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D. P. MUKHERJEE and S. P. SINGH

Summary.

Exposure of male mice to a 1·8/1·0 mixture of air/CO2 for a total of 6 hr reduced the area and breadth of the head and of the midpiece of live spermatozoa in the vasa deferentia. During a total of 26½ hr exposure spread over 6 days, males when test-mated had a low conception rate but the numbers of offspring in the litters produced were normal. The low conception rate of males appeared to persist even 15 days after the end of treatment.

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M. M. Singh, S. Sreenivasulu and V. P. Kamboj

The duration of the anti-implantation action of a single oral post-coital dose (1.25 mg kg−1) of a triphenylethylene anti-oestrogen, centchroman, was determined in adult rats. The effects of centchroman were compared with those of tamoxifen. In rats undergoing delay, centchroman administered orally on day 7 post-coitum prevented the induction of implantation of delayed blastocysts by an implantation inducing dose (1 μg per rat, s.c.) of oestrone which was administered earlier than 120 h after centchroman treatment. In tamoxifen (0.2 mg kg−1, orally) pretreated rats, oestrone administered at 144 h or later induced implantation. In cyclic rats treated with centchroman at intervals of 168 h and mated with males of proven fertility, implantation was prevented only when the interval between centchroman treatment and nidatory oestrogen secretion was less than 120 h. None of the females conceived when treated regularly at intervals of 120 h during exposure to fertile males. Discontinuation of treatment resulted in the occurrence of normal implantations in rats that mated 48 h or later after the last dose of centchroman, since in these animals the interval between anti-oestrogen treatment and nidatory oestrogen secretion was greater than 120 h. These findings suggest that the duration of the anti-implantation action of a single oral antifertility dose of centchroman in rats is about 120 h. Recovery of normal blastocysts from rats treated continuously with this dose of centchroman at these intervals suggests lack of significant effect on follicular maturation, ovulation, fertilization, preimplantation development or mating behaviour. Tamoxifen appears to be slightly longer acting and the duration of its action was about 144 h.

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G. Singh, M. M. Singh, S. C. Maitra, W. Elger, V. Kalra, S. N. Upadhyay, S. R. Chowdhury and V. P. Kamboj

Summary. RU-38486 or ZK-98734 treatment (3 mg/day, s.c.) to intact or hysterectomized adult female rats on Days 5–7 post coitum induced changes characteristic of luteolysis. Ultrastructurally, the luteal cells exhibited an extensive vacuolization of the cytoplasm and perinuclear areas, degeneration of mitochondrial cristae, massive accumulation of lipid droplets, increase in number of lysosome like granules and heterochromatinization of the nucleus. In general, RU-38486 induced more marked degeneration of the luteal cells than did ZK-98734. There was also a significant decrease in peripheral plasma progesterone concentrations in treated rats. We suggest that these antiprogestagens act via inhibition of luteal function in addition to their antagonism at the uterine progesterone receptor level.

Keywords: antiprogestagens; ultrastructure; corpora lutea; progesterone; rat

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J. Mohan, J. N. Panda, U. S. Singh and R. P. Moudgal

Summary. Daily oral administration of gossypol acetic acid (40 mg/kg body weight daily) resulted in a gradual decrease in the semen volume and sperm concentration. Fertility dropped to zero at the end of the treatment period. Activities of acrosin, hyaluronidase and angiotensin converting enzyme were also drastically decreased by the end of the treatment period. A loss of appetite, loss of body weight and morphological abnormalities in spermatozoa were noticed in the treated cocks. At 4 weeks after cessation of the treatment, full recovery of the above measures was recorded. Healthy chicks were hatched and were observed for several months.

Keywords: gossypol acetic acid; antifertility; cock spermatozoa; hyaluronidase; acrosin; angiotensin converting enzyme

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S. M. Totey, G. Singh, M. Taneja, C. H. Pawshe and G. P. Talwar

Summary. Cumulus–oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47·4 ± 17·8 and 44·8 ± 25·6, respectively. Addition of luteinizing hormone (LH) (5 μg ml−1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76·8 ± 18·3), but follicle-stimulating hormone (FSH) (0·5 μg ml−1) and oestradiol (1 μg ml−1) failed to synergize with LH (71·7 ± 19·5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42·7 ± 1·4 and 81·7 ± 14·5, respectively; P < 0·05). Frozen–thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine l−1 ± 10 μg heparin showed a higher fertilization rate (29·8%) than those treated in Hepes–Talp and treated with 10 μg heparin ml−1 (19·6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine l−1 and 10 pg heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34·1 and 36·8%, respectively) than with frozen–thawed spermatozoa (27·0 and 22·0%, respectively).

Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28·0%) than when co-cultured on oviductal cell monolayers (8·2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.

Keywords: oocytes; maturation; fertilization; culture; buffalo

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Pritpal S Malhi, Gregg P Adams, Reuben J Mapletoft and Jaswant Singh

The study was designed to test the hypothesis that aging in cattle is associated with reduced developmental competence of oocytes. The hypothesis was tested by comparing embryo production and pregnancy rates between 13- to 16-year-old cows (n = 6 in Year 1 and n = 9 in Year 2) and their 3- to 6-year-old young daughters (n = 8 in Year 1 and n = 9 in Year 2) after superovulation and transfer of embryos into an unrelated group of young recipients. Embryos were transferred into 2- to 5-year-old recipient cows (n = 99) as singletons (n = 45) or in pairs (n = 54 pairs). Embryo survival in recipients was determined by ultrasonography and by the number of calves born. Between old versus young cows, the number of ovulations (31 ± 4 vs 38 ± 3; P = 0.2) and the number of corpora lutea (25 ± 3 vs 29 ± 2; P = 0.3) did not differ, but fewer (P = 0.04) embryos were recovered from old cows (6 ± 2) than their daughters (12 ± 2). A higher proportion (P < 0.0001) of unfertilized oocytes/uncleaved zygotes were recovered from old cows (222/312, 71%) than their daughters (119/316, 38%). Among the embryos recovered, the proportion of International Embryo Transfer Society Grades 1–2 embryos was similar (P = 0.9) between old (59/90, 66%) and young cows (130/194, 67%). The survival of embryos after transfer into recipients, and the proportion of calves born were also similar between old and young cows. In conclusion, recovery of fewer embryos and a greater proportion of unfertilized oocytes/uncleaved zygotes suggest reduced developmental competence of oocytes from old cows, but there was no difference between age groups in embryo survival after the morula/blastocyst stage.

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S G Hannesdóttir, X Han, T Lund, M Singh, R van der Zee, I M Roitt and P J Delves

Immunosterilization is an attractive alternative to surgical castration. Gonadotropin-releasing hormone (GnRH) controls the production of the gonadotropins thereby having an orchestrating effect on the reproductive hormone cascade and spermatogenesis. Induction of neutralizing antibody can abrogate the effect of the hormone. Current GnRH-based vaccines often require strong adjuvants and/or multiple injections of the vaccines to overcome variability in the response. Heat shock proteins (hsp) have been used as carrier molecules because of their powerful intrinsic ability to enhance an immune response to associated antigens. A GnRH-analogue, GnRH-d6-Lys, was conjugated to recombinant Mycobacterium tuberculosis hsp70. Male BALB/c mice were immunized i.p. with GnRH-hsp70 in the mild adjuvant Ribi or in incomplete Freund’s adjuvant (IFA). The initial immunizations were done on pre-pubertal 3-week-old mice, with boosts at 5 and 8 weeks of age. The mice were killed at 10 weeks of age and GnRH-specific antibodies and serum testosterone levels measured. All the immunized mice mounted GnRH-specific antibody responses, with no difference in the mice immunized with GnRH-hsp70/Ribi or with GnRH-hsp70/IFA. There was substantial atrophy of the urogenital complex and significantly (P < 0.0005) reduced levels of testosterone-dependent testicular relaxin-like factor mRNA expression. Mice immunized with GnRH-hsp70/Ribi showed substantially reduced (P < 0.001) serum testosterone levels. These results indicate that hsp70 may serve as a particularly advantageous carrier for GnRH-based vaccines.