Gavin F. Richardson

Growth, rate, body composition and feed digestibility/conversion of growth-enhanced transgenic Atlantic salmon (Salmo salar)

Although dramatic improvements in growth rates have been documented in growth-enhanced transgenic salmonid fish, prior to commercial implementation of this technology, there is a need for further information relating to the physiology of a number of commercially important production traits. Growth rate, feed digestibility, feed conversion, and body composition of F 2 generation growth-enhanced transgenic Atlantic salmon were therefore compared with that of non-genetically modified salmon, over a presmolt growth interval of 8-55 g. The growth-enhanced transgenic fish exhibited a 2.62- to 2.85-fold greater rate of growth relative to non-transgenic salmon over the body weight interval examined. Daily feed consumption over this body weight interval was 2.14- to 2.62-fold greater for the transgenic fish compared to the control fish. Transgenesis did not affect the extent to which protein and energy were digested, with digestibility coefficients 88% and 81%, respectively for transgenic fish, and 90% and 84%, respectively for control fish, both measured over comparable body weight intervals. However, transgenic salmon relative to control fish exhibited a 10% improvement in gross feed conversion efficiency. Body protein, dry matter, ash, lipid and energy were significantly lower in the transgenic salmon relative to controls while moisture content was significantly higher. The transgenic experimental subjects used throughout the present study possessed the physio- logical plasticity necessary to accommodate an acceleration in growth well beyond the normal

Motility, fertility and ultrastructural changes of ocean pout (Macrozoarces americanus L.) sperm after cryopreservation

Abstract The present study examined the possibility of long term storage, by cryopreservation in liquid N 2 , of the sperm of ocean pout ( Macrozoarces americanus L.), an internally fertilizing marine fish, and the changes in motility, fertility and ultrastructure of the sperm after freeze and thaw. Four cryoprotectants, including dimethyl sulfoxide (DMSO), and three semen diluents (A, B and C) were tested for their influence on sperm motility. Since the fresh sperm displayed the highest motility in diluent C, which had the closest chemical composition to that of the seminal plasma of ocean pout, with DMSO, this solution (C-DMSO) was chosen as a diluent for the present study of ocean pout semen cryopreservation. Fresh semen was diluted in three volumes of C-DMSO and filled in 0.25- or 1.7-ml straws, then frozen in liquid N 2 vapor. When the internal temperature of the straws had dropped to −95°C, the straws were plunged into the liquid N 2 . To recover the sperm motility, the frozen semen was thawed in 1 or 30°C water bath for 30 and 7 s, respectively. It was demonstrated that the presence of DMSO in semen extender was essential for protecting the sperm from dying during freeze and thaw, and 20% of DMSO (v/v) yielded the highest post-thawed sperm motility (20–25% of the total cells). A freezing rate of average 9°C/min during the initial freezing phase (in liquid N 2 vapor) produced a higher post-thawed sperm motility than that produced by faster (18°C/min) and slower (6°C/min) freezing rates. Thawing the frozen semen in 30 or 1°C water did not cause any difference in terms of sperm motility. The loss of sperm motility during freeze and thaw might be due to the ultrastructural changes of sperm, e.g., severe swelling of the mitochondria or dehydration of cytoplasm at the midpiece, revealed by scanning electron microscope. The motile sperm in the post-thawed semen possessed fertility because in vitro artificial insemination of fresh eggs using the post-thawed semen yielded a fertilization rate of 33% vs. 48–58% from fresh semen.

Cryopreservation of yellowtail flounder (Pleuronectes ferrugineus) semen in large straws

Abstract Semen from yellowtail flounder (Pleuronectes ferrugineus) was cryopreserved in 0.25 ml and flat 1.7 ml straws. Semen frozen in 1.7 ml straws was collected from males that had been stimulated to increase semen output by treatment with a GnRH analogue. The semen extender consisted of a modification of a diluent that has been used to cryopreserve salmonid semen with 10% propylene glycol added as a cryoprotectant. The semen was frozen in liquid nitrogen vapour and stored in liquid nitrogen. Freeze rates inside the straws were measured by a thermocouple. In a preliminary fertility trial, there were no significant differences in egg fertilization rates and hatch rates between freshly collected semen and semen frozen in 1.7 ml straws. A larger trial compared the fertility of freshly collected semen, semen frozen in 0.25 ml straws and semen frozen in 1.7 ml straws. The mean fertilization rates for these groups were 64.7, 59.2 and 54.4%, respectively, and the hatch rates were 36.7, 52.5, and 42.8%, respectively. The mean fertilization rate for semen frozen in 1.7 ml straws was significantly lower (P

In vitro methods of assessing the viability of rainbow trout spermatozoa

The accuracy of three in vitro methods for estimating the proportion of dead rainbow trout (Onchorhynchus mykiss) spermatozoa was investigated. Motility rating, fluorometry using ethidium bromide, and lactate dehydrogenase (LDH) activity in seminal plasma were compared. Semen samples were prepared to contain 0, 25, 50, 75 and 100% killed spermatozoa. All three methods demonstrated highly significant relationships (P<0.001) with the percentage of killed spermatozoa. Motility rating was found to be quick and accurate but required experienced workers and the results thus could vary between evaluators. Fluorometry was rapid and relatively simple to perform and required only a small amount of semen. Measurement of LDH activity in seminal plasma was accurate but time-consuming and required large amounts of semen.

DIMETHYL-ACETAMIDE AS A CRYOPROTECTANT FOR RAINBOW TROUT SPERMATOZOA

Dimethyl-acetamide was evaluated as a cryoprotectant for rainbow trout (Oncorhynchus mykiss ) semen on the basis of motility, percentage of dead spermatozoa as determined by fluorometry, and fertility of frozen-thawed spermatozoa. Dimethyl-acetamide performed significantly better (P<0.05) than the conventional cryoprotectant, dimethyl sulfoxide, by all evaluation methods. An extender comprised of 0.137 M NaCl, 0.011 M KCl, 0.004 M Na(2)HPO(4) 7H(2)O, 7.5 g/l L-alpha-lecithin and 10 % DMA showed promise for cryopreserving rainbow trout spermatozoa. Future evaluation of new extenders should be carried out by several in vitro techniques and fertility measurements to present a complete assessment of an extenders ability to cryopreserve sperm cells.

Comparison of different extenders for the cryopreservation of Atlantic salmon spermatozoa

Fifteen extenders were produced by adding dimethyl sulfoxide (DMSO) at 8, 10 or 12% of diluent volume to 5 diluents. All extenders were cooled to 4 degrees C. Pooled Atlantic salmon (Salmo salar) semen with greater than 90% progressive motility was kept at 4 degrees C and added to each extender so that the semen was diluted 1:3 (semen:extender). The equilibration time was less than 5 minutes at 4 degrees C. The extended semen was loaded into 0.5-ml straws and was cooled from 4 degrees C to -90 degrees C at a rate of 30 degrees C per minute. The straws were then plunged into liquid nitrogen for storage. Fluorometry was used to determine the viability of the semen in each of the extenders after freezing and thawing. Cryopreservation of Atlantic salmon semen in Extender 3 (0.137 M NaCl, 0.011 M KCl, 0.004 M Na(2)HPO(4).7H(2)O, 7.5 g/l L-alpha-lecithin and 12% dimethyl sulfoxide) and Extender 12 (0.100 M KHCO(3), 0.0065 M reduced glutathione, 0.125 M sucrose and 12% dimethyl sulfoxide) resulted in significantly (P<0.05) lower percentages of dead spermatozoa than for the other extenders. Furthermore, there was a significantly (P<0.05) lower percentage of dead cells in Extender 3 than in Extender 12.

A diluent for prolonged motility of ocean pout (Macrozoarces americanus L.) sperm

The present study describes a new semen diluent (diluent C) which prolongs the sustained motility of ocean pout sperm, a distinguishing feature of the sperm of internally fertilizing teleosts. Sperm motilities were compared in the new diluent (C), based on the ionic composition of ocean pout seminal plasma, vs. four other semen diluents (A, B, D and E) ordinarily used for extending the semen of external fertilizers. While sperm retained motility after extension of ocean pout semen in all of these diluents, motility was significantly reduced following sperm resuspension in diluents B and D. Since sperm motility remained high in diluent C, formulated to closely mimic the composition of ocean pout seminal plasma, it was selected for additional experimentation. Negative effects were observed on sperm motility after dilution (>1:10) of ocean pout semen with diluent C, but sperm motility could be restored by replacement of the seminal plasma. Of practical importance for storage of ocean pout sperm at 4°C, it was shown that semen dilution 1:3 in diluent C preserved sperm motility beyond 5 days. Although ocean pout sperm tolerate a fairly wide range of K+ levels (0–30 mmol/l), the best motility was observed from 10–20 mmol/l [K+], similar to the ionic levels found in seminal plasma. Finally, since no change in fertility of ocean pout sperm occurred following 1:3 dilution of semen in diluent C, we conclude that diluent C is an effective medium for in vitro artificial egg insemination and prolonged motility of ocean pout sperm.

Effect of Seminal Plasma Protein on Postthaw Viability and Fertility of Arctic Char Spermatozoa

Abstract Seminal plasma protein of Arctic char Salvelinus alpinus was characterized using sodium dodecyl sulfate (SDS) gel electrophoresis. Twelve protein bands with molecular weights of 7.2, 12.4, 15.3, 20.0, 20.4, 22.6, 39.4, 66.3, 74.0, 92.0, 94.5, and 130.1 kilodaltons (kDa) were detected. The effect of total seminal plasma protein and protein fractions of three categories ( 100 kDa) on postthaw sperm motility, viability, and fertility was tested. Incorporation of total seminal plasma protein, the fraction greater than 100 kDa, or the fraction less than 50 kDa into the semen extender (300 mmol of glucose/L of water, plus 10% methanol) had a deleterious effect on postthaw sperm motility, viability, and fertility in comparison with spermatozoa frozen in the semen extender only. However, adding the 50-100-kDa fraction of seminal plasma protein to the semen extender did not affect the postthaw sperm motility and fertility relative to spermatozoa frozen in the extender only. Further experi...

Scanning electron microscopy of the endometrium of mares infused with gentamicin.

Scanning electron microscopy (SEM) was used to study the endometrium of nine 1-year-old thoroughbred mares after twice intrauterine infusions of gentamicin, on 2 consecutive days. Five mares were infused on 2 consecutive days with 40 ml gentamicin (50 mg/ml) mixed with 80 ml of normal saline. Four mares served as controls and were infused with 120 ml of saline on 2 consecutive days. Endometrial biopsies were obtained from all mares 3 days after the second intrauterine infusion. Each biopsy was processed for SEM by standard methods. The endometrial epithelium of the gentamicin-infused mares had more cellular perforations than the saline-infused mares. The gentamicin-infused mares had less and shorter microvilli. The ciliated cells were fewer and some ciliated cells had disrupted and some had drooping cilia. The endometrial epithelium of the gentamicin-infused mares had a considerable number of endometrial cells that lost their luminal surfaces and some that lost their microvilli, compared to the saline-infused mares. We suggest that the information gathered in this pilot study should be used as basis for further investigation, on a larger scale basis, of the effects of repeated intrauterine infusion of gentamicin on the endometrial mucosa of mares.

Preservation of rainbow trout (Oncorhynchus mykiss) eyed eggs using a perfluorochemical as an oxygen carrier

The objective of this study was to maintain the viability of chilled rainbow trout (Oncorhynchus mykiss) eyed eggs during storage using oxygenated perfluorochemical (PFC). Three trials were conducted using eggs at 161, 180 or 217 degree days (days from fertilization x incubation temperature in degrees C). A separate trial was conducted for 147 degree day eggs that were not at the eyed stage. For each trial, eggs were stored in a moisture-saturated atmosphere at 1 degrees C in PFC, water, and 1:1 combinations of PFC and PBS, PFC and 0.3 M glucose, PFC and mineral oil, or PFC and water. The PFC was oxygenated before each trial and all media were oxygenated at weekly intervals during the storage period. Eggs from each trial were also incubated without storage to provide Day 0 results. After 3 and 5 weeks of storage, eggs from each medium were incubated at 10 degrees C until hatch. Hatching percentage was expressed as a percentage of Day 0 results. The percentage of normal alevins that hatched was also determined. There were interactions (P < 0.01) between stage of development and treatment for hatching percentage after 3 and 5 weeks of storage. After 3 weeks of storage, eggs stored at 161, 180, or 217 degree days without PFC had hatching rates of 0-14.3% but eggs stored in any medium with PFC had hatching percentages from 75.1 to 106.4% of Day 0 values. After 5 weeks of storage, eggs stored at 161 degree days in PFC plus PBS or PFC plus water, and eggs stored at 217 degree days in PFC or PFC plus water, had higher (P < 0.05) hatching percentages than eggs stored in any of the other media. Eggs stored at 161 degree days for 5 weeks in PFC and water had a higher (P < 0.05) percentage of normal alevins hatching than eggs stored in PFC and PBS. Because of their early developmental stage, eggs stored at 147 degree days had low hatching percentages, except eggs stored for 3 weeks in PFC or PFC plus PBS. Chilling eyed eggs of rainbow trout to 1 degrees C and storing them in water with PFC as an oxygen carrier can preserve their viability for 5 weeks.