Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury,but respond favorably to dimethyl sulfoxide

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000 mOsm), sucrose (369 and 479 mOsm), 1 M glycerol (1086 mOsm) or dimethyl sulfoxide (Me₂SO, 1151 mOsm) for 10 min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me₂SO had no influence (P > 0.05), NaCl and sucrose solutions affected sperm motility (P < 0.05), but not membrane integrity. Motility of sperm exposed to <600 mOsm NaCl or sucrose was less (P < 0.05) than fresh ejaculate, but comparable (P > 0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1 M glycerol or Me₂SO and cooled from 5 °C to −120 °C at −57.8 °C, −124.2 °C or −67.0 °C/min, frozen in LN₂, thawed in a water bath for 30 s at 37 °C or 10 s at 50 °C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P < 0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P < 0.05) post-thaw motility (20.0 ± 1.9% versus 13.5 ± 2.1%) and membrane integrity (51.2 ± 1.7% versus 41.5 ± 2.2%) were observed in samples cryopreserved in Me₂SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with −57.8 °C/min being advantageous (P < 0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me₂SO as a cryoprotectant.     

The vocal repertoires of the bush dog (Speothos venaticus), crab-eating fox (Cerdocyon thous), and maned wolf (Chrysocyon brachyurus)

The vocalizations of the bush dog (Speothos venaticus), crab-eating fox (Cerdocyon thous), and maned wolf (Chrysocyon brachyurus) were studied in a captive setting. The three species' repertoires were similar and included whines, long-distance calls, and growls. The whines and growls varied in amplitude, duration, and repetition rate; these variations and the frequencies of occurrence probably were influenced by both social organization and habitat characteristics. The bush dog, which lives in family groups and inhabits rain forests, had an elaborate whine-scream continuum that at close distances conveyed a wide range of moods. On the other hand, the maned wolf, which is solitary and frequents open pampas, used amplitude variations of the growl to signal hostile intentions over short, medium, and long distances.The long-distance vocalizations of the three species were the least variable in structure; the bush dog and crab-eating fox used the call to promote contact with separated pairmates or family members. Based on recordings during development, these two calls appeared to arise from the repetitive whine, a variant of the basic whine syllable. The maned wolf also had a contact-promoting howl, apparently an extended whine of high amplitude, but it was rarely recorded during the present study. More prominent was the maned wolf roar-bark, which functioned to space individuals over large distances, and appeared to be a growl of high amplitude.     

Induction of fertile oestrus in the bitch using Deslorelin, a GnRH agonist

Oestrus induction in various canine breeds was attempted in 32 bitches. A group of 8 bitches were treated 80–160 d following their previous oestrus (G1) whereas a second group of 24 bitches (G2) were implanted 200–590 d following their previous oestrus. The treatment for each bitch consisted in one Deslorelin implant (Suprelorin®4,7mg, Virbac, France), inserted subcutaneously in the post-umbilical region. Ovulation, pregnancy rate and litter size were recorded. All bitches came in heat 4.3 ± 1.4 d after implantation (2–7 d). Ovulation was reported in 62.5% in G1 and 87.5% in G2. One bitch refused mating and since no AI was performed, she was not considered for further analysis. Pregnancy was obtained in 25% in G1 versus 78.3% in G2. Mean litter size was 6.7 ± 3.5 puppies (1–14). Luteal failure was suspected in 3 bitches, two that remained non-pregnant and one which aborted 58 d post-ovulation since the owner refused progesterone supplementation. Deslorelin implants can therefore be considered as a valuable alternative to induce fertile oestrus in bitches in anoestrus. Follow-up of the luteal phase is recommended, since some bitches might encounter luteal failure.     

Effect of an implant containing the GnRH agonist deslorelin on secretion of LH, ovarian activity and milk yield of postpartum dairy cows

Prevention of high plasma progesterone concentrations in the early postpartum period may improve fertility. Our objective was to determine whether a Deslorelin implant (DESL; 2100 microg, s.c.) would reduce secretion of LH and alter follicle dynamics, plasma concentrations of progesterone, estradiol and PGF2alpha metabolite (PGFM) in postpartum dairy cows. Cows received DESL on Day 7 postpartum (Day 7, n=8) or were untreated (Control, n=9). All cows were injected with GnRH (100 microg, i.m.) on Day 14 to assess LH response. A protocol for synchronization of ovulation with timed AI was initiated on Day 60 (GnRH [Day 60], CIDR [Day 60 to Day 67], PGF2alpha [Day 67, 25 mg and Day 68, 15 mg], GnRH [Day 69] , AI [Day 70]). The LH response to injection of GnRH on Day 14 was blocked in animals treated with DESL. Numbers of Class 1 (<6 mm) follicles were unaffected (P > 0.05) whereas numbers of Class 2 (6 to 9 mm) (P < 0.01) and Class 3 (>9 mm) follicles were less (P < 0.01) in DESL cows between Day 7 and Day 21. From Day 22 to Day 60, DESL-treated cows had more of Class 1 follicles and less Class 2 (P < 0.01) and Class 3 (P < 0.01) follicles, and lower plasma concentrations of progesterone and estradiol (P < 0.01). Concentrations of PGFM between Day 7 and Day 42 were not affected by treatment (P > 0.05). All cows ovulated in response to GnRH on Day 69. Subsequent luteal phase increases in plasma progesterone concentrations (Day 70 to Day 84) did not differ. The use of the DESL implant associated with PGF2alpha given 14 days later suppressed ovarian activity and caused plasma progesterone concentrations to remain < 1 ng/mL between Day 22 and Day 51. The DESL implant did not affect milk production.     

Assessment of ovarian activity in captive goral (Naemorhedus griseus) using noninvasive fecal steroid monitoring

To date, there is no information on gonadal steroidogenic activity of female goral (Naemorhedus griseus), a threatened species of Thailand. Captive goral populations have been established to produce animals for ex situ conservation and reintroduction, but as yet none are self-sustaining. The objectives of the present study were to (1) determine the influence of season on ovarian steriodogenic function; and (2) examine the relationship between gonadal hormone excretion and sexual behaviors throughout the year. Fecal samples were collected 5 to 7 days/wk for 15 months from 8 adult females housed at Omkoi Wildlife Breeding Center in Thailand and analyzed for ovarian steroid metabolites using validated enzyme immunoassays. Observations of sexual behaviors and mating were conducted each morning for 30 min/session. Based on fecal estrogen and progestagen metabolite concentrations, the overall estrous cycle length was about 21 days, with a 2- to 3-day follicular phase and an 18- to 20-day luteal phase. Sexual behaviors, most notably tail-up, increased for 2 to 3 days during the time estrogens were elevated during mating. Fecal progestagens were elevated during luteal phases and increased further during gestation, which lasted approximately 7 months. The lactation period was 5 months, and females were anestrus for 2 to 5 of those months, with the exception of one that cycled continuously throughout. Two females conceived around 2 months postpartum and so were pregnant during lactation. Birth records over the past 21 years indicated young are born throughout the year. This combined with the hormonal data suggests that female gorals are not strongly seasonal, at least in captivity, although there was considerable variation among females in estrogen and progestagen patterns. In conclusion, fecal steroid metabolite monitoring is an effective means of assessing ovarian function in this species and will be a useful tool for breeding management and planned development of assisted reproductive techniques such as artificial insemination and embryo transfer.     

Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori

A cell line BmN-SWU1 was established from the ovarian tissues of 3-day-old fourth instar Bombyx mori larvae of the 21-872nlw variety by performing primary cultures in Grace's medium supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of short spindle cells and round cells. The frequency of cells with chromosome number 2n = 56 was 80.5%; therefore, the cell line was considered to be a diploid cell line. The population-doubling time (PDT) at 45th passage line was 57.7 h. This cell line was susceptible to the B. mori nuclear polyhedrovirus (BmNPV), and the median tissue culture infective dose (TCID₅₀) at a cell density of 10⁵ cells/ml was 16.3 OBs/ml. The transient expression efficiency of the green fluorescent protein (GFP) gene in this cell line was 54.8%. We used the BmN-SWU1 cell line to select and establish a GFP transgenic cell line.     

Induction of lysozymelike activity in the hemolymph and hemocytes of an insect, Spodoptera eridania

Lysozymelike activity is present in the hemocytes and cell-free hemolymph of Spodoptera eridania. Its level remains essentially constant during larval development and can be induced by injection of various foreign materials. Serum bacteriolytic activity rises 24 hr after injection of saline, BSA, bacteria, bacterial endotoxin (LPS), latex particles, or sham injection. However, the magnitude and subsequent duration of the response depends on the nature of the injected material. The response is transient following sham injection or injection of soluble substances, such as saline and BSA, as compared to treatment with latex or bacteria. Both soluble and insoluble fractions of bacterial LPS preparations stimulated the lysozyme response. The response to a single injection of E. coli LPS was dose dependent and persisted for at least 5 days; however, additional injections had no effect on serum lysozyme level. The basal intracellular lysozyme level was significantly increased by E. coli LPS injection. Lysozyme release by hemocytes was proportional to intracellular concentration and did not increase after phagocytic stimulation of hemocytes.     

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila.     

Ultrastructure of the spermatozoon of Anomotaenia quelea (Mettrick, 1961) (Cestoda,Cyclophyllidea, Dilepididae), an intestinal parasite of Quelea quelea (Aves,Ploceidae) in Senegal

The mature spermatozoon of Anomotaenia quelea exhibits an apical cone of electron-dense material and two helicoidal crest-like bodies. The apical cone near its base is surrounded by a lucent cytoplasm and a spiraled layer of cortical microtubules. The crest-like bodies are of different lengths, spiraled and make an angle of 30–40° to the hypothetical spermatozoon axis. The axoneme is of the 9 + ‘1’ trepaxonematan pattern and is surrounded by a periaxonemal sheath of electron-dense material. The cytoplasm contains in regions III and IV numerous electron-dense granules situated between the periaxonemal sheath and the cortical microtubules. The posterior extremity of the spermatozoon of A. quelea exhibits a nucleus and a disorganized axoneme and cortical microtubules. This type of posterior extremity of the mature spermatozoon has never been described previously in a Dilepididae. Similarly, two crest-like bodies have not been observed before in a dilepidid cestode.     

A diapause induction-termination asymmetry in the photoperiodic responses of the Linden bug, Pyrrhocoris apterus and an effect of near-critical photoperiods on development

The critical daylength for the induction of adult (ovarian) diapause in Pyrrhocoris apterus (central Bohemian population) was found to be 15.75 hr per 24 hr, whereas that for diapause termination was 1 hr longer, at 16.75 hr per 24 hr. The termination threshold remained unaltered through 28 to 98 days (4–14 weeks) in diapause, but the need for photoperiodic termination was abolished after 12–16 weeks at 4°C. The rate of nymphal development (25°C) was relatively rapid at both short (< 14.5 hr per 24 hr) and long (> 16.5 hr per 24 hr) photoperiods, but was significantly protracted at intermediate or ‘critical’ daylengths.     

Distyly and incompatibility in Psychotria homalosperma (Rubiaceae), an endemic plant of the oceanic Bonin (Ogasawara) Islands

Distyly rarely occurs on oceanic islands. Indeed, this form of polymorphism is absent on the Hawaiian Islands and in New Zealand. However, Psychotria homalosperma (Rubiaceae), which is endemic to the oceanic Bonin Islands of Japan, was presumed to maintain a distylous breeding system and floral morphology. We investigated the floral and reproductive characteristics of this species and examined incompatibility in self- and intramorph pollination. Psychotria homalosperma is morphologically and functionally distylous, based on findings with the examined populations, but its stigma and anther heights are not exactly reciprocal between the two morphs. This is the second example of distyly among the flora from the Bonin Islands, the other being the endemic P. boninensis. Both of these distylous species contrast with the Hawaiian Psychotria species, which have already become dioecious. The flowers of the long (L)-styled (pin) morph of the Bonin Islands plants are completely self- and intramorph incompatible, while the flowers of the short (S)-styled (thrum) morphs are partially intramorph compatible. The S-styled morph was more abundant in the examined populations from three islands of the archipelago, probably due to the existence of partial S–S intramorph compatibility.     

Effect of salinity variation and pesticide exposure on an estuarine harpacticoid copepod, Microarthridion littorale (Poppe), in the southeastern US

The harpacticoid copepod Microarthridion littorale (Poppe) was tested for interaction effects between salinity change and acute pesticide exposure on the survival and genotypic composition of a South Carolina population. Previous data suggested a significant link between a combined exposure to chlorpyrifos (CHPY) and dichloro-diphenyl-trichloroethane (DDT) and mitochondrial haplotype in the cytochrome b apoenzyme for this euryhaline species when exposed at 12-ppt salinity seawater. Our tests demonstrate a significant non-linear survival response for M. littorale to short-term immersion (24 h) in 3-, 12- and 35-ppt seawater, with copepods transferred to 12-ppt seawater having the lowest survival. There was significant statistical interaction between salinity and pesticide exposure for the dependent variable “survival.” However, changes in genetic composition of survivors were not significant, and they were complicated by extremely low survival in the pesticide/3 ppt and pesticide/36 ppt treatments. As noted for many studies of harpacticoids, males faired worse than females in all treatments, with none surviving pesticide exposure at 45 μg/l CHPY and 6 μg/l DDT.     

AluI polymorphism of the bovine growth hormone (GH) gene, resumption of ovarian cyclicity, milk production and loss of body condition at the onset of lactation in dairy cows

Relationships among GH genotype (AluI polymorphism), parity, metritis and interval from calving to first ovulation, milk production and body condition score (BCS) loss were determined in dairy cows (n = 307) on four large-scale farms in Hungary. Cows with systemic signs of puerperal metritis or mastitis were excluded. Time of the first postpartum (PP) ovulation was obtained from milk progesterone profiles. Based on GH genotype determination, groups of leucine homozygous cows (n = 246) and valine allele carriers (n = 61) were formed. All animals became cyclic during the study period. The average interval to first ovulation was 27.6 ± 0.69-d PP (mean ± S.D.). Genotype had no effect on the commencement of ovarian cyclicity. First ovulation occurred sooner after calving in pluriparous than in primiparous cows. The greater BCS loss cows had during the first 30-d PP, the longer they took to resume cyclic ovarian function. The interval from calving to first ovulation was substantially affected by farm, but not by mild cases of puerperal metritis. Genotype was not related to cumulative 30-d milk yield or BCS loss after calving. Primiparous cows had lower milk yield than pluriparous ones. Cows with metritis lost more body condition than healthy individuals in the first month postpartum. We concluded that, under field conditions, AluI polymorphism of the bovine GH gene had no effect on the interval from calving to first ovulation and could not be directly related to differences in milk yield and to the extent of BCS loss during the first month after calving in Holstein-Friesian cows.     

Assembly and annotation of full mitochondrial genomes for the corn rootworm species, Diabrotica virgifera virgifera and Diabroticabarberi (Insecta: Coleoptera: Chrysomelidae), using Next Generation Sequence data

Complete mitochondrial genomes for two corn rootworm species, Diabrotica virgifera virgifera (16,747 bp) and Diabrotica barberi (16,632; Insecta: Coleoptera: Chrysomelidae), were assembled from Illumina HiSeq2000 read data. Annotation indicated that the order and orientation of 13 protein coding genes (PCGs), and 22 tRNA and 2 rRNA sequences were in typical of insect mitochondrial genomes. Non-standard nad4 and cox3 stop codons were composed of single T nucleotides and likely completed by adenylation, and atypical TTT start codons was predicted for both D. v. virgifera and D. barberinad1 genes. The D. v. virgifera and D. barberi haplotypes showed 819 variable nucleotide positions within PCG regions (7.36% divergence), which suggest that speciation may have occurred ~ 3.68 million years ago assuming a linear rate of short-term substitution. Phylogenetic analyses of Coleopteran MtD genome show clustering based on family level, and may have the capacity to resolve the evolutionary history within this Order of insects.     

Respiratory changes in the euryhaline clam, Mulinialateralis (Say), over a range of temperature and salinity combinations

The combined effects of five temperatures (15, 20, 25, 30, and 32 °C) and five salinities (10, 15, 20, 25, and 35%.) on respiratory metabolism in the euryhaline clam, Mulinia lateralis (Say), were determined from polarographic measurements of oxygen consumption. Although both the effects of temperature and salinity were statistically significant as well as their interaction effects, the effect of temperature was greater. Euryhalinity (previously ascribed to M. lateralis based only on its geographical distributions) was reflected physiologically by the few significant differences between respiration rates in various salinities at a given temperature. Significant differences were most often between rates in 20%. and the 10%. or 35%. extremes. Consistently highest rates in 20%. may reflect a salinity preferendum since this salinity was similar to long-term mean habitat salinity.     

Clinical significance of the NKG2D ligands,MICA/B and ULBP2 in ovarian cancer: high expression of ULBP2 is an indicator of poor prognosis

Objective  To investigate the clinical significance of the expression of the NKG2D ligands MICA/B and ULBP2 in ovarian cancer. Methods  Eighty-two ovarian cancer patients and six patients without ovarian cancer from Department of Obstetrics and Gynecology of Kyoto University Hospital were enrolled in this study between 1993 and 2003. Expression of MICA/B, ULBP2, and CD57 in ovarian cancer tissue and normal ovary tissue was evaluated by immunohistochemical staining, and the relationship of these results to relevant clinical patient data was analyzed. Expression of MICs, ULBP2, and HLA-class I molecules in 33 ovarian cancer cell lines and two normal ovarian epithelial cell lines, as well as levels of soluble MICs and ULBP2 in the culture supernatants, were measured. Results  Expression of MICA/B and ULBP2 was detected in 97.6 and 82.9% of ovarian cancer cells, respectively, whereas neither was expressed on normal ovarian epithelium. The expression of MICA/B in ovarian cancer was highly correlated with that of ULBP2. Strong expression of ULBP2 in ovarian cancer cells was correlated with less intraepithelial infiltration of T cells and bad prognoses for patients, suggesting that ULBP2 expression is a prognostic indicator in ovarian cancer. The expression of NKG2D ligands did not correlate with the levels of the soluble forms of the ligands. Conclusions  High expression of ULBP2 is an indicator of poor prognosis in ovarian cancer and may relate to T cell dysfunction in the tumor microenvironment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by grants from Grant-in-Aid for Scientific Research (19390426, 19591932, 18209052 and 19659421) from the Ministry of Education, Science, Sports, Culture and Technology of Japan.     

PCB contamination of the common barbel,Barbus barbus (pisces,cyprinidae), in the River Meuse in relation to hepatic monooxygenase activity and ultrastructural liver change

To evaluate the impact of PCBs on a wild population of a regressing fish species, we have measured the levels of these toxicants in common barbel (Barbus barbus) from the Belgian part of the river Meuse. We have expressed these levels in terms of the most suitable composition of commercial PCB mixture (Aroclor 1254 and 1260 20/80; v/v), and related them to hepatic xenobiotic-metabolising enzyme activities and to hepatocyte ultrastructure.PCB concentrations in barbel organs were extremely high, with no statistical difference between the two sexes at equal weight. In liver, muscle, and gonads, PCB concentrations increased significantly with age, reaching 20 g g^–1 DW in 12- to 15-year-old individuals. The activities of the monooxygenases (ethoxyresorufin o-deethylase, EROD, and ethoxycoumarin o-deethylase, ECOD) and the cytochrome P-450 content correlated closely with the PCB concentration in fish liver. Moreover, wild fish presented a markedly altered liver ultrastructure, as compared to controls; the rough endoplasmic reticulum (RER) was particularly abundant and the mitochondrial membranes were altered. PCBs thus alter essential metabolic functions in wild barbels, which constitute a highly sensitive tool for biomonitoring wild fish populations. While the effects of PCBs on metabolic pathways may combine additively or synergistically with effects of other xenobiotics, it has been demonstrated elsewhere that they decrease successful reproduction. Their chronic negative effects have thus played a role in reducing barbel populations in highly polluted areas.     

Life history, population dynamics and production of eastern mosquitofish, Gambusia holbrooki (Pisces, Poeciliidae), in rice fields of the lower Mondego River Valley, western Portugal

The introduced population of Gambusia holbrooki from the rice fields of the lower Mondego River Valley, Portugal, was studied for 15 months, relating their life cycle and population dynamics with its production, in order to assess the role of the species in the energy flow and secondary production in this type of agro-ecosystem. Two main annual cohorts (1995 and 1996 cohorts) were identified. The females outnumbered males and the average female/male-ratio was 4. The inspection of ovary developmental stages of this viviparous fish, revealed that the most important reproductive period was between April and August. The first recruits were recorded in June and were present thereafter until October. Males from the parental cohort died before August, whereas parental females could survive until October. Mean adjusted fecundity (number of embryos divided by female standard length) peaked in July 1996 (0.95) and in June 1997 (1.05). Females reached greater sizes, had a higher growth rate and lived longer than males. Annual production was estimated at 3.101 g.m^–2.year^–1 (ash-free dry weight, AFDW), the average biomass at 2.896 g.m^–2 (AFDW), and the P/B ratio was 1.071. A conjugation of life history, population dynamics, production and ecological traits (e.g. fast growth, reduced longevity, viviparity, high productivity, an intermediate position in food chain, and no special habitat requirements for reproduction) clearly show that the populations of G. holbrooki, introduced into rice fields all over the world, may play an important role in the structure and functioning of the biological communities of these important agro-ecosystems.