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Journal of Agricultural Science Vol. 1(5), pp. 89-93. August 2013 Available online a ISSN: 2315-9162 2013 Wyno Academic Journals THE USE OF CHLOROQUINE PHOSPHATE AS A MEANS OF REGENERATING
THE MOTILITY OF POST THAWED CANINE SPERMATOZOA.
Otite, J.R.1*, Sokunbi, O.A1, Ladokun, A. O. 1,2 and Adejumo, D.O1. 1Reproductive Physiology Laboratory, Department of Animal Science, University of Ibadan, Nigeria. 2 Dept. of Animal Physiology, College of Animal Science and Livestock Production, University of Agriculture
ABSTRACT
Semen samples were obtained from six studs, purposely selected for this study. The semen samples were
cryopreserved in four different extenders in a completely randomized design. These extenders were Sodium-Citrate-
Glycine Extender (SCGE), Coconut Milk Extender (CME), Skim Milk Extender (SME), and Zobo Extender (ZE).
Samples were later thawed and analyzed for progressive motility. Chloroquine phosphate (0.2ml of 3mM) was later
introduced into portions of the thawed semen and analyzed hourly for progressive motility. The Skim Milk Extender
had the highest initial progressive motility (69.67%), followed by ZE (68.83%), CWE (68.00%), and SCGE
(12.50%). With the exclusion of SME and ZE, the results revealed that Chloroquine Phosphate has a significant
effect on the progressive motility of canine spermatozoa. The study also confirmed that Chloroquine phosphate had
potentials to rejuvenate canine spermatozoa. However, its inclusion rate still needs to be determined.
Keywords:
Dog, Artificial Insemination, Extenders, Semen.

Non-Standard Abbreviations
CME: Coconut Milk Extender
SME: Skim Milk Extender
SCGE: Sodium Citrate Glycine Extender
ZE: Zobo Extender
INTRODUCTION
The influx of exotic dog breeds into African countries such as Nigeria is on the increase (Ajala et al., 2011).
Although most countries have quarantine laws, these laws are not properly enforced and so dogs are imported into
many countries unchecked either due to the prevalence of poorly trained border staff, or probably the presence of
corrupt border agents who place a flat fee on imported dogs. This should be a major concern as it creates an entry
point for diseases into such countries.
One way of tackling this dilemma is to reduce the dependence on importation of exotic breeds and embark on more
advanced methods of reproduction such as artificial insemination using local or imported semen. This has its
drawbacks as even when semen is imported, its motility drops as a result of the cryopreservation process and hence
breeders are faced with low or zero conception.
Several methods have been used in the past to revitalise already weakened spermatozoa. Amongst these are ascorbic
acid in bull semen (Kumar, 2002), chlorpromazine and catalase in bulls (Paudel et al., 2010) and caffeine as
demonstrated by Satish (2007).
The use of Chloroquine phosphate to boost sperm motility has been widely acclaimed in numerous animal species.
In boars, Egbunike (1989) observed that Chloroquine was able to restore motility to normal levels when motility
drops due to prolonged storage. Kumar (2002) noted that Chloroquine diphosphate which acts as a membrane
stabilizer improved the post-thaw quality of frozen semen of Jersey bulls.
This work aimed to investigate the use of Chloroquine phosphate as a means of revitalizing the motility of post-
thawed canine spermatozoa.


90. Agric. Sci.

MATERIALS AND METHODS
Experimental Dogs
The experiment was carried out in Ibadan Oyo State in Nigeria . Six studs were purposely
selected for this study. The dogs comprised of 3 German Shepherd Dogs (GSD) and 3 Rottweilers. These studs
weighed between 38 and 40kg and were between two and three years of age. All dogs were in good health and had
received all the required vaccinations. The experimental dogs were chosen based on body weight, as the weight of
dogs plays a significant role in semen quality (Rijsselaere et al., 2007). The ejaculates harvested from these studs
were extended in 4 different extenders (Otite and Egbunike, 2002). Namely; Sodium-Citrate-Glycine Extender
(SCGE), Coconut Milk Extender (CME), Skim milk Extender (SME), and Zobo Extender (ZE) in a completely
randomized design.
Semen Collection
Semen samples were collected using the artificial balloon vagina as described by Otite and Egbunike (2000).
All collections were scheduled based on weather predictions as opposed to the time of the day. This was done in
order to ensure that semen was obtained without imposing stress on the dogs.
Semen Evaluation
The following parameters were used for evaluating semen quality of the dogs:
Volume: Ejaculate volume was obtained by means of a graduated measuring cylinder.
Concentration: Sperm concentrations were determined using a Neubeur Haemocytometer (Egbunike et al., 1976).
Percentage Progressive Motility: This was done using Walton’s method of estimating motility (Walton, 1933). A
drop of semen was placed on a warm slide and placed under a cover slip. Estimation of motility was then done by
microscopic examination. In order to increase accuracy, 5 spermatozoa were counted from 4 different fields of the
slide and scored as follows:
5 - All spermatozoa present are motile.
4 - 4/5 spermatozoa present are motile.
3 - 3/5 spermatozoa present are motile.
2 - 2/5 spermatozoa present are motile.
1 - 1/5 spermatozoa present are motile.
0 - Only oscillatory movements.
N- All spermatozoa present are dead.
Semen Extension
The collected semen was gently introduced to the prepared extender at body temperature (37°C) and both were
mixed by gently inverting the mixture. Extension was done at a rate of 100 million spermatozoa to 1mL of extender.
Addition of Penicillin and Streptomycin
Penicillin and streptomycin were also added to all the extenders at a dosage of 105 i.u./1000mL and
100.00mg/100mL respectively.
Cryopreservation
Freezing of the extended semen was done as described by Herman et al. (1994). The extended semen was cooled
alongside glycerol to refrigeration temperature (5°C) and left to equilibrate for 2 hours. Glycerol was gradually
mixed with the extended semen and the mixture further left to equilibrate before freezing in liquid nitrogen.
Freezing was done using a 2L Liquid Nitrogen Flask obtained from Qingdao Ruifeng Gas Co Ltd, Qingdao, China.
Prior to immersion in liquid nitrogen, the semen samples were held in liquid nitrogen vapour for 20 minutes. The
samples where then lowered into the liquid nitrogen tank for complete immersion in liquid nitrogen.
Thawing
Thawing was done by placing the frozen semen samples in a water bath at 37°C for 30 seconds prior to evaluation.
Post Preservation Analysis
All thawed samples were analyzed for progressive motility a described above.
Addition of Chloroquine Phosphate
Chloroquine Phosphate was added at a rate of 0.2mL of 3mM. This low rate of inclusion was chosen as chloroquine
has been known to reduce motility of spermatozoa in other species if added in higher concentrations (Hargreaves et
al., 1998).
Statistical Analysis
The results obtained were analyzed for significant differences using the analysis of variance (p<0.05) and their
means were further subjected to the Duncan’s Multiple Range Test using SAS Version 9.2 (SAS Institute Inc.2010).



RESULTS AND DISCUSSION
Table I: The influence of chloroquine phosphate on the progressive motility of spermatozoa

Means with different superscripts by extender on each row are significantly difference (p<0.05) SEM: Standard Error of Mean SCGE: Sodium Citrate Glycine Extender CWE: Coconut Water Extender SME: Skim Milk Extender NC: No Chloroquine C: Chloroquine With the addition of Chloroquine phosphate to the post thawed samples, SME had the highest initial progressive motility (Table I) (There was no statistical difference between SME, CWE, and ZE). This was followed by ZE, CWE, and SCGE. Chloroquine phosphate significantly affected the progressive motility of spermatozoa extended in SCGE and CWE (Table I). Upon one hour post addition of Chloroquine phosphate, the progressive motility had risen in all extenders (but SCGE) compared to control groups. This inability of Chloroquine phosphate to rejuvenate spermatozoa extended in SCGE may have been due to the low initial post thaw progressive motility which fell far below the recommended minimum value of 60% (England and Lofstedt, 2000)(Table 1). Cryopreservation of canine semen using SCGE might thus be detrimental to the quality of spermatozoa. In addition, the results obtained from SCGE might suggest a minimum percentage progressive motility level for Chloroquine phosphate to be effective. Although CWE, SME, and ZE had their initial post thaw progressive motility within the recommended range of 60%, the results revealed that chloroquine Phosphate has a significant effect on the progressive motility of canine spermatozoa when supplemented in CWE Extender (Table I). Although research carried out on other mammalian species show the potential chloroquine phosphate to improved semen quality the results obtained in this experiment, show that this influence is also extender specific as Chloroquine phosphate did not improve the progressive motility of spermatozoa extended in SME and ZE. This attribute of chloroquine phosphate to boost motility might be due to cyclic adenosine monophosphate (cAMP) reaction and would stimulate the respiration and motility of spermatozoa in vitro (Norman and Gombe, 1974). Studies have shown that stimulation of spermatozoal motility by CAMP is mediated by phosphorylations catalyzed by cAMP-dependent protein kinase (Tash and Means, 1983). Reducing the ratio of ATP to other adenine nucleotides results in an energy charge, improving kinetic activity (displayed by flagella movement) and improving oxygen uptake (Whitehouse and Leader, 1967). Thus, Chloroquine may be working either as a catalyst or partaking in a phosphorylation reaction (Garbers et al., 1971; Lardy et al., 1971; Hoskins, 1973; Tash and Mann, 1973) (Figure I). Although ATP levels were not measured in this experiment, there lies a possible explanation based on the possibility of the spermatozoa using the available ATP from its mitochondria (displayed by the decline in motility) thus having an abundance of ADP (i.e. ATP ADP). Upon addition of Chloroquine phosphate, phosphate is transferred to ADP, thus regenerating ATP and therefore resuming spermatozoa motility. As soon as the available ATP is used, the spermatozoa lose motility again. 92. Agric. Sci. This reaction could result in the production of a hydroxide ion (OH-) as a water molecule breaks into a hydroxide Figure I: Possible reaction showing the Contribution of Chloroquine
Phosphate in ADP to ATP Conversion

ion (Tobin and Dushek, 2005). This hydroxide ion undergoes a neutralization reaction (Masterton et al., 2012) with
the already built up lactic acid thus reversing the drop in pH associated with lactic acid.
One way of further augmenting the life span of the spermatozoa is to inseminate immediately after Chloroquine
phosphate supplementation as the bitch’s reproductive tract is alkaline in nature. This might explain the prolonged
survival time of spermatozoa observed in the bitch. With this, inseminators can: (a) increase the motility of already
weakened spermatozoa and (b) prolong the survival time of spermatozoa through early insemination. The success of
this method will also depend on the time of insemination. It is best to inseminate bitches 48 to 72 hrs after ovulation
as bitches ovulate premature oocyte which must mature before fertilization can occur (Concannon, 2004; Tsutsui et
al., 2009).

CONCLUSION

Although motility is not the sole criteria for evaluating semen quality, the results showed that chloroquine can act as
a booster to already weakened spermatozoa. However, further research needs to be done in order to determine the
optimum inclusion rate of Chloroquine phosphate. The action of Chloroquine Phosphate is dependent on the type of
extender. Chloroquine Phosphate could thus be used to rejuvenate the motility of canine spermatozoa extended in
Coconut Water Extender.
Corresponding Author:
J.R. Otite,
316-710 Concession St,
Hamilton Ontario, L8V1C2, Canada.
Email
Tel: +1 905 531 1092

REFERENCES
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4. Egbunike GN, Holtz W and Smidt D (1976). Reproductive capacity of German Landrace boars. II. Sperm production rates as determined by quantitative testicular histology and from gonadal sperm reserves. Zuchthygiene 11:35-37. 5. England GCW and Lofstedt R (2000). Canine Reproduction Seminar. Atlantic Veterinary College. Prince 6. Garbers DL, First NL, Sullivan JJ and Lardy HA (1971). Stimulation and maintenance of ejaculated bovine spermatozoan respiration and motility by caffeine. Biol. Reprod. 5:336-339. 7. Hargreaves CA, Rogers S, Hills F, Rahman F, Howell RJS and Homa ST (1998). Effects of co-trimoxazole, erythromycin, amoxycillin, tetracycline and chloroquine on sperm function in vitro. Hum. Reprod. 13(7):1878–1886. 8. Herman HA, Mitchell JR and Doak GA (1994). The artificial insemination and embryo transfer of dairy and beef cattle. Eighth Edition. Interstate Publishers, Inc., Danville IL, USA, pp 38-49. 9. Hoskins DD (1973). Adenine nucleotide mediation of fructolysis and motility in bovine epididymal spermatozoa. J. Boil. Chem. 248:1135-1140. 10. Kumar S (2002). Studies on the effect of certain additives on quality and fertility of cryopreserved semen of jersey bulls maintained under sub-temperate climate. PhD thesis. CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur, India. 11. Lardy HA, Garbers DL, Lust WD and First NL (1971). Effects of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility. Biochem. 10(10):1825-1831. 12. Masterton WL, Hurley CN, Neth EJ (2012). Chemistry: Principles and Reactions. Seventh edition. 13. Norman C and Gombe S (1974). Stimulatory effect of the lysosomal stabilizer, chloroquine, on the respiration and motility of fresh and aged bovine spermatozoa. J. Reprod. Fert. 44:481-486. 14. Otite JR and Egbunike GN (2002). Response of canine spermatozoa to different extenders at 4°C. J. Trop. 15. Otite JR and Egbunike GN (2000). Semen collection in the dog using a balloon artificial vagina. J. Trop. Anim. 16. Paudel KP, Kumar S, Meur SK, Kumaresan A (2010). Ascorbic acid catalase and chlorpromazine reduce cryopreservation-induced damages to cross bred bull spermatozoa. Reprod. Domest. Anim6–262. 17.(2007). Effect of body weight, age and breeding history on canine sperm quality parameters measured by the Hamilton-Thorne analyser.42(2):143-8. 18. SAS (2010). SAS/STAT User's Guide Version 9.2 for windows. SAS Institute Inc., SAS Campus Drive, Cary, 19. Satish K (2007). Effect of certain additives in the diluter on post-thaw recovery rate of bovine semen. Indian J. 20. Tash JS and Mann T (1973). Adenosine 3':5'-cyclic monophosphate in relation to motility and senescence of spermatozoa. Proceedings of the royal society of London. Series B. Biol. Sc. 184(1074):109-114. 21. Tash JS and Means AR (1983). Cyclic adenosine 3',5' monophosphate, calcium and protein phospborylation in flagella motility. Biol. Reprod. 28:75-104. 22. Tobin AJ and Dushek J (2005). Asking About Life. Third edition. Brooks/Cole, pp 98. 23. Tsutsui T, Takahashi F, Hori T, Kawakami E and Concannon PW (2009). Prolonged duration of fertility of dog 24. Walton A (1933). The technique of artificial insemination. Imperial Bureau of Animal Genetics, Oliver and 25. Whitehouse NW and Leader JE (1967). Biochemical properties of anti inflammatory drugs. IX. Uncoupling of oxidative phosphorylation and inhibition of a thiol enzyme (papin) by some cyclic B-diones and ninhydrin. Biochem. Pharmacol. 16:537—551.

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