Recovery rates and embryo quality following dominant follicle ablation in superovulated cattle

ELSEVIER

RECOVERY RATES AND EMBRYO QUALITY FOLLOWING DOMINANT FOLLICLE ABLATION IN SUPEROVULATED CATTLE D.W. Shaw’ and T.E. Good’ ‘Departmentof Veterinary Preventive Medicine The Ohio State University, Columbus,OH ‘Select Embryos Inc., Plain City, OH Receivedfor publication: JULY 29, 1998 Accepted: October 8, 1999 ABSTRACT To determinethe associationbetweendominantfollicle ablation and the outcome of a superovulatoryregimen,two data setswere constructedfrom recordsof 171 recoveriesfrom non-ablatedcowsand 1214recoveriesfrom cows that underwent follicular ablationprior to FSH treatment. Data set 1 includedall cows with 2 or more records(n = 1385). Data set2 includedpaireddata for 87 cowswhich had at least2 recordsof both ablatedand non-ablatedsuperovulatoryattempts. Dominantfollicle ablationwasperformedby useof transvaginal,ultrasoundguidedaspiration48 hr prior to the start of FSH. The sameFSH protocolswere usedfor both ablatedand nonablatedcows. For all cows (data set l), more total ova/embryoswere recoveredfrom the ablationgroup (12.1kO.3 vs 10.5kO.8; P=O.O6). This difference could be accountedfor by greater numbersof non-transferableembryosin the ablationgroup (6.5tO.2 vs 5.3kO.6; P>O.Ol). For the paireddata (data set2), greater numbersof total ova/embryosrecoveredfrom the ablationgroup (12.8* 1.0 vs 9.7tO.7; P=O.Ol) could alsobe accountedfor by higher numbersof nontransferableembryosin this group (7.8kO.8 vs 4.5kO.4; P>O.Ol). There were no differencesbetweengroupsfor high quality embryos, percentcowsproducingno ova/embryosor percentcows producingno transferableembryos. Thesedatasupportthe premisethat synchronizationof follicular wavesfollowing dominantfollicle ablation increasestotal ova/embryo output. However, the additionalembryoswere primarily nontranferable thereby negatingpotential economicgains. Q 2000 by Elsevier

Science

Inc.

Key words: embryo transfer, ablation, cattle

Theriogenology 0 2000 Elsevier

53:1521-1526,200O Science Inc.

0093-691X/00/$-see front matter PII SOO93-691X(00)00294-6

1522 INTRODUCTION The discovery of follicular wave dynamics in cattle has led to considerable research targeting manipulation of both dominant follicles and subordinate cohorts. That cohorts of small follicles may be manipulated to begin growth as a synchronous unit is of particular interest to the embryo transfer industry. High variability both within and among cows for superovulatory response has plagued the industry since its inception (9). Little progress toward reducing this variability was made prior to the discovery of follicular waves. Currently the most practical method for synchronization of follicular waves requires elimination of dominant follicles. This can be achieved either mechanically (3) or chemically (4). Both methods appear to be capable of improving the superovulatory response in cattle. Manipulation of the follicular wave is a relatively new technology in the embryo transfer industry. Although private embryo transfer companies and solo embryo transfer practitioners have used the technique for several years, few reports on the efficacy of this strategy under field conditions exist in the scientific literature. In fact, the authors were unable to find any large, retrospective analyses of mechanical ablation in relation to embryo quality and output. We therefore began to compile a database of 2031 recoveries over a 3-yr period. This database included cows which had undergone dominant follicle ablation prior to FSH stimulation, undergone FSH stimmation alone or had undergone both protocols at different times. These retrospective data were then analyzed for the purpose of defining any associations between mechanical ablation of the dominant follicle and subsequent superovulatory response. MATERIALS

AND METHODS

Database Records of FSH stimulation/embryo recovery attempts on cows presented to Select Embryos, Inc. (Plain City, OH) during 1995, 1996 and 1997 supplied the initial database of 2031 observations. The data set was refined by excluding cows with only 1 record. These records included cows from many milk and beef breeds. Two subsets of data were compiled from these records and analyzed retrospectively. Data set 1 consisted of 1385 recovery attempts from cows with at least 2 stimulation records. There were 171 records for cows not receiving ablation and 1214 records for cows which did undergo dominant follicle ablation prior to FSH stimulation. Of the 1385 records, 87 cows had at least 2 recovery records for both ablation and no ablation. These cows are represented in data set 2, and provide the basis for all paired comparisons. Relevant information present in individual recovery records included total ova/embryos recovered, numbers of ova/embryos in each quality grade and ablation or no ablation.

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Stimulation/EmbryoRecovery Protocols All cows were stimulatedby useof commerciallyavailableFSH products administeredin a decreasingdosescheduleover 3 or 4 d. The FSH product, total dosageand length of the regimenwasat the discretionof the embryo transfer clinician. Thesedecisionswere madeindependantlyof ablationor no ablation. FSH stimulation beganbetweenDays 7 and 13 of the estrouscycle, andprostaglandinwasusedto induceluteolysison the last day of FSH treatment. The decisionto useablationor not wasmadeby the clinician during a prestimulationultrasoundexaminationof the reproductive tract. Ablation wasperformedonly when a dominantfollicle ( > 8 mm ) waspresence. Schedulingissuessuchaslabor, facilities and recipient availability also affectedthe decisionto useablation. No prospectiveattempt to either randomizeor biasthis group wasimplemented.Cows which did undergoablationreceived hormone injectionsbeginning48 hr after ablation. The methodinvolved transvaginal, ultrasound-guidedaspirationof follicles andwas not significantly different from previously publishedtechniques(2). Embryoswere recoverednonsurgically7 d post estrusand visually evaluatedfor stageandgrade(11). Grades1, 2 and 3 correspondto excellent, good, and poor quality, respectively. Nontransferableembryoswere subdividedinto fertile deadembryosandnonfertile ova. StatisticalAnalysis Data set 1 wasanalyzedusingtwo mathematicalmodels. The first wasan analysisof variance constructedwithin the Proc GLM proceduresof SAS (14). This modelcontainedthe independentvariables:treatment(ablationvs no ablation)and the nestedeffect, cow within treatment. Cow within treatmentwas a significant component of the model; therefore, the error term for this effect wasusedto generatestandard errors and test for differencesbetweentreatmentleastsquaresmeans. The dependent variablestestedincludedall individual embryo grades,total transferableembryos, total nontransferableova/embryosandtotal ova/embryos. The secondmodel required constructionof frequency tablesusingthe Proc Freq proceduresof SAS. Proportions for recoveriesresulting in 0 or z 1 transferableembryosand recoveriesresulting in 0 or z 1 total ova/embryoswere computedandthe resultsreportedasChi-square probabilities. Data set 2 (paireddata) wasanalyzedby useof a pairedt-test method (Proc TTest, SAS). Dependentvariablesanalyzedwere the sameasthosefor Data Set 1. RESULTS The resultsof analysisof variance for Data Set 1 are compiledin Table 1. There were no differencesbetweentreatmentsfor total transferableembryosor any quality gradedesignatedastransferable. However, the ablationgroup produced1.6 greatertotal ova/embryos(12.1t0.3 vs 10.5t0.8; P=O.O6) than nonablatedcows. This difference was entirely accountedfor by the greaternumberof nontransferable

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ova/embryosrecovered from the ablationgroup. The majority of additional nontransferableembryoswere comprisedof nonfertile ova, accountingfor almost 1 ova per recovery. Table 1. Least squaresmeans,standarderrors (SEM) and probability values for all embryo quality gradesandtotals in Data Set 1 (nonpaired,n = 1385)

Embryo Type Grade 1 Grade2 Grade3 Total transferable Fertile dead Nonfertile ova Total nontransferable Total ova/embryos

LSMeansk SEM Ablation No Ablation 3.6 t 0.1 3.3 f 0.4 1.5 * 0.1 1.4 + 0.2 0.6 k 0.1 0.5 f- 0.1 5.7 f 0.2 5.2 i 0.5 2.4 f 0.1 1.9 f 0.2 4.1 f 0.2 3.4 f 0.5 6.5 k 0.2 5.3 + 0.6 12.1 i 0.3 10.5 + 0.8

P value 0.36 0.55 0.33 0.49 0.07 0.008 0.003 0.06

The remainderincludedfertilized ova in various statesof degeneration. Table 2 containsthe proportionsof recoverieswhich failed to produceeither any ova/embryos or any transferableembryos. This table representsthe absolutefailure ratesamong ablatedand nonablatedcows. Overall, 84% of the recoveriesresultedin at least 1 ova/embryo, while 70% of recoveriesresultedin at least 1 transferableembryo. There were no differencesbetweentreatmentsfor either criteria in this data set. Among cows which underwentat least2 recovery attemptswithin each treatmentgroup, total transferableembryosrecoveredwere not affected by ablation (Table 3). Significantly greater numbersof ova/embryoswere againnoted for total nontransferableova/embryosand nonfertile ova in the ablation group. These differencesaccountedfor a total gain of 3 ova/embryosover that of nonablatedcows (12.8kl.O vs 9.7iO.7; P=O.Ol). There wasa strong tendencyfor quality grades2 (P=O.O8) and 3 (P=O.O6) to be higher in nonablatedcows. However, the magnitude of this effect was insufficient to significantly impacttotal transferableembryos recovered.

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Table 2.

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Proportion of recoveriesresulting in any embryos/ovaor any transferable embryosby treatmentstatus(nonpaireddata, n = 1385)

Category No. of total ova/embryos Zero t One No. of transferableembryos Zero 2 One

Percentof Recoveries Ablation No Ablation 15.7 84.3

15.2 84.8

29.2 70.8

30.4 69.6

x2 probability 0.85

0.73

Table 3. Least squaresmeans,standarderrors (SEM) andprobability valuesfor all embryosquality gradesand totals in Data Set 2 (paired data, n = 87)

Embryo Type Grade 1 Grade 2 Grade 3 Total transferable Fertile dead Nonfertile ova Total nontransferable Total ova/embryos

LSMeans-eSEM Ablation No Ablation 3.0 + 0.3 2.9 t 0.3 1.3 * 0.1 1.8 i 0.2 0.7 f 0.1 0.9 i 0.1 5.0 ?r0.4 5.3 * 0.4 2.5 A0.3 1.9 i 0.2 5.2 k 0.6 2.6 + 0.3 7.8 t 0.8 4.5 + 0.4 12.8 + 1.0 9.7 zk0.7

P value 0.55 0.08 0.06 0.56 0.06 0.0005 0.0004 0.01

DISCUSSION Follicular dynamicsof the bovine estrouscycle are characterizedby multiple wavesof follicular growth andatresia(16). Cohortsof smallfollicles begin synchronousgrowth an averageof 2 to 3 timeseachestrouscycle (6, 16). One or more dominantfollicles arise from eachcohort andsuppressfurther developmentof subordinatefollicles. The commonpractice of beginningFSH injectionsbetweenDays 8 and 12 of the estrouscycle is an attemptto matchthe start of FSH treatmentwith the start of the secondfollicular wave. This strategyis designedto avoid the suppressive effects of the dominantfollicle thereby allowing for greaterefficacy of the exogenous gonadotropin. Although theoretically sound,this strategy is still confoundedby variation amongcows for both length and numberof individual follicular waves. The issueof cow-to-cow variation can be resolvedby useof transrectal ultrasonography. Direct visualization of ovarian follicular dynamicsoffers the

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opportunity to preciselydefine dominantfollicles, developingcohortsand the temporal relationshipsbetweenthese2 populationsof follicles. Although multiple scansprovide more detailedinformation, Bungartz and Niemann(5) have describeda protocol that improved superovulatoryresponsein cattle when FSH was initiated following a single ultrasoundexamination. A secondlabor savingprotocol was offered by Bergfelt et al. (3), who found that ablationof all follicles in combinationwith FSH on the day of ablation (irrespectiveof day of the estrouscycle) resultedin superovulatoryresponses equivalentto thoseachievedby starting FSH betweenDays 8 and 12 of the estrous cycle. Thesereports are in generalagreementwith mostof the literature regardingthe negativeeffect of dominantfollicles on the superovulatoryresponse(1, 8, 10, 13, 17). However, someauthorshave found no difference in the superovulatoryresponsebased on the presenceor absenceof a dominantfollicle at the time of FSH injections(7, 12). Interpretation of resultswere basedon ovarian morphology at initiation of FSH treatment;therefore, neither of theseexperimentsinvolved physical or chemical manipulationof the follicular wave. The lack of a clear, standardizeddefinition of follicular dominance,basedon ultrasoundmorphology, may accountfor the inconsistencybetweentheseandother studies,especiallythosewhich report active manipulationof the follicular wave. Our retrospective analysisis in generalagreementwith mostprospectivestudies which report enhancedsuperovulatory responses in the absenceof a dominantfollicle. Ablation of the dominantfollicle(s) 48 h prior to initiation of FSH treatmentwas associatedwith 2 to 3 additionalova/embryosper recovery attempt. However, this gain was entirely accountedfor by an increasein the numberof nontransferable ova/embryos, primarily nonfertile ova. This finding is unprecedentedby the numerous studiesdiscussedpreviously. A notabledifference betweenour analysisand thoseof previousauthorswasthe study population. Our data setwascompiledfrom primarily dairy cows of high geneticmerit who exhibit above averageproduction traits. Many of thesecows had undergonemultiple stimulationattemptsand may have beenlactating at the time of embryo recovery. Thesecows, althoughprecisely the type of animalupon which the embryo transfer industry is dependent,may not be physiologically comparableto cows typically found at university experimentstations. This supposition is supportedby the low numberof transferableembryosrecovered (averageof 5.4 per cow) from this group of cows relative to other reports (9, 1.5). An unexpectedfinding wasthe lack of associationwith the presenceof a dominantfollicle at initiation of FSH andthe proportion of nonproductiverecoveries. The abundanceof data supportingenhancedstimulationresponses in the absenceof a dominantfollicle lead intuitively to expectationsof fewer absolutefailuresamongcows receiving follicular ablation. A test of that hypothesiswould likely require a larger samplesize than usedin mostof the prospectivestudies. Yet no associationwasfound betweenablation anda completelack of any ova/embryo or transferableembryos recovereddespitethe useof over 1300recovery records. This observationsuggests that while dominantfollicle ablation may increaseovulation rate amongcowsdestined

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to yield ova/embryos, factorsother than follicular dominanceare important determinantsof FSH responsiveness andembryo quality. We have performeda retrospectiveanalysisof 1385embryo recovery records from cows receiving follicular ablation, no ablationor both prior to FSH stimulation. A significant, positive associationbetweendominantfollicle ablation andtotal ova/embryosrecoveredwas found in this populationof cows. However, the observed gain consistedexclusively of nonviableembryosand nonfertile ova which offer no economicreturn. Further, we found no associationbetweendominantfollicle ablation and failure to recover any viable embryos. REFERENCES 1. AdamsGP. Contol of ovarian follicular wave dynamicsin cattle: implicationsfor synchronizationand superstimulation.Theriogenology 1994;41:19-24. 2. Bergfelt DR, Lightfoot KC, AdamsGP. Ovarian synchronizationfollowing ultrasound-guidedtransvaginalfollicle ablationin heifers. Theriogenology 1994;42:895-907. 3. Bergfelt DR, Bo GA, Mapletoft RJ, AdamsGP. Superovualtory response following ablation-inducedfollicular wave emergenceat randomstagesof the oestrouscycle in cattle. Anim ReprodSci 1997;49:1-12. 4. Bo GA, PiersonRA, Mapletoft RJ. The effect of estradiolvalerate on follicular dynamicsand superovulatoryresponsein cows with syncro-mate-Bimplants. Theriogenology 1991;36:169-183. 5. Bungartz L, Niemann.Assessment of the presenceof a dominantfollicle and selectionof dairy cows suitablefor superovulationby a singleultrasound examination.J ReprodFertil 1994;101:583-591. 6. Ginther OJ, Knopf L, KastelicJP. Temporalassociations amongovarian eventsin cattle during oestrouscycleswith two or three follicular waves. J ReprodFertil 1989;87:223-230. 7. Gray BW, CarteeRE, Stringellow DA, et al. The effects of FSH-priming and dominantfollicular regressionon the superovualtoryresponseof cattle. Theriogenology 1992;37:631-639. 8. Guilbault LA, Grass0F, LussierJG, Rouillier P, Matton P. Decreased superovulatoryresponses in heifers superovulatedin the presenceof a dominant follicle. J Reprod Fertil 1991;91:81-89. 9. HaslerJF, McCauley AD, Lathrop WF, Foote RI-I. Effect of donor-embryorecipient interactionson pregnancyrate in a large-scalebovine embryo transfer program. Theriogenology 1987;27:139-168. 10. HuhtinenM, Rainio V, Aalto J, BredbackaP, Maki-Tanila A. Increasedovarian responsesin the absenceof a dominantfollicle in superovulatedCOWS. Theriogenology 1992;37:457-463. 11. Linder GM, Wright RW Jr. Bovine embryo morphology and evaluation. Theriogenology 1983;20:407-416.

Theriogenology 12. Maciel M, Gustafsson H, Rodriguez-Martinez H. Superovualtory response in lactating cows with different follicular dynamics. J Vet Med A 1995;42: 123-129. 13. Nasser LF, Adams GP, Bo GA, Mapletoft RJ. Ovarian superstimulatory response relative to follicular wave emergence in heifers. Theriogenology 1993;40:713-724 14. SAS. SAYSTAT User’s Guide, Release 6.04. Cary NC: Statistical Analysis System Institute Inc. 1988. 15. Shaw DW, Farin PW, Washburn SP, Britt JH. Effect of retinol palmitate on ovulation rate and embryo quality in superovulated cattle. Theriogenology 1995;44:51-58. 16. Shaw DW, Farin PW, Washburn SP, Britt JH. Effect of retinol palmitate on ovulation rate and embryo quality in superovulated cattle. Theriogenology 1995;44:51-58. 17. Stock AE, Ellington JE, Fortune JE. A dominant follicle does not affect follicular recruitment by superovulatory doses of FSH in cattle but can inhibit ovulation. Theriogenology 1996;45: 1091-l 102.