey-scale values of pixels) of images of the follicle antrum and wall increased (P < 0.05) progressively during the early-static to regressing phases. A progressive increase (P < 0.05) in the slope of the regression line of pixel values for the follicle wall was detected from the growing to the regressing phases of the dominant follicle of wave 1. The regression line of the wall of the preovulatory dominant follicle had the lowest (P < 0.05) slope. Oestradiol concentration in the follicular fluid decreased (P < 0.05) from the growing to the late-static phase, while a marked decrease (P < 0.05) in the androstenedione concentration was recorded between the growing and the early-static phases of the dominant follicle. Progesterone content did not increase until follicles were in the final stages of regression. Pixel heterogeneity of the antrum and wall, and the slope of the follicle wall regression line were negatively correlated (P < 0.001) with oestradiol and the oestradiol:progesterone ratio in follicular fluid. The results of this study support the hypothesis that echotexture characteristics of ultrasound images of the follicle antrum and wall are correlated with the functional and endocrine status of a follicle.

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J. Singh, R. A. Pierson and G. P. Adams

Nulliparous heifers (n = 58) were studied to determine whether computer-assisted quantitative echotexture analysis of ultrasound images reflects the functional and histomorphological characteristics of the corpus luteum. The ovaries of heifers were examined daily by transrectal ultrasonography from day −2 (day 0 = ovulation) until the day of ovariectomy during metoestrus (day 3; n = 8), early dioestrus (day 6; n = 9), mid-dioestrus (mean, day 10; n = 7), or pro-oestrus (mean, day 18; n = 8; Expt 1). High resolution ultrasound images of corpora lutea were obtained in vitro, and were digitized and analysed using custom-developed computer algorithms optimized for ultrasonography. Cryostat sections of corpora lutea were examined for lipid distribution, and corpora lutea were homogenized to determine the content of progesterone, total protein, cholesterol and triglyceride. In Expt 2, heifers (n = 26) were ovariectomized as in Expt 1, and ovaries were prepared for histomorphometric evaluation. Pixel values (brightness of picture elements) of ultrasound images of corpora lutea were characterized as high during metoestrus, low during early and mid-dioestrus, and increasing again during pro-oestrus (P < 0.05). Changes (P < 0.001) in volume density of luteal cells were characterized as increasing from metoestrus (40.7 ± 0.4%) to mid-dioestrus (55.8 ± 2.8%) and decreasing again at pro-oestrus (41.5 ± 0.9%). The proportion of blood vascular components decreased (P < 0.001) progressively from 31.0 ± 1.0% in metoestrus to 15.6 ± 1.1% in pro-oestrus. Pixel values of ultrasound images of corpora lutea were correlated with luteal (r = −0.72, P < 0.05) and plasma (r = −0.71, P < 0.03) progesterone concentration, and to the volume densities of luteal cells (r = −0.75, P < 0.02) and connective tissue (r = 0.69, P < 0.03). Estimates of triglyceride, protein and cholesterol content of corpora lutea were not correlated with pixel values of ultrasound images. Protein and cholesterol content did not change while triglyceride concentration increased during pro-oestrus (P < 0.05). Results support the hypothesis that ultrasound images reflect luteal and plasma progesterone content, and histomorphological characteristics of the corpus luteum.

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Kusum Vermat, M. M. Misro, H. Singh, Sudha Mahajan, A. R. Ray and S. K. Guha

Summary. An alternate co-polymer of styrene and maleic anhydride was dissolved in dimethylsulphoxide and injected into the vas deferens of rats. The polymer was retained in the vas deferens and the morphological changes detected were confined to the mucosa. When the polymer was removed by flushing dimethylsulphoxide, the mucosal structure became normal within 2 weeks.

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F C F Dias, M I R Khan, M A Sirard, G P Adams and J Singh

Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2 α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were upregulated. To validate the microarray results, five genes were selected for real-time PCR (NTS, FOS, THBS1, FN1, and IGF2). Expression of four genes increased significantly in the three different animals tested (NTS, FOS, THBS1, and FN1). The upregulated genes are related to matrix remodeling (i.e. tissue proliferation), disturbance of angiogenesis, apoptosis, and oxidative stress response. We conclude that superstimulation treatment i) results in granulosa cells that lag behind in maturation and differentiation (most of the upregulated genes are markers of the follicular growth stage), ii) activates genes involved with the NFE2L2 oxidative stress response and endoplasmic reticulum stress response, and iii) disturbs angiogenesis.

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W Y Kwong, S J Adamiak, A Gwynn, R Singh and K D Sinclair

Maternal B-vitamin status at conception can affect fertility and the health of offspring. This study details transcript expression for genes encoding key enzymes in the linked methionine/folate cycles in the bovine oocyte, somatic cells of the ovarian follicle and pre-implantation embryo. Transcripts for all 12 enzymes that were studied and for the two folate receptors (FOLR1 and FOLR2) and reduced folate carrier (SLC19A1) were expressed in liver cells, but transcripts for betaine-homocysteine methyltransferase and methionine adenosyl transferase 1A were absent in all ovarian cells, and transcripts for FOLR2 were absent in embryonic cells. Transcripts for glycine methyltransferase were also absent/weak in cumulus and granulosa cells. The absence of these enzymes could have a profound effect on single-carbon metabolism within the ovary and pre-implantation embryo. Immunocytochemical analysis revealed SLC19A1 protein expression on the plasma and basal-lateral membranes of the pre-implantation embryo. The folate antagonist methotrexate (MTX) enters the cell via SLC19A1, and in the current study, MTX inclusion in bovine/ovine culture media at either 1 or 10 μM from the 1-cell stage inhibited embryo development beyond the 8-cell stage. Hypoxanthine and thymidine (100 μM) increased the proportion of embryos that developed to blastocysts, but the cell number was reduced by 20%. The reduced uptake of [35S] methionine into intra-cellular S-adenosylmethionine and S-adenosylhomocysteine pools, together with reduced uptake of glutamate and tryptophan, was consistent with depleted intra-cellular pools of reduced folates. These data provide an insight into the importance of maternal dietary folate/B-vitamin status during the peri-conceptional period.