Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 140 Poster Abstracts P341 The effect of various diluents on pigeon semen stored 24h at 5 o C Klimowicz, M 1 *, Batkowski, F 2 1Krakow Agricultural University, Faculty of Animal Breeding and Biology, Department of Animal Reproduction and Anatomy, Al. Mickiewicza24/28, 30- 059 Krakow, Poland; 2 Wroclaw University of Environmental and Live Science, Faculty of Veterinary Medicine, Student Scientific Association, ul. Norwida 31, 50-375 Wroclaw, Poland The aim of the study was to identify a suitable extender for pigeon semen preserved for 24h at 5 o C. Experiment was conducted 10 weeks. Semen was collected twice a week, from 40 fancy pigeons. After macroscopic analysis semen was pooled, diluted in Lake’s solution and BPSE extender, and divided in two equal parts. One part of semen was evaluated immediately and second one was stored at 5 o C and evaluated after 24h. Every sample of semen was stained using 2 different methods - conventional eosin-nigrosin stain to estimate morphology of spermatozoa and SYBR-14/PI to evaluate sperm viability by flow cytometry. Sperm motility and velocity parameters were estimated using computer-assisted semen analyser HTM IVOS 12.2 (CASA). CASA analysis revealed that sperm motility (MOT), percentage of spermatozoa with a progressive motility (PMOT) were significantly higher in BPSE than in Lake’s solution after 24h storage (p≤0.05). Velocity parameters such as VAP, VSL, VCL, LIN, STR and the percentage of viable spermatozoa were not different between extenders. The percentage of morphologically normal spermatozoa was significantly higher at 0h in semen diluted in BPSE than in Lake’s solution (85.48±4.83 and 75.12±3.45; p≤0.01), as well as after 24h in vitro storage - 65.4±10.71 and 52.5±10.44 respectively (p≤0.05). The deformation and damage of spermatozoal acrosome was extremely higher in semen extended in Lake’s solution after in vitro storage (p≤0.001). BPSE is a suitable semen extender for storage of pigeon ejaculates at 5 o C. This diluent has been capable of protection pigeon semen during in vitro storage and could potentially improve methods of cryopreservation. P342 The effect of short-term semen storage temperature and period on South African indigenous cock breeds Mphaphathi, ML*; Raito, MB; Mapeka, MH; Mantiziba, CW; Munyai, PH1; Boshoff, MP; Suzette, F and Nedambale, TL Agricultural Research Council-Livestock Business Division, Germplasm and Reproductive Biotechnologies, Private Bag X2, Irene, 0062, RSA The development of short and long term storage of South African indigenous cock’s semen is needed to ensure a viable reserve of germplasm for artificial reproduction. The aim of this study was to examine the longevity of freshly collected semen of two different indigenous cock breeds at room temperature (25 o C) and low temperature (4 o C) for 4 h, 8 h, and 24 h. Abdominal massaging technique was used to collect cock’s semen of Naked neck (NN) and Venda (V) breed. Following semen collection, spermatozoa was divided equally per treatment group; then microscopic characteristics (motility and survival rate) were evaluated under polarizing BHTU microscope and the sperm concentration was measured by spermacue. Modified Brackett and Oliphant’s (BO) medium was used to dilute (1:2) individual ejaculates per treatment group. The ejaculates volume, concentration, and pH of NN and Venda breed were recorded and evaluated for different time intervals. Data was analyzed by ANOVA. Preliminary results demonstrated a higher concentration of sperm in NN (8.14 x 10 8 /ml) than in Venda (4.46 x 10 8 /ml) breed. In contrast, higher pH was recorded in semen collected from Venda cock breed. However, there were no statistical differences in sperm motility and survival rate of semen stored at 25°C and 4°C between NN and Venda breed for all periods (Table 1). Regardless of time intervals and cock breed, there was an increase in dead sperm percentage and pH over time for semen stored at 4 and 25 o C. In summary, semen collected from NN cock resulted in higher concentration of sperm. This study also indicated changes induced by storage temperature, breed and length of semen storage. Study on cryopreservation of NN and Venda cock semen is on progress. P343 Changes in sperm quality of broiler breeder males supplemented with organic selenium Végi, B*; Váradi, É; Ferencziné Szőke, Zs; Barna, J Avian Reproduction Group, Research Institute for Animal Breeding and Nutrition, Hungary The fertility decline in the second half of reproduction cycle has been a permanent problem in broiler breeders’ production. The aim of the study was to find differences in the effect of inorganic and organic selenium and the higher level of vitamin E on sperm parameters, mainly in the second half of the reproduction cycle in two types of broiler breeders. Selenium and the vitamin E play important role in the antioxidant system of live organism and additionally, of the membrane of avian spermatozoa. Due to the membrane damages the sperm functions failure, which results in reduced fertility. Sperm parameters of 20-20 males from ROSS 308 and Hubbard broiler breeders were compared during the whole reproduction cycle by weekly semen evaluations. The food of experimental groups was supplemented with 0.3 ppm Sel-Plex (Alltech) and 200 ppm vitamin E, while the food of control groups contained sodium selenite in traces and 100 ppm vitamin E. Males of 26 weeks of age were placed in individual cages feeding and keeping according to the management manual, until 61 weeks of age. Sperm collections were made twice a week by dorso-abdominal massage according to Burrows and Quinn (1937). Concentrations were determined by spectrophotometer (Accucell, IMV Technologies), motility by subjective scoring from 0 to 5, morphology of spermatozoa and live/dead cell ratio in stained smears by aniline eosin. As a result, the 0.3 ppm organic selenium and 200 ppm vitamin E affected differently in the two types of males. While in ROSS males they improved significantly the sperm motility, concentration and the ratio of live, morphologically normal spermatozoa in the second half of the production cycle, in the case of Hubbard males there were no any significant improvements in sperm traits. However, significant differences were found between the sperm qualities of the two types regarding to the sperm volumes (.18 - .25 ml/ejaculate), the concentrations (4.9 – 1.9 million/µL), and the dead cells’ ratios (15 – 17.8 %) in ROSS vs. Hubbard control males, respectively. Thus, Hubbard males produced higher semen volume with significantly less sperm concentration, less abnormal sperm cells but significantly more dead cells, than ROSS males. As a consequence, Sel-Plex with vitamin E could maintain the initial good sperm quality of ROSS males until the end of the cycle. However, in the case of Hubbard males the spermatological performance seemed to be more stable during the reproduction cycle and less accessible by exogenous factors. Poster 11 - Reproduction of Other Vertebrates (Fishes, Amphibians, Reptiles) P344 Endogenous opioid system and sharpsnout seabream (Diplodus Puntazzo) milt: detection of mu, delta and kappa opiod receptors on sperm cells Aiudi, G*; De Sandro Salvati, A; Bucci, FA; Micera, E; Albrizio, M Department of Animal Production, University of Bari, Italy Sharpsnout seabream, (Diplodus puntazzo) is the third most farmed marine teleost species in Italy (Taddei et al., Cryobiology 42:244, 2001). Fish reproduction in aquaculture facilities is mainly obtained through environmental/pharmacological conditioning. These techniques may stress fish and decrease reproductive performances (Cleary et al., Aquac. Res. 33:829, 2002). Endogenous opioid peptides (EOPs) system is involved in stress response (Arends et al., J. Endocrinol., 163:149, 1999), and affects several physiological
16 t h International Congress on Animal Reproduction Poster Abstracts 141 functions, both in central nervous system and in peripheral tissues. EOPs act through their specific cell membrane receptors (EOPr), which are classified into three major categories: mu, delta and kappa (Zadina et al., Nature, 386:499, 1997). The EOPs-EOPr binding blocks the cellular calcium channels, giving rise to considerable effects on all calcium-dependent metabolic pathways. Osmolality is one of the most important factors in the regulation of marine Teleosts sperm motility (Morisawa, Zool. Sci., 2:605, 1985). In fish spermatozoa hyperosmolality increases the intracellular calcium levels which allow the activation of flagellar beating (Oda and Morisawa, Cell Motil. Cytoskeleton, 25:171, 1993). Because of the recognized importance of calcium ions in marine Teleost sperm initiation, the high levels of stressors in aquaculture, and effects of EOPs on intracellular calcium balance, we evaluate the expression and localization of mu, delta and κappa opioid receptors in sharpsnout seabream milt by means of indirect immunofluorescence. Briefly, milt was collected by gentle abdominal massage into a depressurized syringe positioned at the genital pore and smeared onto poly-L-lysine coated glass slides. Cells were fixed, washed and incubated in blocking solution. A 1:1250 dilution of primary rabbit specific polyclonal antibodies against each receptor were applied except for the controls and let to react over night. After washing, slides were incubating with an anti rabbit FITC-conjugated IgG secondary antibody diluted 1:200 in Evans blue/PBS to counterstain negative cells. Slides were observed under a fluorescence microscope equipped with a specific filter. Our results show that mu opioid receptor is predominant localized on the tail, while delta and kappa seem to be located on the head. Opioid receptors detection on sharpsnout seabream milt let us suppose that the opioidergic system could modulate sperm motility, so that the use of opioid antagonists in fish farm could be useful to decrease stress effects on reproductive performances. P345 Short-term storage of Abant Trout (Salmo trutta abanticus Tontonese, 1954) semen and fertility trials Hatipoğlu, T 1 ; Akçay, E 2 * 1Ministry of Environment and Forestry, General Directorate of national parks and nature protection, Ankara, Turkey; 2 Department of Animal Reproduction and AI, Faculty of Veterinary Medicine, University of Ankara, Turkey Introduction In developing successful techniques for the preservation of fish sperm, some specific considerations related to the fish species must be taken into account such as biochemical structure and short life span of sperm after their release into water and preservation procedures. Most of the experiments in this field have focussed on finding appropriate extenders and additive agents for salmonids. Generally seminal plasma mimicking media and simple carbohydratebased solutions have been used as extenders. Objective The objective of this experiment was to evaluate spermatological parameters and fertilizing ability of short term stored semen in different extenders from endemic Abant trout (Salmo trutta abanticus T,1954). Materials and methods Semen was collected from 15 adult males by the hand stripping method without anesthesia. Having determined the main spermatological properties (volume, motility, movement duration, concentration and pH), the pooled samples were diluted at a 1:2 ratio with two extenders (0,3 M Glucose and Ringer solution). The diluted semen were stored for 48 hours at 4 °C. Following the cooled storage, spermatological parameters of the semen were evaluated after 24 and 48 hours as regards to the post-cooled period. For the fertilization, dry fertilization technique was used. Eggs were pooled from 10 females. Fertilization took place in dry plastic dishes and 600 eggs were used in each fertilization trial. The sperm-egg ratio was approximately 0,25x10 6 sp/egg. After insemination, 25 ml fertilization solution (0.3% NaCl) were added on sperm-egg mixture. After swelling, eggs were rinsed with hatchery water (7 o C) and batches were placed into vertical incubation trays. Results The experimental success was assessed from sperm motility and the percent of eyed-egg 25 days after fertilization. Fresh semen motility was 81,46±6,39 %. According to the results of the experiment, the highest post-thaw motility (67 % after 24 h, 53 % after 48 h) and movement duration (60 s after 24 h, 42 s after 48 h) were determined by using glucose extender after 24 and 48 hours storage. Fertility rate of fresh semen was 86,3 %. The highest eyedegg rate (80.3%) obtained from semen stored with glucose based extender after 24 h storage. After 48 hour storage fertilization yield for the one diluted with 0,3 M Glucose decreased to 61,92 % and to 43,87 % for the one diluted with Ringer solution. Conclusion Our results indicate that glucose based extender is a better preservative than Ringer solution for short term preservation of Abant trout semen. P346 Comparative study about the use of Oxytocin chlorohydrate administered intravenous or intramuscular for induction of eggs deposition in non obstructive eggs retention in captive breed turtles (Trachemys scripta elegans ) Di Ianni, F*; Parmigiani, E; Bresciani, C; Bigliardi, E; Bertaccini, G Faculty of Veterinary Medicine, Parma University, Italy Introduction Non obstructive eggs retention in turtle may be the result of some different factors including poor husbandry, not appropriate nesting site, improper temperatures, inadequate diet, dehydration, and poor physical condition of the female. Oxytocin, from 1 to 20 IU/kg of body weight stimulates oviductal contraction and induces eggs deposition. The aim of this study is to make a comparison between intravenous (IV) and intramuscular (IM) oxytocin chlorohydrate administration for the induction of eggs deposition in Trachemys scripta elegans turtles. Methods We considered 23 chelonia (Trachemys scripta elegans) recovered in the Teaching Hospital from 2005 to 2007 with signs of anorexia and restlessness. The turtles were radiographed and non obstructive eggs retention was diagnosed. The eggs were counted and the animals were divided in two groups with the same eggs number: group A (10 animals) and group B (13 animals). Oxytocin (2 IU/Kg) was administered IM in all the animals of group A and in the dorsal caudal vein in group B. If the animals did not laide all the eggs, we administered a second dose (2 IU/Kg) 60 minutes later and every 120 minutes up to the end of the deposition for both groups. Results The animals of group A started laiding in a mean of 100.5 ± 54.08 minutes and laided the last egg in a mean of 246± 85.4 minutes. For all animals (100%) was necessary to administer a second bole, for 7 of them (70%) a third for 3 of them (30 %) a fourth one. The animals of group B started laiding in a mean of 51.38 ± 19.80 minutes and laided the last egg in a mean of 141.92± 60.33 minutes. For 10 animals (77.92 %) was necessary a second bole and for 5 (38.46 %) a third one. All the eggs were laided. The statistical comparison ( χ 2 ) was highly significant for the initial laiding time (P < 0.01) and significant for the mean total time from beginning to the end all the depositions (P < 0.05). Conclusions In this study the Authors showed that oxytocin (2 IU/Kg) administered IV induces more quickly the deposition and that this finishes in shorter time. The treatment technique results to be easy to perform with no undesired collateral effects. P347 Induced spermiation in Green Poison Frogs, Dendrobates auratus (Amphibia, Anura, Dendrobatidae), and ultrastructure of the spermatozoa Lipke, C 1 , Meinecke-Tillmann, S 1 *, Meyer, W 2 and Meinecke, B 1 1Department of Reproductive Biology, University of Veterinary Medicine Hannover Foundation, Hannover, Germany; 2 Institute of Anatomy, University of Veterinary Medicine Hannover Foundation, Hannover, Germany The sperm ultrastructure of the Green Poison Frog, Dendrobates auratus, from Panama is described following induced spermiation in living animals. For this purpose a new method for processing of low concentration sperm samples (< 3.8*10 4 sperm/ml) had to be developed. So far only data on testicular spermatozoa were reported in
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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