Activity of phospholipases A and lysophospholipase in turkey semen and oviducal fluid.

Changes in lipid composition of turkey semen have previously been reported to occur during in vitro storage and may be mediated by endogenous hydrolysis of phospholipids. To investigate the presence of phospholipases able to initiate such degradation, phospholipaseA2 (PLA2), phospholipase A1 (PLA1), and lysophospholipase (LPLase) activities were measured in turkey spermatozoa and seminal plasma. These enzymes were also measured in the oviductal fluid because they may be involved in the process prior to fertilization in the female. In spermatozoa and seminal plasma, the major PLA2 was a calcium-dependent and sodium deoxycholate (DOC) stimulated enzyme. However, calcium-independent PLA2 activities were also detected with different characteristics in spermatozoa (DOC inhibited enzyme) and seminal plasma (DOC stimulated enzyme). Additionally, PLA1 activity and high LPLase activity were present in spermatozoa and seminal plasma. In vitro storage of semen for 48 h did not affect PLA2 and LPLase activities. By contrast, PLA1 was the major phospholipase activity detected in oviductal fluid. A PLA2 activity stimulated by calcium or DOC and LPLase activity were also detected, but both were low relative to PLA1. These results showed that turkey semen had several enzymatic activities able to hydrolyze phospholipids. In addition, the phospholipase activities described here in the oviductal fluid could be involved in membrane destabilization prior to fertilization.

Effects of n-3 polyunsaturated dietary supplementation on the reproductive capacity of male turkeys.

To measure the effects of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation on the reproductive capacity of adult male turkeys in industrial flocks, the males of 22 commercial farms were fed either a standard diet or a fish oil diet enriched in n-3 PUFAs. The fatty acid composition of the spermatozoa and reproductive performance were measured throughout the reproductive period. The fish oil diet very effectively increased the percentage of n-3 fatty acids (FA) (22:5n-3 and 22:6n-3) in spermatozoa and correspondingly decreased the percentage of n-6 PUFAs (20:4-6 and 22:4n-6): the n-3/n-6 ratio in spermatozoa fatty acids were 0.04-0.07 with the standard diet and 0.32-0.4 with the fish oil diet. These changes did not affect the spermatozoa content of n-9 PUFAs, particularly of 22:3n-9 which is abundant in turkey spermatozoa (9-12% of the total fatty acids). The supplementation was effective in the middle as at the end of the reproductive period. The reproductive capacity of males was modified by the diet and the positive effect of the n-3 supplemented diet increased with age (increase in hatching rates of nearly 2 points at 48-58 weeks for males fed fish oil diet). These results indicate that an increase in the dietary ratio of n-3/n-6 PUFAs is valuable to sustain the reproductive capacity of male turkeys especially when they are getting older.

Impact of changes in composition of storage medium on lipid content and quality of turkey spermatozoa.

Turkey semen quality is damaged by long term in vitro storage. The objective of the present study was to determine whether changes in energy substrates and antioxidants of semen extender could limit loss of quality and lipid content of turkey spermatozoa during storage. Spermatozoa were incubated in extenders based on Beltsville Poultry Semen Extender (BPSE) to which different energy substrates (acetate, pyruvate and hydroxybutyric acid) or antioxidant (Vitamin E) had been added. Semen was stored at 4 degrees C for 48 h and changes in quality, phospholipid and malondialdehyde (MDA) content of semen were evaluated. Among the different substrates studied, only acetate was able to limit the loss of motility and ATP content after 48 h in vitro storage. Losses of spermatozoal phospholipids were similar when gametes were incubated in an extender without any substrate or in normal BPSE (784-675nmol/10(9) spz versus 837-703 nmol/10(9) spz). However, motility and ATP content were significantly more affected after 48 h of storage in samples incubated without substrates than in BPSE (motility, 2.2 versus 0; ATP, 10 nmol/10(9) spz versus 3 nmol/10(9) spz). The addition of Vitamin E to the extender did not modify either the MDA or phospholipid content of fresh or stored spermatozoa, but increased the motility of stored semen. In conclusion, acetate is an essential substrate for in vitro storage. Spermatozoal phospholipids decreased during storage, but this did not seem to originate from metabolism of endogenous fatty acids. The positive effects of Vitamin E on semen storage did not originate from preservation of lipid oxidation.

Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in Turkeys.

Semen of Turkeys between 31 and 52 weeks of age was analyzed to investigate the cause of reduction in Turkey fertility at the end of the reproductive period. Sperm motility and viability, lipid concentration, fatty acid composition and lipid peroxides were evaluated on fresh spermatozoa or spermatozoa stored for 48h at 4 degrees C. Fertility of fresh semen was also evaluated. Fertility obtained with fresh semen decreased at 44-47 weeks of age. Ageing was also accompanied by a decrease in sperm viability (at 47 weeks) and later by a decrease in motility of spermatozoa (at 52 weeks). Polyunsaturated fatty acids (PUFAs) were the first lipids of fresh spermatozoa affected by age, especially n-3 and n-9 PUFAs. Changes in these PUFAs were followed by a 30% increase in lipid peroxidation at 47 and 52 weeks of age and a reduction in phospholipid content at 52 weeks. In vitro storage did not cause lipid peroxidation in sperm obtained during the first half of the reproductive period but malondialdehyde (MDA) levels significantly increased in sperm obtained during the second half of this period. In vitro storage also decreased phospholipid content of spermatozoa from 41 weeks of age, and viability and motility regardless of age. In conclusion, lipid alteration mainly originating from PUFAs peroxidation could partly explain the decrease in semen quality and fertility observed with ageing. In addition, lipid peroxidation was increased during in vitro storage of spermatozoa from older Turkeys.

Changes in turkey semen lipids during liquid in vitro storage.

The changes in lipid composition of spermatozoa and seminal plasma and changes in motility, viability, and morphological integrity of spermatozoa were measured in turkey semen diluted in Beltsville poultry semen extender and stored for 48 h (4 degrees C). The total phospholipid content of spermatozoa decreased during storage, while no quantitative decrease was observed in seminal plasma. More precisely, significant decreases in phosphatidylcholine, and to a lesser extent in sphingomyeline, phosphatidylserine, and phosphatidylinositol were observed in spermatozoa. The fatty acid profile of turkey spermatozoa partly reflected diet composition and had a high level of n-9 polyunsaturated fatty acids. Neither fatty acid profile nor free cholesterol were affected by storage. The lipid composition of seminal plasma was quite different from that observed in spermatozoa and was similar to the high density lipoprotein composition of chicken seminal plasma. In vitro storage did not significantly affect lipid classes and only small changes were observed in phospholipid classes of seminal plasma. The motility, viability, and morphological integrity of spermatozoa decreased during storage. These changes in phospholipid content may be explained by membrane phospholipid lysis followed by endogenous metabolism or by a complex combination of lysis, metabolism, and peroxidation. They are likely to affect semen quality and the success of in vitro storage severely.

Distribution of glutamic acid decarboxylase mRNA in the forebrain of the rainbow trout as studied by in situ hybridization.

By using degenerate primers designed from glutamate decarboxylase (GAD) sequences of mammals, Xenopus and Drosophila, a 270-bp cDNA fragment was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from cerebellum total RNA of rainbow trout. This partial cDNA shows 90% identity with mammalian GAD 65 and presents the Asn-Pro-His-Lys (NPHK) sequence corresponding to the pyridoxal-binding region of porcine DOPA decarboxylase or mammalian GAD. The distribution of GAD 65 mRNA-expressing neurons in the forebrain of the trout was studied by in situ hybridization using either digoxigenin- or 35S-labeled probes. The results demonstrate that gamma-amino butyric acid (GABA) neurons are widely distributed throughout the forebrain, with a high density in the periventricular regions. In this study, we report their precise distribution in the telencephalon and diencephalon. GAD mRNA-expressing cells were particularly abundant in the preoptic region and the mediobasal hypothalamus, two major neuroendocrine and estrogen-sensitive regions in fish. The presence of GAD mRNA-expressing neurons was observed in visually related structures such as the suprachiasmatic nucleus, the pretectal region, and the thalamus. Immunohistochemistry with antibodies directed against mouse GAD failed to demonstrate the presence of immunoreactive cell bodies, but showed a very high concentration of GAD-immunoreactive fibers in many brain regions, notably in the preoptic area, hypothalamus, and neurohypophyseal digitations of the pituitary, in particular in the proximal pars distalis. These results indicate that GABA neurons are ideally placed to modulate neuroendocrine activities at the hypothalamic and pituitary levels and to participate in the processing of sensorial information.