Bartonella species detection in captive, stranded and free-ranging cetaceans

We present prevalence of Bartonella spp. for multiple cohorts of wild and captive cetaceans. One hundred and six cetaceans including 86 bottlenose dolphins (71 free-ranging, 14 captive in a facility with a dolphin experiencing debility of unknown origin, 1 stranded), 11 striped dolphins, 4 harbor porpoises, 3 Risso's dolphins, 1 dwarf sperm whale and 1 pygmy sperm whale (all stranded) were sampled. Whole blood (n = 95 live animals) and tissues (n = 15 freshly dead animals) were screened by PCR (n = 106 animals), PCR of enrichment cultures (n = 50 animals), and subcultures (n = 50 animals). Bartonella spp. were detected from 17 cetaceans, including 12 by direct extraction PCR of blood or tissues, 6 by PCR of enrichment cultures, and 4 by subculture isolation. Bartonella spp. were more commonly detected from the captive (6/14, 43%) than from free-ranging (2/71, 2.8%) bottlenose dolphins, and were commonly detected from the stranded animals (9/21, 43%; 3/11 striped dolphins, 3/4 harbor porpoises, 2/3 Risso's dolphins, 1/1 pygmy sperm whale, 0/1 dwarf sperm whale, 0/1 bottlenose dolphin). Sequencing identified a Bartonella spp. most similar to B. henselae San Antonio 2 in eight cases (4 bottlenose dolphins, 2 striped dolphins, 2 harbor porpoises), B. henselae Houston 1 in three cases (2 Risso's dolphins, 1 harbor porpoise), and untyped in six cases (4 bottlenose dolphins, 1 striped dolphin, 1 pygmy sperm whale). Although disease causation has not been established, Bartonella species were detected more commonly from cetaceans that were overtly debilitated or were cohabiting in captivity with a debilitated animal than from free-ranging animals. The detection of Bartonella spp. from cetaceans may be of pathophysiological concern.

Testicular development in migrant and spawning bluefin tuna (Thunnus thynnus (L.)) from the eastern Atlantic and Mediterranean

Testis histological structure was studied in bluefin tuna (Thunnus thynnus) from the eastern Atlantic and Mediterranean during the reproductive season (from late April to early June). Testicular maturation was investigated by comparing samples from bluefin tuna caught on their eastward reproductive migration off Barbate (Strait of Gibraltar area) with samples of bluefin tuna fished in spawning grounds around the Balearic Islands. Histological evaluations of cross sections showed that the testis consists of two structurally different regions, an outer proliferative region where germ cells develop synchronously in cysts, and a central region made up of a well-developed system of ducts that convey the spermatozoa produced in the proliferative region to the main sperm duct. Ultrastructural features of the different stages of the male germ cell line are very similar to those described in other teleost species. The bluefin tuna testis is of the unrestricted spermatogonial testicular type, where primary spermatogonia are present all along the germinative portion of the lobules. All stages of spermatogenesis were present in the gonad tissue of migrant and spawning bluefin tuna, although spermatids were more abundant in spawning fish. The testis size was found to increase by a factor of four (on average) during migration to the Mediterranean spawning grounds, whereas the fat bodies (mesenteric lipid stores associated with the gonads) became reduced to half their weight, and the liver mass did not change significantly with sexual maturation. Linear regression analysis of the pooled data of migrant and spawning bluefin tuna revealed a significant negative correlation between the gonad index (IG) and the fat tissue index (IF), and a weaker positive correlation between the gonad index (IG) and the liver index (IL). Our analyses indicate that the liver does not play a significant role in the storage of lipids and that mesenteric lipid reserves constitute an important energy source for gametogenesis in bluefin tuna.

Abundance of cetaceans in the oceanic northern Gulf of Mexico from 2003 and 2004 ship surveys

The Gulf of Mexico (GMx) is a subtropical marginal sea of the western North Atlantic Ocean with a diverse cetacean community. Ship-based, line-transect abundance surveys were conducted in oceanic waters (>200 m deep) of the northern GMx within U.S. waters (380,432 square km) during summer 2003 and spring 2004. Data from these surveys were pooled and minimum abundance estimates were based on 10,933 km of effort and 433 sightings of at least 17 species.The most commonly sighted species (number of groups) were pantropical spotted dolphin, Stenella attenuata (115); sperm whale, Physeter macrocephalus (85); dwarf/pygmy sperm whale, Kogia sima/breviceps (27); Risso’s dolphin, Grampus griseus (26); and bottlenose dolphin, Tursiops truncatus (26). The most abundant species (number of individuals; coefficient of variation) were S. attenuata (34,067; 0.18); Clymene dolphin, S. clymene (6,575; 0.36); T. truncatus (3,708; 0.42); and striped dolphin, S. coeruleoalba (3,325; 0.48). The only large whales sighted were P. macrocephalus (1,665; 0.20) and Bryde’s whale, Balaenoptera edeni (15; 1.98). Abundances for other species or genera ranged from 57 to 2,283 animals. Cetaceanswere sighted throughout the oceanic northern GMx, and whereas many species were widely distributed, some had more regional distributions. Compared to abundance estimates for this area based on 1996-2001 surveys, the estimate for S. attenuata was significantly smaller (P <0.05) and that for the spinner dolphin, S. longirostris, appeared much smaller. Also, P. macrocephalus estimates were based on less negatively biased estimates of group-size using 90-minute counts during 2003 and 2004.

Reproductive biology of male franciscanas (Pontoporia blainvillei) (Mammalia: Cetacea) from Rio Grande do Sul, southern Brazil

The reproductive biology of male franciscanas (Pontoporia blainvillei), based on 121 individuals collected in Rio Grande do Sul State, southern Brazil, was studied. Estimates on age, length, and weight at attainment of sexual maturity are presented. Data on the reproductive seasonality and on the relationship between some testicular characteristics and age, size, and maturity status are provided. Sexual maturity was assessed by histological examination of the testes. Seasonality was determined by changes in relative and total testis weight, and in seminiferous tubule diameters. Testis weight, testicular index of maturity, and seminiferous tubule diameters were reliable indicators of sexual maturity, whereas testis length, age, length, and weight of the dolphin were not. Sexual maturity was estimated to be attained at 3.6 years (CI 95% =2.7–4.5) with the DeMaster method and 3.0 years with the logistic equation. Length and weight at attainment of sexual maturity were 128.2 cm (CI 95%=125.3–131.1 cm) and 26.4 kg (CI 95% =24.7–28.1 kg), respectively. It could not be verified that there was any seasonal change in the testis weight and in the seminiferous tubule diameters in mature males. It is suggested that at least some mature males may remain reproductively active throughout the year. The extremely low relative testis weight indicates that sperm competition does not occur in the species. On the other hand, the absence of secondary sexual characteristics, the reversed sexual size dimorphism, and the small number of scars from intrassexual combats in males reinforce the hypothesis that male combats for female reproductive access may be rare for franciscana. It is hypothesized that P. blainvillei form temporary pairs (one male copulating with only one female) during the reproductive period.

Abundance of cetatceans in the southern U.S. North Atlantic Ocean during summer 1998

The U.S. Marine Mammal Protection Act requires that the abundance of marine mammals in U.S. waters be assessed. Because this requirement had not been met for a large portion of the North Atlantic Ocean (U.S. waters south of Maryland), a ship-based, line-transect survey was conducted with a 68 m research ship between Maryland (38.00°N) and central Florida (28.00°N) from the 10-m isobath to the boundary of the U.S. Exclusive Economic Zone. The study area (573,000 km2) was surveyed between 8 July and 17 August 1998. Minimum abundance estimates were based on 4163 km of effort and 217 sightings of at least 13 cetacean species and other taxonomic categories. The most commonly sighted species (number of groups) were bottlenose dolphins, Tursiops truncatus (38); sperm whales, Physeter macrocephalus (29); Atlantic spotted dolphins, Stenella frontalis (28); and Risso’s dolphins, Grampus griseus (22). The most abundant species (abundance; coeffi cient of variation) were Atlantic spotted dolphins (14,438; 0.63); bottlenose dolphins (13,085; 0.40); pantropical spotted dolphins, S. attenuate (12,747; 0.56); striped dolphins, S. coeruleoalba (10,225; 0.91); and Risso’s dolphins (9533; 0.50). The abundance estimate for the Clymene dolphin, S. clymene (6086; 0.93), is the first for the U.S. Atlantic Ocean. Sperm whales were the most abundant large whale (1181; 0.51). Abundances for other species or taxonomic categories ranged from 20 to 5109. There were an estimated 77,139 (0.23) cetaceans in the study area. Bottlenose dolphins and Atlantic spotted dolphins were encountered primarily in continental shelf (<200 m) and continental slope waters (200−2000 m). All other species were generally sighted in oceanic waters (>200 m). The distribution of some species varied north to south. Striped dolphins, Clymene dolphins, and sperm whales were sighted primarily in the northern part of the study area; whereas pantropical spotted dolphins were sighted primarily in the southern portion.

Induction of mitotic and meiotic gynogenesis and production of genetic clones in rohu, Labeo rohita Ham.

Studies were undertaken to produce genetic clones derived from all homozygous mitotic gynogenetic individuals in rohu, Labeo rohita Ham. ln view of this, attempts were made to interfere with the normal functioning of the spindle apparatus during the first mitotic cell division of developing eggs using heat shocks, there by leading to the induction of mitotic gynogenetic diploids in the F1 generation. Afterwards, viable mitotic gynogenetic alevins were reared and a selected mature female fish was used to obtain ovulated eggs which were fertilized later with UV-irradiated milt. Milt was diluted with Cortland’s solution and the sperm concentration was maintained at 10⁸/ml. The UV-irradiation was carried out for 2 minutes at the intensity of 200 to 250 µW/cm² at 28± 1°C. The optimal heat shock of 40°C for 2 minutes applied at 25 to 30 minutes a.f. was used to induce mitotic gynogenesis in first (F1) generation and at 3 to 5 minutes a.f. to induce meiotic gynogenesis in the second (F2) generation. The results obtained are presented and the light they shed on the timing of the mitotic and meiotic cell division in this species is discussed.

A histological study of the spermatogenesis in Ompok pabda (Hamilton-Buchanan 1822)

The present study deals with the histological analysis of testicular development in Ompok pabda. For the study, male gonads were collected month wise from January to September at Freshwater Station, BFRI, Mymensingh. From the analysis, 4 stages of sperm formation, namely, spermatogonia, spermatocytes, spermatids and spermatozoa, were distinguished. The percent distribution of spermatozoa was highest in July (about 92%). Maximum GSI value was 1.129±0.271 found in July. By analyzing the histology of spermatogenesis it was established that this species breeds once in a year.

Proteomic Analysis of Proteins Involved in Spermiogenesis in Mouse

Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.

Comparison of effectiveness of heat and cold shocks applied in the induction of gynogenesis in Clarias gariepinus (Burchell)

An experiment was conducted to optimize the procedure of gynogenesis in African catfish, Clarias gariepinus by suppressing meiotic and mitotic cell divisions in fertilized eggs. Gynogensis was conducted by fertilizing normal eggs with UV-irradiated sperm followed by either heat or cold shocking Irradiation of spermatozoa was given for a duration of 1 min and the eggs were fertilized in vitro. Cold shock at a temperature of 3± 1°C for a duration of 30 and 60 min and heat shock at a temperature of 39± 1°C for a duration of 1 and 2 min was applied to induce diploidy. Higher percentage of hatching (68.66) was observed for meiotic gynogens at a shock temperature of 39± 1°C for a duration of 1 min, 5 min after fertilization (af). Higher percentage of mitotic gynogenetic induction (15.33) was observed at a temperature shock of 39± 1°C for a duration of 1 min, 30 min af.

Development of artificial breeding techniques for long-whiskered catfish, Sperata aor and giant river catfish, Sperata seenghala of Bangladesh

Sperata aor and S. seenghala are the two important native catfishes of Bangladesh but commercial farming of these species is not possible due to lack of naturally collected or artificially produced seeds for stocking. Attempts were made to develop techniques for seed production by artificial breeding and nursery-rearing of fries of these catfishes. A total of 60 S. seenghala (750-1,500 g) and 10 S. aor (600-1,000 g) broods were collected from the Brahmaputra river-basin and floodplains in Mymensingh region four months prior to their breeding season. The collected brood fishes were reared in separate earthen ponds with supplementary feeds comprising of rice bran (40%), mustard oil cake (29%), fish meal (30%) and vitamin-premix (1 %). Three experiments were conducted to optimize the hormone dose. A total of nine S. seenghala females weighing from 750 to 1,500 g were given an initial and resolving dose of 12-20 and 16-24 mg PG/kg body weight, respectively. The males weighing from 650-950 g were administered a single dose of 18-26 mg PG/kg body weight at the time of the time of administering the resolving dose to the females. The females ovulated partially and the eggs were examined under a compound microscope, but most of them were found to be less ripe or damaged. Collection of milt by stripping the males was not successful. The testes were taken out and sperm were observed to be non-motile and less developed. In view of stimulating natural propagation of S. seenghala, artificial holes (nests) were constructed in the pond bottom. Each hole was 0.7 m in diameter and 0.3 m in depth. A total of 10 holes were made and then 10 pairs of S. seenghala breeders (800-1,200 g) were stocked in the pond. In mid February, 3,000 fry of S. seenghala with a mean length of 4.60 cm and weight of 0.36 g were collected by repeated netting followed by drying of the pond. The fry were then stocked in a nursery pond and fed with commercial feed (SABINCO starter-1). The average length and weight of the fingerlings were 9.01 cm and 3.95 g, respectively and the estimated survival was 60% after two months of rearing. S. aor did not respond to natural spawning. Further study is essential to develop techniques for their successful artificial and natural breeding.

Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris-egg yolk freezing media. TEST and TTE. which have: been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa From four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization or oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes. 82 +/- 13% were fertilized and 63 +/- 22 and 39 +/- 21% of the resulting zygotes developed into morulae and blastocysts. respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species. (C) 2001 Academic Press.

Identification of novel semenogelin I-derived antimicrobial peptide from liquefied human seminal plasma

Semenogelin I (SgI) is one of the most abundant proteins in human seminal plasma. SgI plays a key role in sperm coagulation and spermatozoon immobilization. in addition, SgI and/or its proteolytic fragments are involved in regulating spermatozoon motility

A nerve growth factor from the venom of Chinese cobra (Naja naja atra) and its effects on male reproductive system in rats

A nerve growth factor (NGF) was isolated from the venom of Chinese cobra (Naja naja ntr a) by ion exchange chromatography, gel filtration and fast protein liquid chromatography (FPLC). The N-terminal sequence of 22 amino acid residues was identical with other NGFs previously purified from the venom of the same genus. The NGF monomer molecular weight was estimated to be 13 500 by reducing SDS-PAGE and the isoelectric point was determined to be 7.2 by isoelectric focusing electrophoresis. NGF improved the epididymal sperm motility of male rats and increased the pregnancy rate and fetus number of mated female rats. The serum levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) of male rats administrated NGF + gossypol was lower than that of male rats administrated gossypol. Histological sections of testes and epididymides showed that NGF reduced the destructive effects of gossypol on rat testes. (C) 1999 Elsevier Science Inc. All rights reserved.

Single UV excitation of Hoechst 33342 and propidium iodide for viability assessment of rhesus monkey spermatozoa using flow cytometry

Many fluorescent probes excited by visible light have been used to assess sperm quality by flow cytometry. Developing a viability evaluation method using UV excited stains would be useful for multiparameter analysis of sperm function. This investigation was conducted to determine the efficacy of Hoechst 33342 (H342) and propidium iodide (PI) dual staining for evaluating rhesus monkey sperm viability through use of flow cytometry and excited by a single UV laser. The results showed that the live cells stained only with H342 strongly correlated with expected sperm viability, and flow cytometric analyses were highly correlated with fluorescence microscopic observation. Using H342/PI/SYBR-14 triple staining method, it was found that the live/dead sperm distributions were completely concordant in both H342/PI and SYBR-14/PI assays. In addition, this dual staining was extended with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) to simultaneously analyze viability and acrosome integrity of sperm cryopreserved using two different extenders, TTE and TEST, and indicated that TTE offered better Preservation of plasma and acrosome integrity than TEST Therefore, the H342/PI dual staining provides an accurate technique for evaluating viability of rhesus monkey sperm and should be valuable for multiparameter flow cytometric analysis of sperm function.

Effects of exogenous hormone injections on milt consistency in newly caught, wild milkfish

A study was conducted to determine the effects of single injections of human chorionic gonadotropin (HCG) and Durandron Forte 250 on sperm motility, vitality and density and also on the consistency of milt in newly caught, wild, mature milkfish (Chanos chanos). In contrast to HCG, single injections of Durandron Forte 250 were effective not only in inducing spermiation but also in maintaining newly caught mature males in good running condition for a maximum of 7 days, despite daily handling and collection of approximately 3ml milt.