Twelve pony mares were randomly assigned to either a control or a treatment group and inseminated with fresh, raw semen from a single stallion of known fertility in a cross-over trial design. Pregnancy was diagnosed by transrectal ultrasound 12-14 days post-ovulation and then terminated by administration of a luteolytic dose of cloprostenol. Treatment mares received a uterine instillation of 100 m of electrochemically activated (ECA) saline 4-12 hours post-insemination. Control mares received no treatment post-insemination. Per cycle pregnancy rate was 58.3 % in the control group and 50 % in the treatment group. There was no statistical difference (P = 1.000) in pregnancy rate between the 2 groups. The principles of ECA and applications of ECA saline are discussed

Annandale, C.H.

Kirkpatrick, R.D.

Schulman, M.L.

English

Annandale, C.H. (Cornelius Henry)

Schulman, M.L. (Martin)

UPSpace at the University of Pretoria

The use of electrochemically activated saline as a uterine instillation in pony mares

Research Repository

Article — ArtikelThe use of electrochemically activated saline as a uterine instillation inpony maresC H Annandalea*, M L Schulmana and R D KirkpatrickbINTRODUCTIONEndometritis in mares is of considerableclinical significance in equine practice. Ithas been cited4 as the 3rd most commonmedical condition in mares. LeBlanc7 clas-sifies endometritis into 3 categories: 1)persistent mating-induced endometritis(PMIE), 2) chronic infectious endometritisand 3) sexually transmitted endometritis.In mares, both natural breeding and artifi-cial insemination initiates a uterine in-flammatory response14. Current thinkingholds that transient uterine inflammationfollowing breeding is a normal physiolog-ical process assisting evacuation of theuterus of bacteria, dead sperm and excessseminal plasma20. Normal mares can beexpected to clear uterine inflammationwithin 12 hours post-breeding6. Persis-tence of inflammatory products occurs inmares with impaired uterine motility andfunction15,19 and may lead to accumula-tion of fluid in the uterus and increasedearly embryonic death rates18. The latterconsequence results in a lower overallpregnancy rate and hence reduction ofpersistent post-breeding uterine inflam-mation post-breeding could potentiallyyield considerable clinical and economicbenefits.Common therapeutic strategies forendometritis include: 1) intrauterine ther-apy via uterine lavage, irrigation and infu-sions of varying temperatures usingsaline, plasma and other solutions with orwithout the addition of antimicrobialagents (antibiotics and iodinate com-pounds), pH-altering compounds andother agents including colostrum, plasmaand disinfectants3,9,13,21,22; 2) systemicecbolics, principally oxytocin and prosta-glandins17; and 3) systemic antimicro-bials16. These therapeutic modalities areused alone or in various combinations.The combination of uterine lavage, antibi-otic infusion and an ecbolic agent is possi-bly the most commonly applied strategyin current practice17. Some concern aboutantimicrobial resistance has led to numer-ous investigations into therapeutic mo-dalities that can replace antimicrobialagents. Electrochemically activated salineholds promise in this regard10.Electrochemical activation (ECA) is anovel refinement of established electro-lytic procedures for the electroactivationof aqueous solutions. The original reportsclaim it has applications in agriculture,dermatology, dressing and cleaning ofwounds and disinfection of instru-ments1,8. During ECA of water, a dilutesaline solution is ‘activated’ by passingthrough a cylindrical electrolytic cell inwhich the anode and cathode chambersare separated by a permeable membrane.Two separate streams of activated waterare produced: ‘anolyte’ with a pH rangeof 2–9 and an oxidation-reduction poten-tial (ORP) of +400 to +1200 mV and‘catholyte’ with a pH of 12–13 and an ORPof about –900 mV. ‘Anolyte’ is an oxidis-ing agent due to a mixture of free oxidis-ing radicals and has an antimicrobialeffect12, while ‘catholyte’ is reducing withsurfactant properties and is an antioxi-dant. Some of the oxidant species in ‘ano-lyte’ are ClO; ClO–; HClO; OH–; HO2–;H2O2; O3; S2O82– and Cl2O62–. The use of theoxidising solution ‘Anolyte’ as an anti-microbial agent is well established11,12 andthe solution has been extensively as-sessed for its mammalian toxicity profilewithout any adverse evidence of acute orchronic deviations from the norm1,8. ‘Ano-lyte’ may assist abolishment of the inflam-matory process post-mating via its freeoxidising radicals and hence contribute tothe establishment of a favourable uterineenvironment. The effect of activatedphysiological saline on pregnancy rates ofmares is as yet unknown.The purpose of the present study was todetermine and evaluate the effects ofpost-breeding intrauterine infusion ofelectrochemically activated saline on theper cycle pregnancy rate in pony mares.MATERIALS AND METHODSAnimalsTwelve Nooitgedacht pony mares, anindigenous South African breed, wereused. The mean age of the mares was6.9 years (range: 3–20 years). Mares werefed grass hay and were on mixed pasturefields. They were all bred to a singleNooitgedacht stallion of known fertilityas part of routine practical teaching of un-dergraduate students. The trial was con-ducted during the summer of 2005.Experimental designA randomised, prospective, cross-overmodel was used. At time of breeding,mares were randomly allocated to a treat-ment group (n = 12) or a control group(n = 12). Mares from the control group36 0038-2809 Tydskr.S.Afr.vet.Ver. (2008) 79(1): 36–38aSection of Reproduction, Department of Production Ani-mal Studies, Faculty of Veterinary Science, University ofPretoria, Private Bag X04, Onderstepoort, 0110 SouthAfrica.bRadical Waters, PO Box 6482, Kyalami, 1685 South Africa.*Author for correspondence.E-mail: henry.annandale@up.ac.zaReceived: August 2007. Accepted: March 2008.ABSTRACTTwelve pony mares were randomly assigned to either a control or a treatment group andinseminated with fresh, raw semen from a single stallion of known fertility in a cross-overtrial design. Pregnancy was diagnosed by transrectal ultrasound 12–14 days post-ovulationand then terminated by administration of a luteolytic dose of cloprostenol. Treatmentmares received a uterine instillation of 100 m of electrochemically activated (ECA) saline4–12 hours post-insemination. Control mares received no treatment post-insemination. Percycle pregnancy rate was 58.3 % in the control group and 50 % in the treatment group.There was no statistical difference (P = 1.000) in pregnancy rate between the 2 groups. Theprinciples of ECA and applications of ECA saline are discussed.Key words: electrochemical, endometritis, fresh semen, mare, saline.Annandale C H, Schulman M L, Kirkpatrick R D The use of electrochemically activatedsaline as a uterine instillation in pony mares. Journal of the South African Veterinary Associa-tion (2008) 79(1): 36–38 (En.). Section of Reproduction, Department of Production AnimalStudies, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderste-poort, 0110 South Africa.0038-2809 Jl S.Afr.vet.Ass. (2008) 79(1): 36–38 37were re-allocated to the treatment groupat the next breeding and vice versa.Breeding managementThe 12 mares selected for the trial wereevaluated for breeding soundness duringdioestrus prior to breeding. Breedingsoundness evaluation included trans-rectal ultrasound examination, using anAloka SSD-500 ultrasound machine and a5 MHz linear array transducer (Axim (Pty)Ltd., Midrand, South Africa) and guardedendometrial swabs (Minitüb, Tieffen-bach, Germany) for cytology. Any cytol-ogy sample positive for signs of inflam-mation was also submitted for micro-biological culture.All mares were teased daily. Maresshowing positive signs of oestrus behav-iour were identified and their genitaltracts evaluated by transrectal palpationand ultrasonographic examination.Mares were bred when the predictedpre-ovulatory follicle had reached a mini-mum diameter of 35 mm. Semen was col-lected by artificial vagina. Aliquots fromeach ejaculate were evaluated for spermconcentration, individual progressivemotility in a modified Kenney’s motilitydiluent at 35 °C and normal sperm mor-phology using eosin-nigrosin-stainedsmears. Mares in both groups were in-seminated into the uterine body withfresh, raw semen within 15 minutes of col-lection. The dose for insemination wasstandardised at 500 million progressivelymotile sperm. The insemination volumeranged from 10 to 20 m . Mares were onlyinseminated once per cycle.At the time of insemination mareswere randomly allocated to either thecontrol or treatment group and 1500 IU ofhCG (Chorulon®, Intervet, Isando, SouthAfrica) was administered by intravenousinjection to all mares to induce ovulation.All mares were examined ultrasono-graphically until ovulation was verified.Any intraluminal fluid accumulation wasalso recorded. Mares in the treatmentgroup were additionally examined bytransrectal ultrasound 4–12 hours afterinsemination and the presence of intra-luminal uterine fluid recorded. At thistime, mares in the treatment group re-ceived an intra-uterine infusion of 100 mof electrochemically activated saline atambient temperature. The solution wasgenerated less than 24 hours previouslyand deposited directly into the uterinebody using an appropriate sterile plasticpipette (Minitüb, Tieffenbach, Germany),preceded by standard aseptic preparationof the perineum. No additional intra-uterine or parenteral treatments weregiven to any of the mares during thecourse of the trial, regardless of either theaccumulation of fluid or the treatmentgroup allocation.Pregnancy was diagnosed by trans-rectal ultrasound examination of theuterus 12–14 days after ovulation. Afterrecording of pregnancy status, a luteo-lytic dose of cloprostenol (Estrumate®,Schering-Plough, Isando, South Africa)was administered by intramuscular injec-tion and the mare was returned to theteasing programme. The same procedurewas repeated during the course of theensuing oestrus.Statistical analysisThe pregnancy results in the groupswere compared with the Fischer’s exacttest to account for the small numbers ofsubjects in each group. A 2-sided P-valueof 0.1 was taken as statistically significant.Preparation of ECA salineThe oxidant ‘anolyte’ solution was pro-duced in an electrochemical cell with acurrent of 5–7 A and a voltage of 24 V,yielding electric field intensity at the in-terface between the electrode surface andelectrolyte of about 105 V/cm. An influentsalt solution (2.5 g/ NaCl) was electro-lysed in the denominated chamber of theelectrochemical cell. The resultant oxi-dant ECA saline was generated to have apH of 7.4, and was bottled in a sterile con-tainer and delivered for utilisation in thetrial within 24 hours of production.RESULTSThe pregnancy rates for the mares in the2 groups are presented in Table 1. Thepregnancy rates were similar for the 2groups and no statistically significant dif-ference could be demonstrated (P =1.000). In the control group, 7 pregnancies(n = 12; 58.3 %) were diagnosed. In thetreatment group 6 pregnancies (n = 12;50 %) were diagnosed. The cumulativepregnancy rate was 54.2 %. Four maresfailed to conceive during the course of thetrial. These 4 mares included 1 mare thatconsistently had post-breeding uterinefluid accumulation. None of the othermares had any post-breeding fluid accu-mulation.DISCUSSIONOur results demonstrate no detrimentaleffect of post-breeding instillation of elec-trochemically activated saline upon theper cycle pregnancy rates of pony mares.The cumulative pregnancy rate and theper cycle pregnancy rate for the treatmentand the control group in this study islower than published reports for fertilitywith fresh semen AI5. This can in part beexplained by the inclusion of 4 mares thatfailed to conceive, irrespective of treat-ment group allocation, over the course ofthe trial. Included among these 4 mareswas 1 mare that had suffered a traumaticinjury to the hock joint and underwentarthroscopy 1 week post-ovulation. It ispossible that the anaesthetic and surgicalprocedure could have influenced herpregnancy status, but this remains unde-termined. Accumulation of fluid post-breeding can account for failure to con-ceive in the mare in which it occurred, butthe reason(s) for pregnancy failure in theother 2 mares were not determined. Re-moval of the 4 mares that failed to con-ceive during the course of the trial yieldspregnancy rates of 87.5 % (7/8) and 75 %(6/8) for the control and treatment groupsof mares, respectively. This conformsfavourably to published results.While numerous studies have looked atpost-breeding instillations as a treatmentfor PMIE, the focus of most of these stud-ies have been on the effect of the agent onthe endometrial histology or the bacterio-logical population in the uterus2,13. Thestudy reported here focused exclusivelyon pregnancy rate as the outcome afterpost-breeding instillation. The clinicalefficacy of ECA saline as an irrigationagent in mares with endometritis and itsantimicrobial effect against equine uter-ine pathogens associated with endo-metritis warrants further investigation.REFERENCES1. Bakhir V M 1997 Electrochemical activa-tion: theory and practice. First InternationalSymposium on Electrochemical Activation,Moscow: 38–452. Bennett D G, Poland H J, Kaneps A J,Snyder S P 1981 Histologic effect of infusionsolutions on the equine endometrium.Equine Practice 3: 37–42, 443. Brinsko S P, Varner D D, Blanchard T L,Meyers S A 1990 The effect of postbreedinguterine lavage on pregnancy rate in mares.Theriogenology 33: 465–4754. Card C 2005 Post-breeding inflammationand endometrial cytology in mares. Therio-genology 64: 580–5885. Gahne S, Ganheim A, Malmgren L 1997Effect of insemination dose on pregnancyrate in mares. Theriogenology 49: 1071–10746. Katila T 1995 Onset and duration of uterineinflammatory response of mares after in-semination with fresh semen. Biology of Re-production Monograph 1: Equine Reproduc-tion: 515–5177. LeBlanc M M, Lopate C, Knottenbelt D,Pascoe R 2003 Endometritis. In KnottenbeltD (eds) Equine stud farm medicine and surgery.Saunders, Elsevier Science, Edinburgh:178–184Table 1: Pregnancy rates for mares in con-trol and treatment groups.Group Pregnancy rateControl 58.3 % (7/12)Treatment 50.0 % (6/12)38 0038-2809 Tydskr.S.Afr.vet.Ver. (2008) 79(1): 36–388. Leonov B I 1997 Electrochemical activationof water and aqueous solutions: past, pres-ent and future. First International Sympo-sium on Electrochemical Activation, Moscow:11–279. Ley W B, Bowen J M, Sponenberg D P,Lessard P N 1989 Dimethyl sulfoxide intra-uterine therapy in the mare: effects uponendometrial histological features and bi-opsy classification. Theriogenology 32: 263–27610. Marais J T 2000 Cleaning efficacy of a newroot canal irrigation solution: a preliminaryevaluation. International Endodontic Journal33: 32011. Marais J T, Brozel V S 1999 Electro-chemi-cally activated water in dental unit waterlines. British Dental Journal 187: 154–15812. Marais J T, Williams W P 2001 Antimicrobialeffectiveness of electro-chemically acti-vated water as an endodontic irrigationsolution. International Endodontic Journal 34:23713. Mattos R, Castilho L F F, Malschitzky E,Neves A P, Keller A, Gregory R M, MattosR C 1997 W Die Endometritisbehandlungbei der Stute Mittels Intrauteriner NaCl-Lavage. Pferdeheilkunde 13: 521–52414. Nikolakopoulos E, Watson E D 1997 Doesartificial insemination with chilled, ex-tended semen reduce the antigenic chal-lenge to the mare’s uterus compared withnatural service? Theriogenology 47: 583–59015. Nikolakopoulos E, Watson E D 1997 Uterinemotility in mares resistant and susceptibleto endometritis. J. Reprod. Fertil. Abstr. Ser.20: 3216. Ott R S 1986 The efficacy of uterine treat-ment with antimicrobial drugs. In MorrowD A (eds) Current therapy in theriogenology.W B Saunders, Philadelphia: 39–4217. Pycock J F, Newcombe J R 1996 Assessmentof the effect of three treatments to removeintrauterine fluid on pregnancy rate in themare. Veterinary Record 138: 320–32318. Pycock J F, Newcombe J R 1996 The relation-ship between intraluminal uterine fluid,endometritis, and pregnancy rate in themare. Equine Practice 18: 19–2219. Troedsson M H T, Liu I K M 1991 Uterineclearance of non-antigenic markers in re-sponse to bacterial challenge in marespotentially susceptible and resistant tochronic uterine infection. J. Reprod. Fertil.Suppl. 44: 283–28820. Troedsson M H T, Loset K, Alghamdi A M,Dahms B, Crabo B G 2001 Interactionbetween equine semen and the endo-metrium: the inflammatory response tosemen. Proceedings of the 4th InternationalSymposium on Stallion Reproduction 68: 27321. Waelchli R O, Corboz L, Winder N C 1987Effect of intrauterine plasma infusion in themare: histological, bacteriological and cyto-logical findings. Theriogenology 28: 861–86922. Youngquist R S, Blanchard T L, Lapin D,Klein W 1984 The Effects of Edta-Tris infu-sion on the equine endometrium. Therio-genology 22: 593–599

https://repository.up.ac.za/bitstream/2263/8667/1/Annandale_Use%282008%29.pdf

The use of electrochemically activated saline as a uterine instillation in pony mares

Abstract

Similar works

Full text

Available Versions

UPSpace at the University of Pretoria

Research Repository