Livestock is the major source of income in agricultural countries. It boosts economic status by producing valuable food products and earning huge amount of Foreign exchange. Pakistan is rich in livestock population having more than 37 goat breeds. Artificial insemination is the most widely used ART and has made the most significant contribution to genetic improvement worldwide. It provides accurate means of progeny testing and spread of genetics to wide area with minimum cost. In contrast to its large scale use in bovine breeding, it is not routinely practiced in small ruminants. It may be due to effects of cryopreservation procedures on sperm plasma membrane, cytoskeleton, motility, mitochondrial membrane, acrosome and the nucleus of spermatozoa, which results in lower fertility rates and discourages the goat breeders to get benefit from this biotechnology. As mitochondria are power house of sperm cell, it provides energy to sperm cell and maintains motility. Therefore, the intactness of mitochondrial membrane must be maintained to keep the sperm cell motile after freezing and thawing procedure. In recent years, a pharmacologic agent, Adenosine 5’-triphosphate (ATP) has been used to enhance the quality and fertility of cryopreserved sperm. Previous reports have shown the freezing of mammalian sperm in hypertonic extenders to improve the rate of sperm viability and acrosome integrity. However, the impact of extracellular ATP supplementation on the quality of goat sperm after freezing and thawing still unknown. Thus the present study was conducted to evaluate the effects of Adenosine 5’ triphosphate (ATP) supplementation in pre-freeze and post thawed buck semen. The semen of six adult Beetal bucks was collected by artificial vagina maintained at 42ºC. After initial evaluation of percentage motility and concentration the semen was pooled and diluted with TCEY extender to the final concentration
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of 200 × 106 sperms/mL. The study was conducted in two experiments, in first experiment the extended samples were divided in four equal aliquots and treated with one of the ATP concentrations (0 mM, 0.5 mM, 1 mM, 2 mM). All the samples were incubated at 35 ºC for 15 min. The samples were filled in 0.5 mL French straws cooled to 4ºC within 1 hr, equilibrated at 4oC for a period of 2hr and cryopreserved using standard freezing protocol. After freezing of minimum one week two straws from each aliquot were evaluated for percentages of post thaw motility, sperm viability, acrosome, plasma membrane, mitochondrial and DNA integrities. In second experiment the extended semen samples were cryopreserved with standard freezing protocols. Ten doses were thawed, evaluated individually for post thaw motility and pooled. Now the pooled samples was divided in four equal aliquots and treated with one of the ATP concentrations (0 mM, 0.5 mM, 1 mM, 2 mM). All the samples were incubated at 37 ºC and evaluated at 10 min, 1 hr, 2 hr, 3 hr and 6 hr for percentages of post thaw motility, sperm viability, acrosome, plasma membrane, mitochondrial and DNA integrities. The results of first experiment were analyzed by using one way ANOVA with SPSS. The results indicated that mitochondrial integrity % and DNA integrity % were higher (p < 0.05) in ATP treated groups. Sperm viability % was higher in 0.5 mM group as compared to all other groups. However, post thaw motility %, acrosome integrity % and plasma membrane integrity % remained similar in all groups. The results of second experiment were analyzed using GLM univariate procedure of SPSS. The results revealed that mitochondrial integrity % and DNA integrity % remained higher (p < 0.05) in ATP treated samples throughout the incubation time. Sperm viability % was higher (p < 0.05) in 2 mM ATP group while acrosome integrity % was higher (p < 0.05) in 1 mM ATP group throughout the incubation time. Post thaw motility % and plasma membrane integrity % remained similar (p > 0.05) throughout the incubation time. The interaction between time and
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treatment was found non-significant (p>0.05). However, significant (p<0.05) decline in percentages of all parameters was observed with the increasing incubation time. In conclusion, addition of ATP enhances the post thaw quality of buck sperm by maintaining its motility, enhancing sperm viability, mitochondrial and DNA integrities. It also effects positively on post thaw quality of buck sperm. Moreover, is recommended in future to conduct research to elaborate the effects of ATP supplementation in semen on fertility of goats which is also primary objective of cryopreservation of semen.