The aim of this research was to evaluate two different diluents for sperm cryopreservation and to study functional parameters in relation to the response to heparin, lysophosphatidylcholine and progesterone, in frozen-thawed semen of fallow deer (Dama dama) during the reproductive season (brama). In this way, fallow deer can be used as a biological model of endangered cervids. Semen was obtained by electroejaculation. Heparin, progesterone and lysophosphatidylcholine were used as capacitation and acrosome reaction inducers, respectively. Capacitation and acrosome reaction were evaluated by chlorotetracycline epifluorescence technique (CTC), membrane integrity by Hypo-osmotic swelling test (HOS) and viability and acrosome integrity by trypan blue stain/DIC. Data was analyzed by ANOVA and Tukey Test (P < 0.05). Semen was cryopreserved in different diluents and Fructose-Tris-Glycine extender was selected. Capacitation with heparin at different incubation times determined that the highest capacitation percentage was obtained at 45 minutes incubation. Progesterone (1 'M) and lysophosphatidylcholine in heparin capacitated sperm induced acrosome reaction (P < 0.05). This study contributes to improve cryopreservation methods and to increase the knowledge about capacitation and acrosome reaction in vitro in deer spermatozoa, allowing an advance in the development of reproductive biotechnologies.
Correspondence toProfessor Cisale THERE are few reports describing the electroejaculation of boars (Fraser 1971, Evans 1980, Evans and Ko 1990 or of related wild species (Gilmore and others 1998). Obtaining semen from boars using a dummy sow or even a sow in oestrus is not always feasible under field conditions, particularly when the subject is a mature, intractable boar (Fraser 1971). This is particularly true with wild boars. Nevertheless, it has been reported that the use ofelectroejaculation to collect boar semen is unsatisfactory, either because of a failure to obtain a representative sample (Hurtgen and others 1977) or because only small volumes of semen are collected (Foote 1974). Wild boars have a strict seasonal reproductive behaviour, and mate during autumn to winter (Mauget 1982, Schopper and others 1984). For wild boar farms where wild boars are mated with domestic gilts, it is necessary to develop a method for conserving wild boar semen, to allow insemination in the spring to summer months. This short communication describes the collection of semen from wild boar by electroejaculation.Four wild boars were used in the study. A combined anaesthetic was administered to the animals in two phases: first, xylazine (Sedomin 10 per cent; Konig) and diazepam (Diazepan 5 mg/ml; Lamar) were administered intramuscularly in the lumbar or neck region, and then ketamine (Ketamina 50 mg/ml; Holliday) was administered intravenously in the ear or caudal saphenous lateral vein. Individual variations in response were detected, and so the doses were adjusted for each male. Anaesthesia was reversed with dexamethasone 21-phosphate (Cortivet Shock 20 mg/ml; Viterra). Cardiac and respiratory frequencies were monitored continuously and the animals' temperatures were constantly measured with an ear thermocouple.Before electroejaculation stimulation, the penis was exteriorised using surgical forceps inserted into the preputial cavity. Adequate probe size has been shown to be a critical factor for proper stimulation with electroejaculation (BasurtoKuba and Evans 1981), thus, two different probes were used: a custom-designed rectal cylindrical probe, of 2-6 cm in diameter and 35 cm long, with four electrode rings of 0-5 cm width, and a rectal probe number 4 (Model 304; PT tlectronics), which had a diameter of 1-7 cm and length of 22-5 cm, with three linear electrodes of 0-5 cm wide and 4-5 cm long. The rectal probe number 4 was used with the electrodes oriented ventrally. Faeces were removed with the probe before stimulation. Electrical stimuli were applied in steadily increasing steps, using a 50 Hz electronic electroejaculator (Liluform; PT Electronics). The erection and ejaculation zones were found approximately 30 cm from the anus. No additives were necessary for good electrical contact.Optimal stimulation was obtained using potential differences between 5V and 8V; higher or lower values did not cause ejaculation. The frequency of stimulation was variable for each male. The best results were obtained when two or three series ...
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