Morphological observations on frozen and thawed Muscovy spermatozoa

1. Fresh Muscovy drake spermatozoa were examined using a scanning electron microscope (SEM). The average lengths of the segments were: acrosome 1·8 μm, nucleus 10‐9 μm, midpiece 3·6 μm and flagellum (exclusive of midpiece) 71 μm.

2. Under the light microscope, the incidence of abnormal spermatozoa in Muscovy semen subjected to freezing and thawing (almost all with crooked necks) was about 5% higher than that in diluted unfrozen semen.

3. In thawed semen, various abnormalities of the acrosome were observed under the SEM. It seemed that the most radical change was the complete separation of the acrosome from the apical part of the nucleus.

4. The incidence of abnormal acrosomes was increased more than 20% by freezing and thawing.

5. These results suggest that low fertility in thawed semen may be related to increases in the proportion of spermatozoa with crooked necks and acrosomal damage.

     

Genetics of the hypoplasia of feathers of the tail region in the Ingie fowl

1. A unique breed of fowls called Ingie, found on the Tanegashima Island of Kagoshima Prefecture in Japan, is characterised by the incomplete development of the tail feathers.

2. No abnormality of the caudal skeleton was found in the Ingie fowls but the structure of anterior and posterior barbules of the tail feathers differed from those of the normal fowl.

3. Genetical analysis indicated that the hypoplasia of the tail feathers is inherited as an autosomal dominant character: the symbol Hy is proposed for the gene.     

Relationship between type 1/type 2 immune responses and occurrence of vertical transmission in BALB/c mice infected with Neospora caninum

To examine the relationship between occurrence of vertical transmission and type 1/type 2 immune responses induced by Neospora caninum infection in BALB/c mice, pregnant (group 1 p) and non-pregnant mice (group 1 np) were inoculated with 2 x 10(6) of the N. caninum parasites and then we examined the vertical transmission rate and production of IFN-gamma and IL-4. We also studied chronically infected mice, which were bred at 4 weeks or more after infection (group 2), and mice inoculated during pregnancy and re-bred at 4 weeks or more after delivery (group 3). In groups 1p, 2 and 3, vertical transmission was observed in 27.4, 41.4, and 50% of the offspring, respectively. The serum IFN-gamma level increased on days 1 and 5 post-inoculation (p.i.) in groups 1 p and 1 np, while no increase level was observed in groups 2 and 3 during pregnancy or after delivery. When the mice in groups 2 and 3 were re-inoculated, all mice showed a transient increase in serum IFN-gamma on day 1 post-re-inoculation. The serum IL-4 level in both of groups 1p and np increased in a similar manner following infection. In group 3, the serum IL-4 level was somewhat higher than that in group 2 after re-inoculation. The anti-N. caninum antibody IgG1 titer in group 3 increased on day 10 post-re-inoculation. These results suggest that the mice infected during pregnancy may acquire a weaker immune response to the parasite than mice infected when they are not pregnant, and that mice infected during pregnancy may show an enhanced type 2 immune response in the recrudescence of the infection.     

Studies of the factors causing abnormal acrosomes and crooked-necks in fowl spermatozoa during freezing and thawing

In order to investigate the factors causing crooked-necked spermatozoa (CNS) or those with abnormal acrosomes during freezing and thawing, fowl spermatozoa in NaCl or glucose solutions containing 92 ml glycerol/l were examined using a scanning electron microscope before and after freezing and thawing. The incidence of CNS in NaCl solution significantly increased after freezing and thawing, but not in glucose solution. The acrosomal damage caused by freezing and thawing was considerable in both solutions, and the incidence of damage in glucose solution was significantly higher than that in NaCl solution. In neither solution was there a significant difference between the incidence of acrosomal damage in CNS and in non-CNS. The ratios of incidences of abnormal acrosome after, versus before, freezing were higher in non-CNS than in CNS. It appears from these results that the factors during freezing and thawing which cause CNS may differ from those causing acrosomal damage.     

Effect of active immunization of pony mares against recombinant porcine inhibin alpha subunit on ovarian follicular development and plasma steroids and gonadotropins

Two pony mares were immunized against recombinant porcine inhibin alpha subunit three times with 39 day intervals. Clinical findings and endocrinological changes before immunization were taken as the control. The first significant rise in the anti-inhibin titre (P<0.05) in the circulation was found 27 days after the first injection. Maximum binding activity was reached by the 12th day after the second booster dose. The number of small, medium and large sized follicles had increased significantly compared to before immunization (11.75 +/- 4.30, 2.75 +/- 0.69 and 2.51 +/- 0.63 vs 6.50 +/- 1.43, 1.83 +/- 0.44 and 1.33 +/- 0.38, respectively), but the ovulation rate remained unchanged after immunization. The average plasma concentration of FSH and estradiol-17beta during the estrous cycle increased significantly (P<0.05) after immunization. These results suggest that immunization against inhibin is a useful tool to increase the number of ovarian follicles during the estrous cycle of pony mares. Moreover, the present study supported the concept that inhibin plays a major role in the control of follicular growth through its inhibitory effect on FSH secretion synergistically with steroid hormones.     

Expression of LacZ gene in canine muscle by intramuscular inoculation of a plasmid DNA

DNA immunization induces systemic humoral and cellular immune responses to the antigen encoded by cDNA in a plasmid DNA. In the present study, a plasmid DNA encoding cDNA of beta-galactosidase (beta-gal), pCAGGS-lacZ, was inoculated intramuscularly to a healthy dog in order to evaluate location and duration of the gene expression. On day 7, the plasmid DNA was found by PCR in the muscle where the plasmid was injected. Furthermore, beta-gal expression was detected in the same muscle sample by beta-gal staining. However, the plasmid DNA was not detected in any samples collected on days 14, 21 and 28. The present results suggest that duration of the gene expression of beta-gal by the plasmid DNA is limited in the muscle in dogs and an efficacy for a gene expression should be evaluated depending on the gene inserted in the plasmid DNA for immunotherapy.     

Immunological findings in 3 dogs clinically diagnosed with allergic rhinitis

Three dogs clinically diagnosed with allergic rhinitis (AR) were examined for their immunological findings. House dust mites (HDM) such as Dermatophagoides farinae (DF) and D. pteronyssinus (DP) were identified as positive allergens in the 3 dogs with both intradermal skin test and serum antigen-specific IgE test. Lymphocyte blastogenic response of peripheral blood mononuclear cells (PBMCs) under stimulation with DF antigen in dogs with AR was higher than that in 4 healthy control dogs. Expression level of IL-4 mRNA in PBMCs obtained from the 3 AR dogs was higher than that in PBMCs obtained from 4 healthy control dogs before and after stimulation with DF antigen. Expression level of IFN-gamma mRNA in PBMCs was not different between the AR and control dogs before and after stimulation with DF antigen. These results suggested that allergic reaction to HDM antigen and T(H)2-type immune response were associated with the development of AR in 3 dogs examined in this study.     

Regulation mechanism of selective atresia in porcine follicles: regulation of granulosa cell apoptosis during atresia

More than 99% of follicles undergo a degenerative process known as "atresia", in mammalian ovaries, and only a few follicles ovulate during ovarian follicular development. We have investigated the molecular mechanism of selective follicular atresia in mammalian ovaries, and have reported that follicular selection dominantly depends on granulosa cell apoptosis. However, we have little knowledge of the molecular mechanisms that control apoptotic cell death in granulosa cells during follicle selection. To date, at least five cell death ligand-receptor systems [tumor necrosis factor (TNF)alpha and receptors, Fas (also called APO-1/CD95) ligand and receptors, TNF-related apoptosis-inducing ligand (TRAIL; also called APO-2) and receptors, APO-3 ligand and receptors, and PFG-5 ligand and receptors] have been reported in granulosa cells of porcine ovaries. Some cell death ligand-receptor systems have "decoy" receptors, which act as inhibitors of cell death ligand-induced apoptosis in granulosa cells. Moreover, we showed that the porcine granulosa cell is a type II apoptotic cell, which has the mitochondrion-dependent apoptosis-signaling pathway. Briefly, the cell death receptor-mediated apoptosis signaling pathway in granulosa cells has been suggested to be as follows. (1) A cell death ligand binds to the extracellular domain of a cell death receptor, which contains an intracellular death domain (DD). (2) The intracellular DD of the cell death receptor interacts with the DD of the adaptor protein (Fas-associated death domain: FADD) through a homophilic DD interaction. (3) FADD activates an initiator caspase (procaspase-8; also called FLICE), which is a bipartite molecule, containing an N-terminal death effector domain (DED) and a C-terminal DD. (4) Procaspase-8 begins auto-proteolytic cleavage and activation. (5) The auto-activated caspase-8 cleaves Bid protein. (6) The truncated Bid releases cytochrome c from mitochondrion. (7) Cytochrome c and ATP-dependent oligimerization of apoptotic protease-activating factor-1 (Apaf-1) allows recruitment of procaspase-9 into the apoptosome complex. Activation of procaspase-9 is mediated by means of a conformational change. (8) The activated caspase-9 cleaves downstream effector caspases (caspase-3). (9) Finally, apoptosis is induced. Recently, we found two intracellular inhibitor proteins [cellular FLICE-like inhibitory protein short form (cFLIPS) and long form (cFLIPL)], which were strongly expressed in granulosa cells, and they may act as anti-apoptotic/survival factors. Further in vivo and in vitro studies will elucidate the largely unknown molecular mechanisms, e. g. which cell death ligand-receptor system is the dominant factor controlling the granulosa cell apoptosis of selective follicular atresia in mammalian ovaries. If we could elucidate the molecular mechanism of granulosa cell apoptosis (follicular selection), we could accurately diagnose the healthy ovulating follicles and precisely evaluate the oocyte quality. We hope that the mechanism will be clarified and lead to an integrated understanding of the regulation mechanism.     

Prominent expression of spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1 in LH-producing cells, LbetaT2

The LH-producing cell line, LbetaT2, and non LH-producing cell line, alphaT3-1 cells, established from a pituitary tumor, were employed for cDNA subtraction cloning to identify genes with expression unique to LH producing cells. Several cDNAs that code for known as well as for many unidentified clones were discovered. Most clones were the spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1, the abnormality of which causes neurodegeneration and loss of cerebellar Purkinje cells. We examined whether the expression of SCA1 gene in LbetaT2 cells is related to hormone production. We also compared the expression of SCA1 with that in various other pituitary tumor derived cell lines, and confirmed the prominent expression of SCA1 in LbetaT2 cells. The effect of gonadal factor(s) for SCA1 gene expression was examined. The expression level in female rats was low and did not change during the estrus cycle, but increased significantly after ovariectomy and did not return to the normal level under low and high doses of estrogen. In the male pituitary SCA1 gene expression increased markedly after castration and was not decreased by estrogen or testosterone. The Ontogeny of SCA1 gene expression was investigated in porcine fetal and postnatal pituitaries and revealed biphasic and sexually dimorphic expression. Transient expression of SCA1 gene was observed at fetal day 50 and 65 in males and day 40 in females, followed by a decline and increased expression before birth in both genders. Thus the expression of SCA1 gene is prominent in LH-producing cells and is not under direct control of gonadal factor(s) in both genders. In addition to the variable expression of SCA1 gene during the fetus period, the present results provide a novel aspect to the understanding of Boucher-Neuhauser syndrome (Ataxia Hypogonadism Choroidal Dystrophy).     

Vertical transmission of Neospora caninum in BALB/c mice in both acute and chronic infection

To examine the frequency of congenital infection by Neospora caninum, BALB/c mice were inoculated intraperitoneally with tachyzoites of N. caninum either during pregnancy (Group 1) or 4 weeks or more before pregnancy (Group 2). Further, the mice inoculated during pregnancy were bred at 4 weeks or more after delivery to form Group 3. Congenital transmission was observed in 76% of the neonates of the mice in Group 1 and in 50% of the neonates of the mice in Group 2. Interestingly, congenital transmission was observed in 86% of the neonates from Group 3. These results suggest that chronically-infected BALB/c mice efficiently transmit N. caninum infection to their offspring.