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P. HEALEY

Summary.

Spermatozoa of bull, ram, boar, stallion and chinchilla were examined in the electron microscope before and after freezing of samples of semen in liquid nitrogen. The results showed that the ultrastructure of bull spermatozoa was almost identical before and after treatment, but ram and chinchilla spermatozoa showed consistent damage to the outer membrane and acrosome complex. In the ram, damage ranged from slight swelling of the acrosome to total removal of the cytoplasmic regions and most chinchilla spermatozoa were grossly affected. The spermatozoa of boar presented special difficulties at the fixation stage but gross damage to the acrosome was seen. There appeared to be minimal damage to the stallion spermatozoa, although only one sample of this species was examined.

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P. HEALEY and BARBARA J. WEIR

During the search for a suitable medium in which to freeze spermatozoa of the chinchilla (Chinchilla laniger), samples taken from ten males by electro-ejaculation (Healey & Weir, 1967) were pooled and studied after treatment with different diluents. All the samples contained a large proportion of motile spermatozoa and no samples were discarded before treatment. One aliquot of undiluted semen was dropped into 2 % buffered osmium tetroxide at pH 7·4 (Palade, 1952) and other aliquots were diluted in 0·5 ml of Baker's fluid (Baker, 1931), normal Ringer (8·5 g sodium chloride, 0·42 g potassium chloride and 0·25 g calcium chloride/litre water), half-strength Ringer or double-strength Ringer. The undiluted semen solidified immediately in the fixative and only the outer layer was used. The sperm suspensions in the different diluents were left at room temperature
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P. HEALEY and BARBARA J. WEIR

This technique was standardized on chinchilla (Chinchilla laniger) as part of two wider projects: (a) to investigate a method for artificial insemination (Weir, 1966), and (b) an electron microscope study of the spermatozoa.

Dalziel & Phillips (1948) used unipolar lumbar-anal electrodes on both guinea-pigs and chinchillas, Scott & Dziuk (1959) reported success with bipolar rectal electrodes on rats, mice and guinea-pigs. Hillemann, Gaynor & Dorsch (1963) described an electro-ejaculation method in chinchilla, but gave no details of the electrode or of the voltage used (Table 1).

Some of the sixty-four chinchillas used were those already in our colony and the rest were kindly lent by two chinchilla ranchers. The electrode was a multiring type (Healey & Sadleir, 1966) 12 cm long × 6·5 mm in diameter with 2·5 mm brass rings separated by

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P. HEALEY and R. M. F. S. SADLEIR

The technique of electro-ejaculation is now commonly used for the collection of semen from some domesticated species and is currently being applied in this Institute to exotic species of differing sizes. This note describes the construction of several types of rectal electrodes which we are using in the development of semen collection techniques. Originally a single rectal pole in combination with a needle pole inserted in the lumbar muscles was used by Gunn (1936) for electro-ejaculation of the ram. Laplaud & Cassou (1945) did not describe the bipolar rectal electrode which they used on rams but later Thibault, Laplaud & Ortavant (1948) gave details of a multipolar electrode having thirty brass rings, separated by vulcanite rings, for use on bulls. Blackshaw (1954) described a four-electrode pole for rams made from copper tubing covered with

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João P E Saut, Gareth D Healey, Alan M Borges and I Martin Sheldon

The risk of bacterial infection of the endometrium causing uterine disease in cattle is increased in the progesterone-dominated luteal phase of the ovarian cycle, while oestrogens or oestrus are therapeutic or protective against disease. The first line of defence against bacteria, such as Escherichia coli that cause inflammation of the endometrium, is the innate immune system, which recognises bacterial lipopolysaccharide (LPS). This study tested the hypothesis that cyclic variation in ovarian hormone concentrations alters innate immune responses within the bovine endometrium. Ex vivo organ cultures of endometrium, and in vitro cultures of endometrial epithelial and stromal cells, and peripheral blood mononuclear cells (PBMCs), all mounted inflammatory responses to E. coli or LPS, with secretion of inflammatory mediators interleukin 1β (IL1β), IL6 and IL8, and increased expression of mRNA encoding IL1B, IL6, CXCL8 (IL8) and CCL5. However, these inflammatory responses, typical of innate immunity, were not affected by the stage of ovarian cycle in which the endometrium was collected for organ culture, or by exogenous oestradiol or progesterone. Although a dexamethasone-positive control reduced inflammation stimulated by E. coli or LPS, treatment with oestradiol or progesterone, or inhibitors of oestradiol or progesterone nuclear receptors, did not affect endometrial cell or PBMC secretion of IL1β, IL6 or IL8, or IL1B, IL6, CXCL8 and CCL5 gene expression. In conclusion, the stage of the oestrus cycle or ovarian steroids did not modulate the innate immune response in the bovine endometrium in vitro.

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A Vitorino Carvalho, P Reinaud, N Forde, G D Healey, C Eozenou, C Giraud-Delville, N Mansouri-Attia, L Gall, C Richard, P Lonergan, I M Sheldon, R G Lea and O Sandra

In mammals, suppressor of cytokine signalling (CISH, SOCS1 to SOCS7) factors control signalling pathways involved in the regulation of numerous physiological processes including pregnancy. In order to gain new insights into the biological functions of SOCS in the endometrium, a comprehensive analysis of SOCS gene expression was carried out in bovine caruncular (CAR) and intercaruncular (ICAR) tissues collected i) during the oestrous cycle, ii) at the time of maternal recognition of pregnancy and at implantation in inseminated females, iii) following uterine interferon-tau (IFNT) infusion at day 14 post-oestrus, iv) following a period of controlled intravaginal progesterone release and v) following transfer of embryos by somatic-cell nuclear transfer (SCNT). The regulatory effects of IFNT on in vitro cultured epithelial and stromal cells were also examined. Altogether, our data showed that CISH, SOCS4, SOCS5 and SOCS7 mRNA levels were poorly affected during luteolysis and pregnancy. In contrast, SOCS1, SOCS2, SOCS3 and SOCS6 mRNA levels were strongly up-regulated at implantation (day 20 of pregnancy). Experimental in vitro and in vivo models demonstrated that only CISH, SOCS1, SOCS2 and SOCS3 were IFNT-induced genes. Immunohistochemistry showed an intense SOCS3 and SOCS6 staining in the nucleus of luminal and glandular epithelium and of stromal cells of pregnant endometrium. Finally, SOCS3 expression was significantly increased in SCNT pregnancies in keeping with the altered immune function previously reported in this model of compromised implantation. Collectively, our data suggest that spatio-temporal changes in endometrial SOCS gene expression reflect the acquisition of receptivity, maternal recognition of pregnancy and implantation.

Free French abstract

A French translation of this abstract is freely available at http://www.reproduction-online.org/content/148/6/545/suppl/DC1.