An in-vitro investigation of effects of acetylcholine on attenuate and low motile 4 diluted rooster semen quality

This study was conducted to evaluate the effects of acetylcholine (ACh) on 24 hours preserved 2 diluted rooster semen at 4 o C with a low motility percentage during 2 hours at room temperature 3 incubation. Ten indigenous same-aged roosters were used in this study, adapted by Dorso-abdominal 4 massage for semen collection for one month before starting main work. Semen was collected weekly 5 twice by the Dorso-abdominal massage technique, collected semen transferred to the laboratory in 6 less than 10 minutes, and took a previous qualifying examination. Qualify semen from each rooster 7 polled together and diluted by the Lake extender and preserved for 24 hours at 4 o C in the refrigerator. 8 Different ACh concentrations (10mM-100mM) were added to Low motile semen and stored for 24 h 9 at 4 o C, and the quality parameters such as motility, viability, acrosome, and plasma membrane 10 integrity were measured evaluated. Adding ACh in 10mM for the low motile semen significantly 11 increased semen motility from 50% to 78.5% (P<0.05) at time zero, but other ACh concentrations 12 did not have any significant differences compared to control. During two hours' incubation of 13 recovered semen at room temperature, 10mM ACh prevented declining semen motility compared to 14 control and other ACh concentrations significantly (P<0.05), which motility in 10mM ACh 15 concentration 59%. In contrast, control, 1mM ACh, and 100μM ACh group was 2.5%, 4%, and 1%, 16 respectively. Semen viability after two hours of recovery at room temperature significantly 3.5% was 17 less than the control group (P<0.05), but acrosome and plasma membrane integrity has not had any 18 differences between all experimental groups (P>0.05). We can conclude that 10mM ACh can recover 19 semen motility and not have toxicity and side effects on semen quality. this study to evaluate the in-vitro effect of the cholinergic agent (ACh) on semen quality in roosters.


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Cholinergic systems are one of the vital systems in the body's nervous system. Neurotransmitter 23 acetylcholine (ACh), synthesis of molecules, receptors, transporters, and enzymes for its degradation 24 are parts of this system. Otto Loewi and Henry Dale, for the first time in 1926, discovered the ACh and, for this foundation, received a Nobel prize in 1936. After discovering ACh in the nervous system: 26 Identified ACh as a post-ganglionic neurotransmitter in parasympathetic neurons and a preganglionic-27 neurotransmitter in sympathetic neurons in several parts of the brain and body [1]. According to 28 phylogenesis, in non-neuronal cells such as monera, protozoa, algae, tubellariae, and primitive plants, 29 fortunately, are shown the cholinergic system; for this reason, this kind of cholinergic system is called

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Because animal spermatozoa access all cholinergic system compartments, we hypothesized that 45 direct adding ACh increases sperm motility in roosters. Therefore, this study aimed to evaluate the 46 in-vitro effect of the cholinergic agent (ACh) on semen quality in roosters.

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This study used ten same-aged indigenous roosters for the sample collection and roosters habituated 51 by abdominal massage for semen collection over one month before starting the experiment. Animals 52 eat a balanced diet (metabolizable energy 2800kcal, crude protein 12%, crude fiber 3.5%, available 53 phosphor 0.35%, calcium 0.7%, salt 1%, and vitamin premix 0.25%, and mineral premix 0.25%) of 54 90-100g per day. Water was continuously available for it in 16:8 light/dark photoperiods, and the 55 temperature was 20-25°C. 56 The semen collection takes place twice a week. The semen is collected in a degraded microtube after 57 evaluating the primary semen parameter and pooled together to discards differences between the 58 roosters. Semen with total motility lower than 85% and progressive motility lower than 65% was 59 discarded.

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Extender preparation 62 Used a Lake extender for rooster semen dilution and shown their components in Table 1.

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Collected semen was diluted by Lake extender 1:10 ratio and preserved in the refrigerator at 4 o C.

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This is tha Table 1   parts of it change their color to pink color have died spermatozoa (Fig 1), finally, expressed the data 81 as a percentage.    1000X magnification. This is the Fig 2 legend  97 hypo-osmotic solution needs 9 g fructose with 4.9 g sodium citrate dissolved in 1L of distilling water. 101 10uL of diluted semen added for 100uL of hypo-osmotic solution in 0.5cc micro-tube mixed it and 102 incubated in the water bath for 60 min at 37°C. Added 10uL of incubated sample on a clean slide and 103 mixed with 10uL of eosin-nigrosine stain, finally prepare a smear and late it for drying at room 104 temperature. Seen ten fields under the light microscope at 1000X magnification, non-crawled 105 flagellum spermatozoa are lost their plasma membrane integrity, and vice versa expressed the data as 106 a percentage (Fig 3).  Used the utterly random design with ten replicate, and the obtained data were submitted to 122 analysis of variance using the SAS statistical package's general linear model (GLM) procedure (SAS means of treatments. The differences were considered significant at P < 0.05.  adding 10mM acetylcholine to low motile rooster semen (50%) can increase and recover motility of 175 the spermatozoa and remained this increase of motility (nearly 75%) for 30min more. Therefore, this 176 finding can help the parent stock breeders to use the low motile semen from their roosters when it 177 decreases the semen quality and is used in the artificial insemination technique.