"Land and Food Systems, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Calder, Michele D."@en . "2008-12-20T00:00:00"@en . "1992"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "The presence of a dominant follicle has been shown to reduce response to superovulatory treatment. Studies were carried out to assess the superovulatory response to FSH/PGF2atreatment using ultrasound scanning and progesterone profiles in the absence of a dominant follicle. The first experiment examined the effect of initiating superovulatory treatment at Day 2 of the estrous cycle, before a dominant follicle was identifiable. Although adequate follicular development occurred after administration of superovulatory hormones, few animals demonstrated estrus and ovulation rates and the number of embryos recovered was low. In the second experiment, humanchorionic gonadotrophin (hCG) was used to remove the dominant follicle present at Day 7 of the estrous cycle prior to induction of superovulation on Day 9. Cows treated with hCG tended to have higher numbers of follicles, corpora lutea and embryos recovered after treatment, however values were not significantly different from control cows superovulated at mid-cycle.\r\nBecause the cost of superovulation and embryo transfer is high, economically it may be necessary to produce only calves of the desired sex. Several methods have been used to select for calves of the desired sex, including: production of sexed semen; determination of fetal sex in early pregnancy; and detection and selection of the sex of preimplantation bovine embryos prior to embryo transfer. Two methods of selection of embryonic sex were investigated. Karyotyping was done to\r\ndirectly visualize the sex chromosomes of bovine preimplantation embryos. Previously, anti-H-Y antisera has been used in vitro and in vivo to select against male cells. Antibodies to H-Yantigen were produced in female mice after several weeks of immunization against male cells. Anti-H-Y antibody titre was assessed in enzyme-linked immunosorbent assays (ELISAs).Immunized females were then bred to study the effect of H-Y antibodies on litter size and sex ratio. Unexpectedly, increases in the male offspring were noted in the first litters of immunized females."@en . "https://circle.library.ubc.ca/rest/handle/2429/3256?expand=metadata"@en . "11284984 bytes"@en . "application/pdf"@en . "STUDIES ON SUPEROVULATION AND EMBRYO SEXING IN DAIRY CATTLEByMichele D. CalderB.Sc., The University of British Columbia, 1987A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF ANIMAL SCIENCEWe accept this thesis as conforming to the standardUNIVERSITY OF BRITISH COLUMBIAAPRIL, 1992\u00A9 MICHELE D. CALDERIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department ofThe University of British ColumbiaVancouver, CanadaDate DE-6 (2/88)ABSTRACTThe presence of a dominant follicle has been shown toreduce response to superovulatory treatment. Studies werecarried out to assess the superovulatory response to FSH/PGF 2atreatment using ultrasound scanning and progesterone profiles inthe absence of a dominant follicle. The first experimentexamined the effect of initiating superovulatory treatment atDay 2 of the estrous cycle, before a dominant follicle wasidentifiable. Although adequate follicular development occurredafter administration of superovulatory hormones, few animalsdemonstrated estrus and ovulation rates and the number ofembryos recovered was low. In the second experiment, humanchorionic gonadotrophin (hCG) was used to remove the dominantfollicle present at Day 7 of the estrous cycle prior toinduction of superovulation on Day 9. Cows treated with hCGtended to have higher numbers of follicles, corpora lutea andembryos recovered after treatment, however values were notsignificantly different from control cows superovulated at mid-cycle.Because the cost of superovulation and embryo transfer ishigh, economically it may be necessary to produce only calves ofthe desired sex. Several methods have been used to select forcalves of the desired sex, including: production of sexedsemen; determination of fetal sex in early pregnancy; anddetection and selection of the sex of preimplantation bovineembryos prior to embryo transfer. Two methods of selection ofiiembryonic sex were investigated. Karyotyping was done todirectly visualize the sex chromosomes of bovine preimplantationembryos. Previously, anti-H-Y antisera has been used in vitroand in vivo to select against male cells. Antibodies to H-Yantigen were produced in female mice after several weeks ofimmunization against male cells. Anti-H-Y antibody titre wasassessed in enzyme-linked immunosorbent assays (ELISAs).Immunized females were then bred to study the effect of H-Yantibodies on litter size and sex ratio. Unexpectedly,increases in the male offspring were noted in the first littersof immunized females.iiiTABLE OF CONTENTSAbstract^ iiList of Tables^ viiList of Figures viiiList of Plates^ ixAcknowledgements xChapter 1Literature Review^ 1A. Superovulation and Embryo Transfer^ 1I. History of Superovulation 2II. History of Embryo Collection and Embryo TransferTechniques^ 6III. Variability of Response^ 12B. Methods used for Semen and Embryo Sexing to produceOffspring of the Desired Sex 16I. Methods to separate X- and Y- spermatozoa^ 17II. Methods to detect Sex of the Fetus in EarlyPregnancy^ 23III. Methods of Sexing Preimplantation Embryos priorto Embryo Transfer^ 24Aims and Objectives^ 31Chapter 2Experiment One: Follicular growth, ovulation and embryorecovery in dairy cows given FSH at the beginning ormiddle of the estrous cycle^ 34Summary^ 34Introduction 35Materials and Methods^ 37Results^ 39Discussion 45ivExperiment Two: Superovulatory responses of dairycows following ovulation of the dominant follicleof the first wave^ 49Summary^ 49Introduction 50Materials and Methods^ 51Results^ 55Discussion 60Chapter 3Experiment One: Immunization of female mice againstmale spleen cells and determination of antibodytitre against H-Y antigen^ 66Summary^ 66Introduction 66Materials and Methods^ 67Results^ 72Discussion 73Experiment Two: Effect of maternal immunizationagainst H-Y antigen on litter size and sex ratio^ 76Summary^ 76Materials and Methods^ 76Results^ 77Discussion 78Experiment Three: Sexing the bovine embryo bykaryotype analysis^ 85Summary^ 85Introduction 85Materials and Methods^ 86Results^ 88Discussion 92vChapter 4Concluding Discussion^ 95Superovulation^ 95Sexing^ 99References 101viLIST OF TABLESTable 2.1^Estrus, ovulation and embryorecovery following superovulation at thebeginning of middle of the estrous cycle indairy cows 44Table 2.2.1^Mean (\u00B1 s.e.) numbers of follicles,corpora lutea, total ova and transferrable ovarecovered in control and hCG-treated cows followingFSH/PGF2a treatment^ 59Table 2.2.2^Mean (\u00B1 s.e.) plasma progesteroneconcentration (ng/mL) in control and hCG-treatedcows during superovulatory treatment^ 59Table 3.2.1 The number of males and females inthe first litter of control and H-Y immunizedfemales^ 79viiLIST OF FIGURESFigure 2.1^The mean numbers (\u00B1 s.e.) of follicles > 10mm,corpora lutea and unovulated follicles in cows treatedwith FSH/PGF2a. in the middle of the estrous cycle orat the beginning of the estrous cycle^ 43viiiLIST OF PLATESPlate 2.1.1^Follicles, corpora lutea andunovulated follicles in a cow superovulatedat Day 2 of the estrous cycle^ 41Plate 2.1.2^Follicles, corpora lutea andunovulated follicles in a cow superovulatedin the mid-luteal phase^ 42Plate 2.2.1^Superovulatory responses of acow superovulated in the mid-luteal phase ofthe estrous cycle^ 56Plate 2.2.1^Superovulatory responses of acow treated with hCG on Day 7 of the estrouscycle to ovulate the dominant follicle priorto superovulation 57Plate 3.3.1^Photomicrograph of lymphocytesdemonstrating the female bovine karyotype^ 89Plate 3.3.2 Photomicrograph of lymphocytesdemonstating the male bovine karyotype^ 90Plate 3.3.3^Photomicrograph of chromosomesprepared from a bovine embryo^ 91ixACKNOWLEDGEMENTSI would like to thank the many people who helped with myprojects over the last two and a half years:Dr. Rajamahendran for being a supportive supervisor and sourceof good ideas, as well as helping with ultrasound scanning, micebleeding and collection of embryos.Chris Taylor and Collins Sianangama for performing most of theultrasound scanning during the course of the projects.The people in Dr. Lee's lab for help in maintaining cellcultures, developing ELISA systems and taking photomicrographs.Sylvia Leung and Dr. Cheng for help with statistics.Staff members at the UBC Dairy unit, who put up with my demandsand interference.My parents, who gave me encouragement.B.C. Science Council which funded the H-Y antibody developmentproject.xChapter 1Literature ReviewA. Superovulation and Embryo TransferSuperovulation is a method of treating a female withhormones to increase the number of follicles developing and thusincrease the number of ovulations and eggs recoverable perfemale. Embryo collection involves surgical or non-surgicalcollection of ova from the female reproductive tract. Embryotransfer is the surgical or non-surgical procedure to place anembryo into the reproductive tract of a recipient.Superovulation in combination with embryo transfer can allow:a decrease in the generation interval, as females may besuperovulated and embryos recovered before the animal couldcarry a pregnancy; progeny testing of females; use of superiorfemales as embryo donors; increases in the number of progenyfrom superior females; the transport of embryos; research intoearly embryonic development; and study of maternal effects onembryo development (Foote, et al., 1970). The birth of thefirst embryo transfer calf was reported in 1951 (Willett et al.,1951). Since this time the embryo transfer industry has growntremendously, such that 4% of the total calf registrations inthe United States in 1987 were embryo transfer calves (Seidel,1991). In addition, 27.5% of the top type-production indexdairy cows in 1990 and 44% of the top indexing artificialinsemination sires were born through the use ofsuperovulation/embryo transfer techniques.1I. History of SuperovulationOne of the first hormones that was discovered which canelicit superovulation was pregnant mare serum gonadotrophin(PMSG) by Cole and Hart in 1930. The hormone was discoveredusing a bioassay system in prepuberal rats. The rats wereinjected with varying quantities of serum from mares atdifferent stages of pregnancy. When injected into prepuberalrats, PMSG was found to increase the numbers of follicles andovarian weight and at higher doses could cause luteinization orovulation of these follicles. The amount of serum required toproduce these effects varied with the mare and stage ofpregnancy. Since that time, PMSG has been widely used to elicitsuperovulation responses in cattle.Willett et al. (1948) investigated the effect of timing ofinitiation of superovulation in relation to the day of theestrous cycle, the amount administered and the hormone used forovulation induction in the superovulation of heifers. Theregimen used to cause superovulation consisted of one injectionof follicle stimulating hormone (FSH) subcutaneously daily forfive days, followed by ovulation induction on the sixth day andartificial insemination was performed on the sixth and seventhday. No fertilized ova were recovered from heifers which beganhormonal treatments on day 4 of the estrous cycle. Failure wasattributed to uterine infections caused by attempting to performartificial insemination in the luteal phase of the cycle. Thebest responses to superovulation induction occured whentreatment was initiated in the follicular phase of the estrous2cycle. Heifers treated on day 16 with FSH extracted from 30 or40g of sheep pituitaries had better responses than those treatedwith 20g.In a review, Foote et al. (1970) noted that many regimes ofhormone treatment had been tried to induce superovulation, themost successful being the use of PMSG or porcine FSH. Betterovulation rates were observed if the superovulation treatmentwas conducted in the absence of an active corpus luteum (CL).The best method to avoid an active CL was to beginsuperovulation treatment on day 15-16 of the estrous cycleto ormanually or surgically remove the CL. Avery et al. (1962a) usedpurified porcine FSH and luteinizing hormone (LH) to stimulatesuperovulation in calves, cows and pregnant animals and was ableto induce ovulations in 61 of 75 animals treated. For cows, thedosage used was 20mg FSH and 5mg LH subcutaneously on the firstday, 10mg FSH and 5mg LH on the second day, 10mg FSH and 5mg LHon the third and fourth days, and 100mg LH i.v. on the fifthday. Twenty-eight of thirty-two cows ovulated in response totreatment. The use of progestogens to synchronize the cycle orremoval of the CL caused better estrus responses. Hafez et al.(1963) used 3000i.u. PMSG followed by 2000i.u. hCG 5-6 dayslater to superovulate cows. Estradio1-1713 (E 2) given after PMSGincreased the number of follicles ovulating. The protocol usedfor superovulation consisted of 3000i.u. PMSG on day 16 of thecycle, 20mg E2 on day 19 and 20 and 2000i.u. human chorionicgonadotrophin (hCG) on day 21. Hafez et al. (1963) also foundthat performing several inseminations ensured better3fertilization rates. Scanlon et al. (1968) superovulated cowswith 3000i.u. PMSG on day 16 followed by 2000 i.u. hCG at estrusor 5 days after PMSG if estrus was not seen. Of eighty-ninecows treated, 88 were successfully superovulated, however only79/89 had shown estrus. When the reproductive tracts wereflushed at slaughter, 74% of the cows treated produced more thanone egg, and the eggs recovered represented 70% of the corporalutea. From the 79 cows which showed estrus, an average of 4.4fertile eggs were recovered. Foote et al. (1970) noted that forgood responses to superovulation, FSH had to be given once ortwice a day for five days while PMSG could be given in oneinjection.Early work in attempting to increase the efficiency of beefproduction by hormonal induction of twinning increased knowledgeabout superovulation. Bellows et al. (1968) attempted toproduce multiple births in progestogen- synchronized beef cattleusing several regimes of hormone treatment. The treatmentsconsisted of 75mg FSH in one injection; twice daily injectionsat constant doses for a total of 6.25-75mg FSH; or a decreasingdose schedule twice daily for a total of 50mg FSH. Laparotomieswere performed to count the number of ovulations. Fifty mg FSHproduced an average of 17.8 CL at a constant dose and 23 CL whenadministered at a declining dose. Injected twice daily, FSHproduced a greater response than the same amount of FSH given inone injection. Total dosages of 25-75mg FSH produced similareffects, although there was a large amount of variability ateach dose. Twenty-five mg FSH produced an average of 13.0 CL4with a range of 1-30 CL. A total dosage of 6.25mg FSH producedan average of 2.1 CL with good fertilization rates of eggs, andin this experiment was the best dosage for induction oftwinning. Laster et al. (1970) induced twinning using 2000i.u.PMSG on day 5 and 1500i.u. on day 17 of the estrous cycle andfound that 80% of the synchronized heifers treated had 1-3 CLbut 13% had over 6 CL. Laster et al. (1973) compared PMSG andFSH in the induction of twinning in heifers. The number of CLwas higher and more variable with FSH, ranging from 1-19, andthe average was 4.4 \u00B1 4.9 CL vs. 1.6 \u00B1 1.0 CL after PMSGtreatment. Many of the heifers aborted, which was probably dueto carrying excessive fetuses.Researchers have noted that there is a lot of variation insuperovulation response to a given amount of hormone, and thusmany attempts have been made to investigate the causes ofvariation in response. Elsden et al. (1974) suggested that thehighly variable responses were likely related to the difficultyin selecting the time of administering PMSG in relation to thenext predicted estrus. Rowson et al. (1972) were among thefirst to use prostaglandin in combination with superovulationtreatment. Prostaglandin F2 (0.5mg) could be administered on 2consecutive days into the uterine horn adjacent to the corpusluteum on day 5-16 of the estrous cycle and would induce estrususually by the morning of the third day. Rowson et al. (1972)found this to be an efficient method of synchronization ofestrus and resulted in normal fertility.Elsden et al. (1974) administered 1500-2000 i.u. PMSG in5the mid-luteal phase of the cycle, followed 48h later by 1mgdoses of prostaglandin Fla (PGFu ) for two consecutive days intothe uterine lumen ipsilateral to the CL. Eighteen oftwenty-four cows exhibited heat within five days of PMSGtreatment, and all 24 ovulated to produce an average of 13.2 \u00B11.9 CL. Of 35 cows which were given 2000i.u. PMSG alone on day16 of the cycle, only 24 had responded to treatment and only17/24 had ovulated for an average of 8.0 \u00B1 1.5 CL. Use of PGF uto synchronize estrus in superovulation protocols, increased thenumber of cows which ovulated, increased the number ofovulations per cow and resulted in good fertility.Prostaglandin Fu is now an integral part of superovulation andestrus synchronization regimes in cattle.II. History of Embryo Collection and Embryo TransferTechniques The first successful embryo transfer was reported inrabbits in 1890 (Heape, 1890). After that time few successeswere reported until almost the 1950's. In 1949, Warwick andBerry reported experiments done using embryo transfer in sheepand goats. Goat embryos surgically transferred to the sheeputerus did not survive past 22 days, although sheep embryossurvived for at least 45 days in the goat uterus but nonesurvived to term. Some sheep-sheep and goat-goat transfers didsurvive to produce live young. Both saline and Tyrode's mediumwere used for transfers with limited successes, then the use ofaqueous humour from the eye discovered to be a better transfermedium.6Chang (1950) reported the importance of synchrony betweenthe stage of the embryo and stage of the recipient's estrouscycle. One day old rabbit embryos could only be successfullytransferred to oviducts. However, two day old rabbit embryoscould be transferred either to a two day post-estrus uterus ortwo day oviducts, but not a one day uterus. Four day oldembryos could only be successfully transferred to the uterus,but could tolerate up to two days of asynchrony (i.e. two to sixday post-estrus uterus). Chang (1950) used homologous serumalone or a mixture of 1:1 serum and saline for embryocollections and transfers. Similar requirements for synchronybetween the stage of embryo and recipient uterus was later notedin cattle. Newcomb et al. (1975) found that few pregnancieswere achieved after transfer of two or three day old bovineembryos into the uterus, but four day embryos could tolerate atleast \u00B11 day asynchrony when transferred into the uterus. Thenecessity for synchrony was believed to be an embryo requirementfor a progesterone-primed uterus and for appropriate signallingbetween uterus and embryos to initiate pregnancy.The first successful embryo transfer (E.T.) in cattle wasreported in 1951 (Willett et al., 1951). The estrous cycles ofdonors and recipients were synchronized with progestogens.Embryos were recovered at slaughter from superovulated donorsand were flushed from the reproductive tracts using homologousblood serum. Embryos were surgically transferred in serum to arecipient through mid-ventral laparotomy and placed into theuterine horn using a glass micropipette. Two more calves were7born using the same method in 1953 (Willett et al., 1953).Most of the early efforts to collect embryos for embryotransfer were done by flushing the reproductive tract afterslaughter of the donor cow, although some embryo recoveries wereperformed surgically. Several attempts were then made to designnon-surgical methods of embryo collection to lower costs; toincrease the repeatability of the procedure, because multiplesurgeries caused adhesions of the reproductive tract; to reducethe time required to complete the collection, and to reduce theperiod of recovery for the animals. Dracy and Petersen (1950)used two methods to attempt to collect fertilized embryos. Inthe first method, a rubber catheter was passed through thecervix through the uterine horn to the utero-tubal junction, toattempt to catch the egg as it passed from the oviduct into theuterus. This method was unsuccessful because the uterusunderwent violent contractions to force the catheter out and atleast one egg was able to bypass the catheter and causepregnancy. The second method, used to collect embryos from theuterus seven days after estrus, was more successful. A probewas used to dilate the cervix and then a steel cannula waspassed into the uterus and directed to either uterine horn. Abicycle pump was used to flush one litre of warm physiologicalsaline into the uterus. The returning fluid was collected andallowed to settle in separation funnels before eggs werelocated. In 37 flushes, embryos were recovered 12 times. Dziukand Petersen (1954) attempted non-surgical embryo collection andembryo transfer. A self-retaining catheter with 10 holes was8placed into the uterus and 200m1 of homologous serum was usedfor flushing.^In 13 attempts to collect embryos, fivecollections were successful.^Elsden et al. (1976) reportedsuccess using non-surgical embryo recovery.^The cow wasprepared by giving epidural anaesthesia, clearing the rectum offeces, tying the tail out of the way and washing the perinealarea. A cervical expander was placed into the cervix then aFoley catheter with large holes, stiffened with a metalstylette, could be guided into the uterus. The balloon on theFoley catheter was inflated with 20-30m1 air and the styletteremoved. Eight hundred millilitres of phosphate buffered saline(PBS) with 1% serum was used for flushing the uterus, and thefluid recovered was collected into 250m1 sedimentation funnels.Elsden et al. (1976) was able to collect an egg from 36/51unsuperovulated donors. Twenty-four of twenty-six attempts tocollect embryos from superovulated donors were successful.Although it is more difficult to locate eggs due to the largevolume of flushing medium, the non-surgical technique is moreefficient than surgical methods of embryo collection because itmay be used repeatedly, causes little damage and can allowsingle egg collections between superovulations. The non-surgical method of embryo collection is now used almostexclusively, with only a few modifications from the originalprocedure.There were also several attempts to design methods toachieve non-surgical embryo transfers with similar reasons asfor performing non-surgical recoveries. Avery et al. (1962b)9tried several methods of embryo transfer. Non-surgical embryotransfers were done by three methods: a) the use of anartificial insemination pipette to pass through the cervix onday 4, and air displacement of the embryo in a small volume offluid; b) the use of a steel cannula to pass through the cervixand then passage of a capillary tube through the steel cannulaand displacement of the embryo in a small volume of fluid; c)the use of a long needle to puncture the rectal wall then theuterine lumen. Tubing and a small needle were passed throughthe long needle and the embryo displaced using a 20mL syringe.No pregnancies resulted through any of these procedures.However, one pregnancy was achieved after four surgicaltransfers by laparotomy. Sugie et al. (1965) designed one ofthe first successful methods for nonsurgical embryo transfer.Three plastic tubes were designed which fit inside each other.The A tube was placed into the vagina and was directed adjacentto the cervix. The B tube, which had a long hypodermic needle,was guided so that the needle punctured the vaginal wall andentered the uterine lumen. The C tube had a small diameterhypodermic needle which contained the embryos, the embryos weredisplaced by slight pressure on the bulb. Carbon dioxide gaswas then passed into the uterus to stop uterine contractions.Two pregnancies and one live calf resulted from this method ofnon-surgical transfer. The use of a similar apparatus in fivegoats produced four pregnancies. The advantage of Sugie'smethod of non-surgical transfer was that it avoided passingthrough the cervix. Rowson et al. (1966) reported that the twomain causes for failure to establish pregnancy after nonsurgical10embryo transfer were the high rates of inducing uterineinfections during manipulations and by expulsion of thetransferred eggs via the cervix due to violent contractions ofthe uterus. Inflation of the uterus with CO2 allowed betterretention of transferred eggs. Successful non-surgicaltransfers were achieved by passing a sterile speculum throughthe cervix, followed by deposition of 2-3 eggs in 0.5mL serumusing an artificial insemination pipette. The uterus was thendistended with CO 2 gas. In six transfers with more than two daysasynchrony, no pregnancies resulted. However, when synchronywas \u00B11 day, 3 pregnancies from 8 transfers were achieved.Rowson et al. (1969) reported that the method of collection andtransfer, medium used for transfer and synchrony were importantin establishing pregnancies through embryo transfer. Of 33 eggstransferred in serum, half surgically and half non-surgically,no pregnancies resulted. When embryos were transferred intissue culture medium (TCM) using surgical embryo transfer, morepregnancies resulted from embryos collected surgically than fromembryos collected after slaughter of the donor. A pregnancyrate of 20% was achieved after non-surgical embryo transfer.Most embryo transfer procedures are now done nonsurgicaaly.Non-surgical embryo transfers are currently performed using anembryo transfer pipette which is much the same as an artificialinsemination pipette, except that it is longer so that embryosmay be placed deeper into the uterus. Similar procedures areused to prepare recipients as for donors at embryo collection.An epidural block is now used to anaesthetize the uterus instead11of inflation with CO2 gas. The tail is tied out of the way andthe vulva washed. Then the E.T. pipette is passed through thecervix into the uterine horn ipsilateral to the corpus luteumand the plunger depressed to release the embryos. Over theyears, pregnancy rates from non-surgical transfer have improvedto 50-60% (Picard et al., 1985).III. Variability of response to superovulation inductionSuperovulation and embryo transfer are used widely forresearch and as a method to increase the number of progeny fromsuperior females. Despite advances in the last twenty years indeveloping better drugs for superovulation induction, techniquesfor non-surgical embryo collection and transfer; superovulationis still hampered by the unpredictability of superovulationresponse. After discovery and widespread use of prostaglandinF2, or its analogues to control timing of estrus in superovulatedcattle; the biggest problem which remained in inducingsuperovulation was the high variability in reponse to treatment.Donaldson (1984) reported in over 1200 superovulation attemptsthat one third of the donors treated produced no transferrableembryos.Response to superovulation depends on many factors, bothextrinsic and intrinsic to the animal. Response tosuperovulation is known to depend on the amount of drugadministered (Bellows et al., 1969); the type of drug used,response to FSH is generally higher than PMSG (Monniaux et al.,1983); the purity of the drug preparation, as high amounts of LH12contamination in FSH preparations decrease superovulationresponse (Murphy et al., 1984); schedule of administering thedrug, giving FSH 2 or 3x daily produces better responses thanonce daily injections (Chupin and Procureur, 1982); and givingFSH at a decreasing dose produces better responses than aconstant dose (Bellows et al., 1969; Chupin and Procureur,1982).Intrinsic factors also influence superovulation response.Romero et al. (1991) found that the number of small follicles onthe ovaries before treatment affected the number of folliclesgenerated after hormonal induction of superovulation. Monniauxet al. (1983) noted that one factor which affects superovulationsuccess is the ovarian status at the time of initiation oftreatment. Several researchers have attempted superovulationinduction at different times of the cycle. Philippo and Rowson(1975) adminstered PMSG, in combination with PGF 2a , at fourdifferent times of the cycle and found the best responses whensuperovulation was initiated between Days 8-12. Sreenan andGosling (1977) and Lindsell et al. (1985) and also found betterresults when superovulation was initiated on Days 8-12 of theestrous cycle.Much research has been conducted into studying folliculargrowth and atresia in cattle. Moor et al. (1984) reported thatmost of the small follicles present in the ovary are atretic.Monniaux et al. (1983) reported that superovulation does notincrease the number of small follicles present in the ovary, but13instead rescues follicles normally destined for atresia.Rajakoski (1960) studied follicular populations in heiferovaries recovered at slaughter from animals at known stages ofthe estrous cycle and concluded that follicular growth occurs inwaves. However, early work on follicular dynamics was hamperedby lack of a method to repeatedly survey follicular developmentin individual animals and thus relied on specimens recoveredafter slaughter or after laparotomy. Matton et al. (1981)performed laparotomies to mark the largest follicles present onthe ovaries with india ink. At slaughter a few days later, thefate of the marked follicles was determined. It was concludedthat there were several periods of follicle turnover during theestrous cycle and that early in the estrous cycle there waslittle or no turnover of the largest follicle. Ireland andRoche (1983) examined ovaries collected at slaughter at knowndays of the cycle and classified follicles > 6mm histologicallyand endocrinologically as healthy or atretic. It was found thatthere were several periods of follicular growth. On days 3-7 ofthe estrous cycle, all heifers had one large non-atreticfollicle in a pair of ovaries, but by day 9-11 only one largeatretic follicle was found. After day 13 another large non-atretic follicle was found. Moor et al. (1984) reported thatmedium sized follicles are most common days 0-5 and 9-13 of thecycle, and this may explain why superovulation response is beston days 8 to 10 of the estrous cycle.Recently, ultrasonography has allowed monitoring cattleovaries on a frequent, often daily, basis. Pierson and Ginther14(1984) first reported monitoring follicular growth throughoutthe estrous cycle, and concluded that the numbers of folliclesin different size classes varied with day of the estrous cycle.Pierson and Ginther (1987) reported that there were two waves offollicular activity in heifers. Savio et al. (1988) and Siroisand Fortune (1988) reported that most heifers have three wavesof follicular growth; however most cows have two waves (Taylorand Rajamahendran, 1991). A follicular wave is characterized bythe appearance of a cohort of small follicles, followed by theselection of one follicle which becomes dominant and continuesto grow to preovulatory size while the rest of the cohortregress. The first dominant follicle can be detected on day 4,reaches maximum size on day 6 and remains stable between days 6-10. Then the first dominant follicle begins to regress and isnot detectable by day 15. A second dominant follicle isdetectable by day 12, reaches maximum size on day 16, and willovulate in a two wave cycle. In three wave cycles, the thirddominant follicle is detectable on day 16, grows to maximumdiameter on day 21 and ovulates (Savio et al., 1988).Ultrasonography has been used to monitor folliculardevelopment and corpus luteum formation during superovulationinduction (Pierson and Ginther, 1984; Goulding et al., 1990).Goulding et al. (1990) have postulated that the current optimaltime to initiate superovulation treatment is at mid-cycle, whenan active dominant follicle may not be present. However theexact timing this period may vary depending on whether the cyclewould have two or three waves of follicular growth. Recently,15the presence of a dominant follicle at the time of induction ofsuperovulation has been shown to decrease superovulatoryresponses (Guilbault et al., 1991; Huhtinen et al., 1992).Grasso et al. (1989) stated that the presence of a dominantfollicle at the initiation of hormone treatment will bothdecrease the number of large follicles stimulated by thetreatment and delay the appearance of these large follicles.However, Wilson et al. (1990) did not find any effect of adominant follicle on superovulatory response.B. Methods used for semen and embryo sexing to produceoffspring of the desired sexNormally the sex ratio at birth is approximately 1 male:lfemale. However, the ability to choose the sex of offspring hasbeen desired for many years both in the human population, andfor important livestock species. In livestock, females may bedesired for herd replacements, while in other situations, malesmay be preferred for more efficient meat production or for salecontracts with artificial insemination organizations. Inmammals, males are the heterogametic sex. When an X-chromosomebearing spermatozoa fertilizes the egg, a female offspring willresult, but when a Y-chromosome bearing sperm fertilizes, a malewill result. Therefore, the most efficient place to act inorder to produce embryos and pregnancies of the desired sex isto selectively increase the numbers or impair the function ofeither the X- or Y-chromosome bearing sperm. In practical andeconomic terms, semen would have to be enriched to produce >80%males or females (Pinkel et al., 1985). Many attempts have been16made to alter the sex ratio by selecting for the sex of spermprior to fertilization.I. Methods to separate X- and Y-spermatozoa Modififying DH of the female reproductive tract One of the earliest used methods to select for the sex ofthe fertilizing spermatozoa came from attempts to alter the pHof the female reproductive tract by using acidic or basicdouches. Increases in the numbers of females born have beenreported after acidic douches and increased males after basicdouches in rabbits (Roberts et al., 1940). Using similar pHprotocols, no sex ratio differences have also been reported inrats or rabbits (Cole et al., 1940).Density gradients The X-chromosome is one of the largest of the chromosomesbut the Y-chromosome is among the smallest chromosomes inmammals. Therefore sperm which carry the X-chromosome, may beheavier than sperm which carry a Y-chromosome. Sumner et al.(1976) found that sperm had two peaks of dry mass. BecauseY-sperm carry less DNA, it is believed that they may be moremotile (Goodall et al., 1976) or less dense, therefore therehave been many attempts to separate X- and Y-sperm based onthese two characteristics. The most commonly employed methodhas been the use of albumin gradients. The use of adiscontinuous gradient or several layers of bovine or humanserum albumin are believed to select for Y-sperm, as these spermshould be able to move faster and farther into the viscous17medium.^Many researchers have claimed success using thismethod. A commonly used method in which 2- or 3-layers ofalbumin are employed to separate spermatozoa, has been used inseveral clinics in the United States to select for Y sperm(Ericsson et al., 1973; Dmowski et al., 1979; Beernink et al.,1982) to increase male births. Other researchers demonstrate noseparation in X- or Y-chromosome bearing sperm (Evans et al.,1975; Ross et al., 1975). The use of bovine serum gradients toselect for sex in cattle has been attempted but with littlesuccess (Beal et al., 1984). Numerous other types of densitygradients have been used to try to separate X- and Y-spermthrough expected differences in swimming behavior or bycentrifugation in Percoll (Iwasaki et al., 1988; Upreti et al,1988), egg yolk (Schilling et al., 1966) or Sephadex (Quinlivanet al., 1982) but with no consistent effects on sex ratio. Anattempt was made to filter sperm through cervical mucus andclaimed to increase the proportion of male sperm but not enoughto enrich X- or Y-sperm populations (Broer et al., 1978).Surface chargeSeveral attempts were made in attempting to separate X- andY-sperm via electrophoresis or galvanization, on the basis ofpossible differences in surface charge. Some workers have foundincreased numbers of Y-sperm at the anode (Shishito et al.,1975; Bhattacharya et al., 1977) while others did not (Hagele etal., 1984). Many other experiments were designed to separatesperm through thermal convection or laminar flow throughsupposed differences in swimming behavior (Hagele et al., 1984;18Sarkar et al., 1984), also with no consistent success.H-Y antigenIn 1955, a male-specific antigen coded by the Y-chromosomewas discovered to be present on male mouse skin (Eichwald andSilmser, 1955). Since that time the antigen, H-Y antigen, hasbeen localized to many mammalian male tissues includingspermatozoa (Goldberg et al., 1971). It has been believed thatbecause male determining sperm have the Y-chromosome whichencodes the H-Y antigen gene, that more H-Y antigen may bepresent on Y-sperm. There have been several attempts to alterthe sex ratio by combining antisera raised against H-Y antigenwith sperm prior to insemination with some reports of increasesin the number of females born (Bennett and Boyse, 1973; Zavos,1983). However others, (Hoppe and Koo, 1984) found nodifferences in sex ratio when sperm had been reacted withmonoclonal anti-H-Y antibodies and complement prior to use inin-vitro fertilization of mouse eggs. Some experiments havetested the effect of maternal H-Y immunization in vivo.Immunized females have been bred and produced increases infemales (Pechan, 1985; Singh and Verma, 1988), no change in sexratio (McLaren, 1962; Rao et al., 1981) or increases in males ifthe immunized female had been previously splenectomized (Lappeand Schalk, 1971; Shalev et al., 1980). It therefore seemsunlikely that H-Y antigen is expressed on Y-spermpreferentially. In fact, H-Y antigen expression on sperm hasbeen found to decrease as the sperm ages (Hoppe and Koo, 1984),therefore maturational state of sperm could explain differences19in H-Y antigen expression.Evaluation of X- and Y-chromosome bearing sperm separationSome of the difficulties in measuring whether there havebeen significant shifts in the proportions of X- and Y-sperm,lie with the methods used to measure separation. Differencesare known to exist between X- and Y-spermatozoa in mammals.Pearson et al. (1970) discovered that the human Y chromosomestains very brightly with the fluorescent dye quinacrinemustard. This bright spot is labelled the fluorescent orF-body. Approximately 50% sperm carry the F-body (Barlow andVosa, 1970) and thus the presence of an F-body has been widelyused to quantify the number of sperm with Y-chromosomes in humansperm sexing regimens. However, researchers report that oftenless than 50% sperm exhibit F-bodies, and also F-bodies are notfound in all species (Hagele et al., 1984; Ogawa et al., 1988).Counting the number of sperm with F-bodies may be rathersubjective. Also other chromosomes such as human chromosome 3and the D group chromosomes (Pearson 1970) also show areas ofintense fluorescent staining which may be confused with the F-body.Zona-free Hamster Oocyte assayRudak et al. (1978) developed the zona-free hamster oocyteassay to be able to visualize sperm chromosomes. Hamsteroocytes, after removal of the zona pellucida, can be penetratedby sperm of other species. After several hours of culture, thesperm head will decondense and the chromosomal complement of the20sperm can be analyzed. Tateno et al. (1987) used zona-freehamster oocytes to study the proportions of X- and Y- bull spermafter attempts at sperm separation. They found that 73-92%hamster oocytes were penetrated, with about 42% of the eggshaving analyzable sperm chromosome complements. Due to the highincidence in polyspermy, for 100 eggs there were 56 analyzablesperm complements.Flow CvtometryMammalian sperm differ in the amount of DNA they contain,because there is a large difference in X- and Y-chromosomalsize. Therefore it may be possible to separate sperm on thebasis of the amount of DNA. Some fluorescent dyes are known tobind stoichiometrically to DNA. The use of a fluorescence-activated flow cytometer to measure the amount of DNA in sperminvolved several modifications. The flow cytometer requiredmodifications in order to control the introduction of sperm sothat orthogonal flow was achieved, to reduce the refraction dueto sperm head shape and orientation (Van Dilla et al., 1977).Unfortunately, to achieve good separation of peaks with high(more DNA, X-sperm) and lower fluorescence (less DNA, Y sperm),which only differ by 3-4% in total DNA, several modifications ofthe sperm were necessary. Sperm have been treated with DMSO,ethanol, papain and dithioerythritol to allow stoichiometricfluorescent DNA stain uptake as well as removing sperm tails toallow better orientation (Otto et al., 1979; Garner et al.,1984). These treatments are quite harsh, and leave spermlargely nonviable. These sorted spermatozoa may be used if21microinjected into oocytes, although only a low proportion ofeggs will become activated (Johnson et al., 1988a). Morerecently, methods have been modified in using sonication and astain which is less damaging to the sperm (Johnson et al.,1987). Sperm which were sorted once and then resorted (due todifficulty in getting good orientations with intact sperm withtails) produced X-sperm with 86% purity and 81% purity of Y-sperm (Johnson et al., 1989). These sperm were used forintrauterine insemination of rabbits and produced 94% femalesfrom the X-sorted sperm and 81% males from the Y-sortedspermatozoa. However, Johnson et al. (1989) state that the costof the flow cytometer is too high and the semen sorting speedtoo slow to produce large amounts of sexed semen for artificialinsemination. Morrell et al. (1988) report pregnancy rates tobe low using flow-sorted semen. However, flow cytometry may beused successfully to quickly produce counts on the percentagesof X- and Y-sperm in semen samples that have been separatedusing other sexing methods, thus could save the time and expensein performing fertility trials. Flow cytometry has been used tocount numbers of X- and Y- sperm from sperm separated byalbumin, laminar flow, Percoll, Sephadex, H-Y antibodies,magnetism and electrophoresis (Pinkel et al., 1985; Johnson etal., 1988b). In over 200 samples analyzed, no differences inX:Y ratio have been reported. Although flow cytometry can notmeasure any differences in fertilizing capacity of X- and Y-spermatozoa in sexed semen; no method of semen sexing tested hasreliably changed the relative proportions of X- and Y-sperm.22II. Methods to detect the sex of the fetus in early pregnancySome methods have been used successfully to determine thesex of the fetus during early pregnancy. However, determiningthe sex at this time may be quite an inefficient process becauseby this time the pregnancy is well underway and time and moneywill be lost in inducing abortion of fetuses of the wrong sexand trying to get the female pregnant again.Hormonal DeterminationOne of the methods used to determine the sex of the fetus,is by obtaining allantoic fluids to measure the concentration oftestosterone (Bongso et al., 1976). Testosterone levels inallantoic fluid of over 320pg/mL indicate the presence of a malefetus in cattle.KaryotypinqAnother method to sex the fetus, is by collecting amnioticfluid. Cells contained in the amniotic fluid must be cultureduntil there are enough to perform chromosomal analysis. Iffluid is collected at d70-90 of pregancy, results could take 2-3weeks (Hare et al., 1978).Use of UltrasoundUltrasonic evaluation of fetal sex, by attempting tovisualize scrotal swellings of the male, can be done in cattleafter day 57 of pregnancy. Muller et al. (1986) reported thismethod to be very accurate.23Use of DNA probes for the Y-chromosomeChorionic biopsy is a technique that has been applied tohumans and may be useful in sexing cattle pregnancies. DNAprobes have developed which are specific for the Y-chromosome ofman (Kunkel et al., 1976; Bishop et al., 1983; Handyside et al.,1989). Others have been developed for identification of the Y-chromosome in mice (Nishioka et al., 1986) and cattle (Leonardet al., 1987). Gosden et al. (1984) used the techniques ofchorionic biopsy and in situ Y-probe hybridization to sex humanfetuses and had the results within a few days.III. Methods of sexing preimplantation embryos prior to embryotransferDue to the facts that semen sexing is unreliable and sexingthe fetus during pregnancy is inefficient; recently more effortin obtaining pregnancies of the desired sex has focused ondetecting the sex of preimplantion embryos prior to embryotransfer. In 1987, 4% of the total U.S. cattle registrationswere born through embryo transfer techniques (Seidel, 1991).Therefore, there may be a relatively large market for sexingpre-implantation embryos. A catalogue from British ColumbiaArtificial Insemination Centre (1990) advertises frozen embryosfor sale at three hundred dollars each. The cost of embryos ishigh, therefore it may be well worth the money to ensure thatthe desired sex of calf will be born. Some commercial embryotransfer companies offer embryo sexing (Bondioli et al., 1989)as a service. There are two main categories of sexing embryos;noninvasive and invasive techniques.24Non-invasive techniquesEvaluation of cleavage rates Non-invasive methods of embryo sexing are less detrimentalto the embryo, usually easier to perform, but may be lessaccurate. One method to assess sex of embryos non-invasively isto examine the stages of embryos recovered at collection. Dueto the fact that males are heavier than females at birth andthereafter, it is believed that males have faster growth rates.Several different embryo stages may be recovered at the time ofembryo collection at Day 6-8 after estrus. Embryos at themorula stage have fewer cells than blastocysts. Some of thedifferences in the developmental stages found at embryo recoverymay be due to asynchronous ovulations and fertilization of theoocytes, although some differences in growth rate of the embryosmay be due to inherent differences in embryo cleavage rates.Avery et al. (1989) found that in the 1/3 of embryos whichdeveloped the fastest (most cells) 71% will be male, the middlethird will be 50% male and the slowest third will be 20% male.However, it was noted that this tendency is only significant ifthree different developmental stages are found in the embryoflush, yet only 24% embryo recoveries produce embryos at threedevelopmental stages.Measurement of X-linked enzymes Another method to sex preimplantion embryos non-invasivelyinvolves assessing X-chromosome linked enzyme activity. The X-chromosome is larger than the Y-chromosome and carries geneswhich are not found on the Y-chromosome. Therefore an early25embryo with two X-chromosomes (female) will be more likely toexpress those X-linked gene products than a male embryo withonly one X-chromosome. Assessing the metabolic activity of X-linked enzymes has been used to diagnose the sex of embryos.This usually involves a period of culture in a medium containingthe substrate for a particular X-linked enzyme, then theconversion rate of the substrate to a coloured or radiolabelledproduct can be measured. Williams (1976) assayed the activityof glucose-6-phosphate dehydrogenase in mice embryos. Thoseembryos scored as having two copies of the X-linked enzymeproduced 72% females, while those with scored as having one copyproduced 57% males. Monk and Handyside (1988) compared X-linkedenzyme activity to an autosomal enzyme activity in mice embryosand then sexed the resulting fetuses at d15 of gestation.Fourteen of fifteen fetuses were correctly sexed. Somedifficulties exist in measuring X-linked enzyme activities: oneis that because females have two X-chromosomes and males onlyone X-chromosome, females would have excess production of X-linked genes. Nature has compensated for this by having one X-chromosome become inactivated in females. It is unclear at whatstage X-inactivation occurs during embryonic development andthus may affect the amount of X-linked genes being expressed.Also, Rieger et al. (1984) had previously tested X-linkedenzymes and stated that it was necessary to compare the X-linkedenzyme activity to autosomal enzyme activity to standardize forthe metabolic rate of individual embryos.26Detection of H-Y antigenH-Y antigen has been localized on 50% of mammalian embryosafter the 8-cell stage (Krco and Goldberg, 1976). There havebeen many attempts to detect H-Y antigen on male embryos as amethod of embryo sexing. The cytotoxic assay was developedfirst and involves culturing embryos with H-Y antisera (Krco andGoldberg, 1975) or monoclonal H-Y antibodies with embryos in thepresence of complement for several hours. Affected embryos havelysed cells or are retarded in development and are considered tobe males. When unaffected embryos have been karyotyped ortransferred to recipients, 80-90% are females (Epstein et al.,1980; White et al., 1983, 1984; Shelton et al., 1984). Howevercytotoxic assays destroy most of the male embryos, therefore thefluorescent immunoassay for detecting H-Y antigen on embryos hasbeen employed. Embryos are cultured first with anti-H-Yantibodies, washed and cultured in media containing a secondantibody (bound to a fluorescent dye) which can bind to theanti-H-Y antibody. Embryos are washed again and then evaluatedusing a fluorescent microscope. Fluorescent embryos areconsidered to be males. Seventy-eight to eighty-five percent ofthe fluorescent embryos have been karyotyped to be males and 83-97% of the nonfluorescent embryos have been karyotyped to befemale in cattle (White et al., 1984, 1987). Some difficultiesin sexing using the H-Y antibody are H-Y antigen is weak and incytotoxicity assays some males may not be affected, whereas inimmunofluorescent assays dead cells may take up the stain non-specifically. Piedrahita and Anderson (1985) report that thetitre of the H-Y antisera affects the accuracy of embryo sexing.27Invasive methodsDetection of the Barr BodyInvasive methods have the advantage of being very accurate,although one or more cells must be removed from the embryo toperform sexing and this may affect embryo viability. One of theearliest methods to diagnose embryonic sex was by the use ofBarr body identification. In females, one X-chromosome isinactivated in most cells so that excess X-gene expression doesnot occur, thus both males and females have one active X-chromosome. The inactivated X-chromosome has been detected asthe Barr body, a triangular or ovoid structure in contact withthe nuclear membrane in 5 3/4 day old rabbit embryos (Edwards andGarner, 1967). When karyotyped, 41/47 embryos were correctlysexed. However due to the nature of the chromatin, it may notbe possible to detect Barr bodies in other species (King, 1984).KaryotypingAnother method for embryo sexing is to directly visualizethe chromosomes to identify the sex chromosomes. Melander(1959) reported that the chromosomal complement in cattle was2n=60 with all autosomes acrocentric while the X-chromosome isa large metacentric and the Y-chromosome a small metacentric.McFeely (1960) developed a method of preparing karyotypes fromDay 10 pig embryos. Whole embryos were cultured for lh at 37Cin colcemid to arrest cells in metaphase, followed by culture inhypotonic solution of one part serum to five parts distilledwater. Cells were then fixed, agitated and centrifuged using afixative of three parts methanol to one part acetic acid in28siliconized glassware, cells were then dropped onto a cleangrease-free slide and dried before examination. Initiallyinvestigators karyotyped large numbers of cells excised fromtrophoblasts of expanded d12-18 bovine embryos. Hare et al.(1976) sexed two week old bovine embryos using this method andwas able to sex 20/34 embryos. However, the pregnancy rateafter transfer of excised embryos was only 37.5%.Due to the ease of collection, transfer and freezingtechniques, most embryo transfer work is now done on d7-8embryos. Most of the methods used to karyotype zona-enclosedembryos have been developed from the air-drying method(Tarkowsky, 1966). The technique involves exposure of theembryos to colchicine for a few hours to arrest dividing cellsin metaphase when the chromosomes are contracted and easy toidentify. Embryos are then cultured in a hypotonic solution of1% sodium citrate to cause swelling of the cells and dispersalof chromosomes in the nucleus, followed by the placement of theembryo on a clean slide and addition of a few drops of 3:1ethanol-acetic acid to dissolve the zona pellucida and fix theembryo on the slide. The embryo is then stained. Similarmethods have been successfully applied to sexing bovine embryos.King et al. (1979) used a solution of 1:1 methanol-acetic acidto soften the zona pellucida prior to 3:1 methanol-acetic acidto fix the embryo with good results. With the low numbers ofcells available, it was important to obtain cells with goodquality metaphases; the culture time, concentration and timeexposed to colchicine was very important. Dyban et al. (1983)29modified Tarkowsky's air-drying method in the culturing embryoswith colcemid for 2h, the use of cold 0.9% sodium citrate for 20minutes to three hours, the use of 1:1 methanol-acetic acid asa zona softening solution and 3:1 methanol-acetic acid as afixative, followed by Giemsa staining, with good results.However, Murray et al. (1985) found that after 10h culture in0.8pg/mL colchicine only 40% of the embryos processed hadmetaphases and in only 25% was the sex analyzable. Picard etal. (1984) used one half of bisected embryos for sexing andafter 4h culture in 0.05pg/mL colcemid 60% embryos were sexable.Furthermore, the pregnancy rate from fresh bisected embryos was60%. Rall and Leibo (1987) obtained an average of 2.64analyzable metaphases per embryo with 62% sexable afterovernight culture of bisected embryos exposed to colcemid in thelast 2 hours of culture. Using the method of King (1984),whole or part embryos may be processed for sexing within 5-6hours.Although karyotyping is an easy technique to perform, itmay be often limited by the quantity and quality of metaphasesavailable for analysis.Use of probes for Y-chromosomal DNADNA probes have been developed which can specificallydetect the presence of Y-chromosomal DNA in man (Bishop et al.,1983), mice (Nishioka et al., 1986) and in cattle (Leonard etal., 1987). Leonard et al. (1987) removed 10-20 cells frombovine embryos and used a radiolabelled Y-probe for in-situ30hybridization. The results from sexing were obtained within 30hours. Bondioli et al. (1989) claimed that in order for aY-probe to be useful, the probe needs to be a) male-specific andb) detect repetitive DNA on the Y-chromosome, which isparticularly important when the number of cells assayed issmall. Biopsies of 10-20 cells have been used while theremainder of the d6-7 embryos can be frozen to await the resultsof the sexing assay and transferred when convenient. Afterbiopsy of Day 6-7 embryos, 90% were sexable at 97% accuracy;biopsied embryos gave a 40% pregnancy rate. Sexing bovineembryos using a Y-probe was also performed on a commercial basiswith 41% pregnancy rate and 100% accuracy. Polymerase chainreaction (PCR) is a technique that has been used to amplify DNAprobing (Saiki et al., 1985). Sexing embryos with Y probes inconjunction with PCR will take less time to obtain results andmay require as little as one cell to be removed from an embryo(Handyside et al., 1989).Aims and Objectives1) Two experiments were carried out in the course of theMaster's degree program to attempt to improve superovulatoryresponses and decrease response variability in dairy cattle.These two experiments are described in chapter 2 and are basedon reports that show the presence of a dominant follicle at thetime of initiation of superovulation decreases superovulatoryresponse (Guilbault et al., 1991; Huhtinen et al., 1992). Theexperiments were designed to study the response tosuperovulation, using ultrasound scanning, at times in the31estrous cycle when a dominant follicle would not be present.The first experiment (Chapter 2.1), studied the initiation ofsuperovulation at the beginning of the cycle, before a dominantfollicle is evident, in comparison to the traditional mid-lutealtreatment. The second experiment (Chapter 2.2) was based onreports that the dominant follicle present on day 7 of the cyclehas large numbers of hCG-receptors (Ireland and Roche, 1983) andcan be induced to ovulate following administration of 1000i.u.hCG (Rajamahendran and Sianangama, 1992). Superovulatoryresponses were compared between cows which had the dominantfollicle eliminated before superovulation induction and controlcows which were superovulated in the mid-luteal phase when adominant follicle may have been present. Both experiments werecarried out to determine whether initiation of superovulation inthe absence of a dominant follicle may improve superovulatoryresponses.2) In many livestock enterprises, offspring of one sex maybe preferred over another. In dairy cattle, females may bepreferred for herd replacements while males may have littlevalue except as veal calves. However, superior dairy cows mayhave large monetary contracts to produce males for artificialinsemination organizations. In beef cattle, males may bepreferred for their more efficient growth rates. When the costsof superovulation and embryo transfer are high, it may becomenecessary to ensure the desired sex of calf is born. In chapter3, three experiments on embryo sexing have been described.Chapter 3.1 discusses the development of H-Y antibodies, and32development of an ELISA to measure anti-H-Y antibody titre.Chapter 3.2 studies the effect of maternal anti-H-Y antibodyproduction on the in-vivo sex ratio. Chapter 3.3 provides adescription of bovine embryo karyotyping.33Chapter 2 Experiment OneFOLLICULAR GROWTH, OVULATION AND EMBRYO RECOVERY IN DAIRY COWSGIVEN FSH AT THE BEGINNING OR MIDDLE OF THE ESTROUS CYCLESummary The variability of the superovulation response is animportant problem to the embryo transfer industry. Theobjective of this study was to determine whether FSH treatmentat the beginning of the cycle would improve the ovulation rateand embryo yield in dairy cows. Twenty-eight cycling postpartumcows were allocated at random to four treatment groups: A, B, Cand D. Control cows (group A) received FSH (35mg) at adecreasing dose starting Day 9-13 of the estrous cycle (DOstanding estrus) for 5 days and PGFu (25mg) was given on thefourth day of FSH treatment. Cows assigned to treatments B,Cand D (n=6 cows, respectively) were given 35mg FSH at adecreasing dose from Days 2 to 6 and PGF2, on Day 7. Group C andD cows, in addition, received a progesterone releasingintravaginal device (PRID) from Day 3 to 7. Group D cows alsoreceived 1000 i.u. hCG 60h after PGF Ovaries were scannedever other day using a real-time ultrasound scanner from thebeginning of FSH treatment until the time of embryo recovery tomonitior follicular development, ovulations and number ofunovulated follicles. Embryos were recovered from the uterus bya non-surgical flushing technique seven days after breeding.There were no differences (p > 0.05) in the number of follicles>10mm 48h after PGF2, among treatment groups. The mean number offollicles were 10.6 \u00B1 1.2, 9.3 \u00B1 1.3, 12.2 \u00B1 1.3 and 15.0 \u00B1 2.9for groups A, B, C and D, respectively. Significantly (p <340.05) more ovulations were observed and embryos recovered ingroup A cows. Results of this study indicate that treatmentwith FSH at the beginning of the cycle caused sufficientfollicular development but resulted in a very poor ovulation andembryo recovery rates.IntroductionAn effective procedure for superovulation of cattle at thepresent time appears to be administration of gonadotrophin (FSH)during mid-cycle followed by administration of prostaglandin Fla(PGF2a) 48h later to induce luteal regression, estrus andovulation (Elsden et al., 1978; Monniaux et al., 1983).However, the variability in response in terms of quantity andquality of the ova produced with the above protocol remains oneof the limiting factors in the embryo transfer industry(Monniaux et al., 1983). Variability in response may be causedby extrinsic factors such as drug dosage (Bellows et al., 1969),method of administration (Chupin and Procureur, 1982), andpurity of the superovulatory homone preparations (Murphy et al.,1984) as well as intrinsic factors such as ovarian status at thetime of treatment (Monniaux et al., 1983). Many studies haveattempted to increase follicular development and ovulation inindividual cows and to reduce the variability of responsebetween cows but with little success (Goulding et al., 1991;Savio et al., 1991).Studies using ultrasound imaging in cattle have describedestrous cycles with two and three waves of follicular growth35(Pierson and Ginther, 1987; Savio et al., 1988; Sirois andFortune, 1988; Rajamahendran and Walton, 1988). A wave offollicular growth is characterized by the appearance of a poolof small follicles and the emergence of a single dominantfollicle while the remainder of its cohorts regress. A pool ofsmall follicles appears on the day after ovulation, in themiddle of the cycle (Days 11-12, Day 0 = day of estrus) and,depending on whether the cycle has two or three waves, at theend of the cycle (Days 15-17). The dominant follicle of thefirst wave in both two and three wave cycles is identifiable byDay 4, reaches a maximum diameter at Day 6, is static in sizeDay 6-10 and is not identifiable by Day 15. The dominantfollicle of the second wave is identifiable on Day 12 and willovulate in a two wave cycle while a third dominant follicle willovulate in a three wave cycle (Savio et al., 1988). Decreasesin ovulatory responses have been reported when superovulationhas been initiated in the presence of a dominant follicle(Guilbault et al., 1991).Based on this information, a logical time to beginsuperovulation treatment to avoid a dominant follicle to improveresponse and reduce variability may be at the beginning of thecycle. Priming with FSH at the beginning of the cycle andsubsequent superovulatory treatment at mid-cycle have been shownto improve response in some studies (Rajamahendran et al., 1987;Ware et al., 1988) but not in others (Rieger et al., 1988).Recently Goulding et al. (1990) reported decreasedsuperovulatory responses and embryo recovery when superovulation36treatments had been initiated at the beginning of the cycle.However, it was not clear from this study whether the decreasedresponse was due to reduced follicular development or inabilityof the induced follicles to ovulate. The objectives of thisstudy were to (a) monitor ovarian follicular development usingultrasound scanning following FSH treatment at the beginning ofthe cycle and (b) examine the effect of administering aprogesterone releasing intra-vaginal device (PRID) and/or humanchorionic gonadotrophin (hCG) along with FSH/PGF 2a at thebeginning of the cycle on ovulatory response and embryo recoveryin dairy cows.Materials and MethodsAnimalsThis study was conducted at the University of BritishColumbia Dairy Unit between September 1989 and March 1991.Holstein cows used in the experiment weighed 450-600kg and wereeither lactating or dry. Dry cows were housed in an open lotwith a shelter and fed hay. When used for the trial, they werehoused in tie-stalls and fed hay or 12kg alfalfa cubes daily.Lactating cows were cycling and at least 40 days postpartum atinitiation of superovulation treatment and were housed in freestalls with limited access to an exercise lot. In addition toalfalfa cubes and hay, they were fed a 16% protein textureddairy ration according to the level of production. Lactatingcows were milked twice daily at 0230 and 1430 h. Cows ranged inage from 2 to 9 years.37TreatmentTwenty-eight cows were assigned at random to fourtreatments A, B, C and D. Cows assigned to treatment A (controln=10) were superovulated starting at day 9-13 (Day 0=estrus) byinjecting intramuscularly (i.m.) with a total dose of 35mgporcine FSH (Folltropin, Vetrepharm Inc., Canada) over five daysat a decreasing dose of 5.25, 5.25, 4.375, 4.375, 3.5, 3.5,2.655, 2.655, and 1.75, 1.75mg (8:30a.m. and p.m. daily). Adose of 25mg prostaglandin F2( (Lutalyse, Upjohn, Canada) wasadministered i.m. at the time of the seventh injection of FSH.Cows assigned to treatments B (n=6), C (n=6) and D (n=6) weregiven FSH (35mg divided dose as in the control group) from Days2 to 6 and PGF2, (25mg) on Day 7. Treatment C and D cowsreceived an progesterone releasing intra-vaginal device (PRID,Ceva Ltd, USA) from Days 3 to 7. Cows in group D, in addition,received 1000i.u. human chorionic gonadotrophin (hCG) (A.P.L.,Ayerst Laboratories, Canada) about 60h after PGF 2a .Ultrasound ExaminationThe ovaries of the cows were scanned every other daycommencing on the first day of FSH treatment and continued untilthe day of embryo recovery, to monitor ovarian status at thecommencement of FSH treatment, follicular growth, ovulations andunovulated follicles. Ovaries were scanned as describedpreviously (Taylor and Rajamahendran, 1991) using a linear arrayultrasound scanner (Tokyo Keiki LS300, Tokyo Keiki Co. Ltd.,Japan) equipped with a 5MHz rectal probe. Images of the desiredstructures could be frozen on the screen and hardcopies could be38made with the printer unit.Estrus Detection, Breeding and Embryo RecoveryCows were observed twice daily after PGF 2, for signs ofestrus with the help of Kamar heat detectors (Kamar Inc,Steamboat Springs, Colorado). Cows were inseminated twice,roughly 12 and 24 hours afer standing estrus. Thawed semenobtained from the British Columbia Artificial InseminationCentre was used for breeding. Cows with at least one corpusluteum (CL) were flushed seven days after breeding using a non-surgical procedure described earlier (Rajamahendran et al.,1987). The flushings were collected into a sterile collectingbottle, siphoned and filtered to remove excess media. Embryoswere located using a dissecting microscope and quality wasevaluated using a system described by Reynard and Heyman (1979).Statistical AnalysesVarious parameters measured such as follicular number 48hafter PGF2a injection, number of ovulations, number of unovulatedfollicles, number of ova and transferrable embryos recoveredwere analysed using the SAS GLM procedure and least square meansusing Tukey's, Duncan's and SNK tests, to determine treatmentdifferences (SAS, 1987).Results Typical ultrasonographic pictures of FSH-stimulatedfollicles 48h post PGF 2, treatment, corpora lutea and unovulatedfollicles observed in cows treated at the beginning of the cycle39and at the middle of the cycle are shown in Plate 2.1.1 and2.1.2, respectively. Plate 2.1.1a demonstrates the left (L) andright (R) ovaries of a cow prior to initiation of FSH treatmenton Day 2 of the estrous cycle. Small follicles are indicated byarrows. Plate 2.1.1b shows follicular development after FSHtreatment. Regressing unovulated follicles, several days afterPGF2a injection are indicated by arrows in Plate 2.1.1c. Plate2.1.2 shows ovarian responses in a cow superovulated at mid-cycle. An arrow indicates the corpus luteum on the left ovaryand a dominant follicle on the right ovary in Plate 2.1.2a.Numerous follicles stimulated by FSH treatment are shown inPlate 2.1.2b. Plate 2.1.2c demonstrates multiple corpora luteain both ovaries at the time of embryo collection. An unovulatedfollicle is marked by an arrow.The mean number of follicles >10mm 48h post PGFu , corporalutea and unovulated follicles at the time of embryo recoveryare shown in Figure 2.1. There were no differences (p > 0.05)in the number of follicles >10mm among treatment groups.However, there was a tendency for increased folliculardevelopment in cows which received exogenous progesterone,groups C and D. The mean (\u00B1 s.e.) number of follicles were 10.6\u00B1 1.2, 9.3 \u00B1 1.3, 12.2 \u00B1 1.3 and 15.0 \u00B1 2.9 for groups A, B, C,and D, respectively. Significantly more corpora lutea (p <0.005) were observed in group A cows (8.7 \u00B1 1.1) compared tocows superovulated at day 2 (0.3 \u00B1 0.3, 0.0 \u00B1 0.0, and 2.5 \u00B1 2.2for groups B, C and D, respectively). The number of unovulatedfollicles at the expected time of embryo collection was40Plate 2.1.1 Follicles, corpora lutea and unovulated folliclesin a cow superovulated at day 2 of the estrouscycle.LPlate 2.1.2 Follicles, corpora lutea and unovulated folliclesin a cow superovulated at mid-cycle.a^LCb15 -N^1m 10 -er5 -020 -I S.E.folCLunov folb^bA B C D^ABCD ABCDdd daFigure 2.1 The mean number (\u00B1 s.e.) follicles >10mm,corpora lutea and unovulated folliclesin cows treated with FSH/PGF2a in the middle (A) orat the beginning of the estrous cycle (B, C, D).Treatment C and D cows received a PRID during FSHtreatment. D cows in addition received 1000i.u. hCG60h after PGFac Values with different superscriptsdiffer (a,b: p < 0.001; c,d p < 0.01).Treatment43Table 2.1 Estrus, ovulation and embryo recovery followingsuperovulation at the beginning or middle of theestrous cycle in dairy cows.Treatment No. of cowsexhibitingstandingestrusNo. of cowswith >2ovulationsMean^(\u00B1 s.e.)no. oftransferrableembryosGroup A:FSH, Days 9-13^PGF2,, 10 9 4.30 \u00B1^0.66 aDay 12 n=10Group B:FSH, Days 2-6^PGF2,,, 1 1 0.17 \u00B1 0.85 bDay 7n=6Group C:FSH, Days 2-6^PRID, 0 0 0.00bDays 3-7PGF2a/ Day 7n=6Group D:FSH, Days 2-6^PRID,Days 3-7 1 1 1.00 \u00B1 0.85 bPGF2a , Day 7hCG, Day 9n=6a,b p < 0.0544Values with different superscripts are significantly differentsignificantly lower in group A cows (p < 0.01) than groups B, C,and D.The incidence of standing estrus, numbers of cows with morethan two ovulations and the mean numbers of transferrableembryos are shown in Table 2.1. Estrus response was very poorin cows superovulated in the beginning of the cycle, whereasall ten of the cows superovulated in the mid-luteal phaseexhibited standing estrus about 48h post PGF 2a injection. Nineof ten cows in group A possessed more than two CL andsignificantly more transferrable embryos were recovered fromthis group (p < 0.05).DiscussionA PRID was given to cows in groups C and D to maintainearly luteal phase progesterone levels (Rajamahendran et al.,1981) and to prevent premature ovulation of FSH-stimulatedfollicles in the absence of a functional CL at the beginning ofthe cycle. PRID treatment did not improve ovulation or embryorecovery rates. Some evidence exists in the literature thatprogesterone levels at the time of initiation affects thesuccess of superovulation treatments (Yadav et al., 1986; Gotoet al., 1987), although other researchers have found nosignificant relationships (Sreenan and Gosling, 1977; Tambouraet al., 1985). It is unlikely that a single PRID could have hadany detrimental effects on stimulated follicles as treatmentwith a PRID or norgestomet have been used successfully in45superovulation regimens (Kunkel et al., 1979).The presence of a dominant follicle at the time ofinitiation of superovulation treatment has been shown todecrease superovulatory response (Guilbault et al., 1991). Ithas been hypothesized that adminstration of gonadotrophins atthe beginning of the estrous cycle, when there is no dominantfollicle to exert its inhibitory effect, would result inincreased superovulatory responses and embryo recoverycompared to mid-cycle superovulated cows. However, theresults observed clearly demonstrate that the ovulatoryresponse and embryo recovery were significantly lower in cowstreated at the beginning of the cycle, compared to the mid-luteal phase treated cows. This observation is in agreementwith other reports (Sreenan and Gosling, 1977; Lindsell etal., 1985; Goulding et al., 1990) although very few cowstreated at the beginning of the study exhibited estrus,ovulated and produced embryos in this study.Poor superovulatory response and embryo yield in cowstreated at the beginning of the cycle was not due to poorfollicular devlopment, as the number of ovulatory-sizedfollicles present 48h after PGF 2, injection, measured usingultrasound, were not different between early or mid-cycletreated cows. Superovulation treatment at the beginning of thecycle, in the absence of a dominant follicle was expected toincrease the numbers of follicles generated. However, recentreports indicating high levels of inhibin in small follicles at46the beginning of the cycle (Goulding et al., 1991b) could partlyexplain the follicular response observed.Therefore, the poor superovulatory response and embryoyield observed in cows treated at the beginning of the cyclecould be attributable to ovulatory failure, as significantlyhigher numbers of unovulated follicles were present at thenormal time of embryo collection in these groups. Inability ofthe CL to respond to PGFu , failure of the follicles to secretesufficient estrogen to cause the pre-ovulatory LH surge,inability of the follicles to respond to LH and atresia of thestimulated follicles are possible causes of failure to ovulate.Based on the ultrasound data, in the majority of cows treated atthe beginning of the cycle, the CL was maintained or underwentonly partial luteolysis after PGF 2a on Day 7. This implies thatFSH treatment and subsequent follicular development may havealtered the susceptibility of the CL to PGF u , as adminstrationof PGF2a on Day 7 of the normal estrous cycle causes completeluteolysis (King et al., 1982; Momont et al., 1984; Savio etal., 1990).Adminstration of hCG 60h after PGF2, also did not improveovulation or embryo recovery rates. This implies that largefollicles stimulated by FSH administration at the beginning ofthe cycle (a) did not have sufficient LH receptors, as inprevious experiments hCG given on Day 7 of the cycle causedovulation of large follicles (Rajamahendran and Sianangama,1992) or (b) were atretic, because large follicles present in47the early luteal phase of the cycle are able to ovulatefollowing PGF2a adminstration (Savio et al., 1990).Administration of PMSG on Day 2 or 3 of the sheep estrous cyclehas been shown to cause large atretic follicles to develop(Cran, 1983).In conclusion, the results of this study show thatsuperovulation with FSH at the beginning of the cycle causessufficient follicular development but results in very poorovulation and embryo recovery rates. Administration of a PRIDor hCG did not improve results obtained from early cyclesuperovulation attempts. Reduced sensitivity of the CL to PGF 2aand/or abnormal follicular development are possible causes ofovulatory failure in cows superovulated at the beginning of thecycle.48Chapter 2 Experiment TwoSUPEROVULATORY RESPONSES IN DAIRY COWS FOLLOWING OVULATION OFTHE DOMINANT FOLLICLE OF THE FIRST WAVESummaryThis experiment investigated using hCG on Day 7 of theestrous cycle (Day 0=Day of standing oestrus) to ovulate thedominant follicle prior to FSH treatment and the associatedincrease in progesterone as a method of improving superovulatoryresponse in dairy cows. Twenty cycling lactating cows wereallocated at random to two groups each of ten animals, controland hCG-treated. Ovaries of each cow were scanned usingultrasound equipment on Day 7 to confirm the presence of thedominant follicle and thereafter every other day until embryorecovery. All cows received a total dose of 400mg FSH indecreasing amounts for five days (Day 9-13) and 35mg PGF 2a on Day12. Treated cows, in addition, received 1000i.u. hCG on Day 7.Cows were inseminated twice during estrus and embryos wererecovered seven days later by a non-surgical procedure. Bloodsamples were taken several times during the treatment period forprogesterone determination. All cows on Day 7 possessed adominant follicle and all but one of the hCG-treated cowsovulated the dominant follicle and formed an accessory corpusluteum in response to hCG treatment. Plasma progesteroneconcentrations were not different (p > 0.05) between control andhCG-treated cows at Day 7. Plasma progesterone concentrationswere significantly higher (p < 0.01) on Day 9, the first day ofFSH treatment; and on Day 12, the day of PGF 2a (p < 0.005) inhCG-treated cows. Mean follicular number at estrus, ovulations,49total and transferrable embryos were not different (p > 0.05)between control and hCG-treated cows. However, there was atendency towards increased superovulatory responses and embryoyield in hCG-treated cows.IntroductionSuperovulation is a method of increasing the number of eggsovulated by the female. An effective procedure forsuperovulation in cattle at the present time appears to beadministration of follicle stimulating hormone (FSH) during mid-cycle followed by prostaglandin Fla 48h later to induceluteal regression, estrus and ovulation (Elsden et al., 1978;Monniaux et al., 1983). However, the variability in terms ofthe quality and quantity of embryos produced with the aboveprotocol, remains one of the major limiting factors in theembryo transfer industry (Monniaux et al., 1983). Variabilityin response may be caused by both extrinsic factors such as drugpurity (Murphy et al., 1984), drug dosage (Bellows et al., 1969)and drug administration (Chupin and Procureur, 1982); andintrinsic factors such as ovarian status at the time oftreatment (Monniaux et al., 1983).Studies using ultrasound imaging in heifers and cows havedescribed the estrous cycle as having two or three waves offollicular growth (Roche and Boland, 1991). However, in cows,two waves of follicular growth appear to be the norm (Taylor andRajamahendran, 1991). A wave is characterized by the appearanceof a pool of follicles and the emergence of a single dominant50follicle while the remainder of its cohort regress, probably dueto the inhibitory effects of substances produced by the dominantfollicle (Ireland and Roche, 1987; Ko et al., 1991). Thedominant follicle of the first wave of follicular growth, incycles with two or three waves, is identifiable by Day 3 (Day0=Day of estrus), reaches a maximum diameter on Days 6-7, is ina static phase from Day 6-10 and is not identifiable by Day 15(Savio et al., 1988). Recently, there have been reports statingthat the presence of a dominant follicle at the time ofinitiation of FSH treatment during mid-cycle reduces thesuperovulatory response (Guilbault et al., 1991; Huhtinen etal., 1992).Recently it has been reported that administration of1000i.u. human chorionic gonadotrophin (hCG) on Day 7 of theestrous cycle results in the ovulation of the dominant follicle,accessory corpus luteum formation and significant increase inplasma and milk progesterone concentrations (Rajamahendran andSianangama, 1992). Therefore, the objectives of this study werea) to examine the effects of eliminating of the dominantfollicle with hCG prior to FSH treatment and b) examining thehCG-induced increase in progesterone concentration on folliculardevelopment, ovulations and transferrable embryos recovered insuperovulated lactating dairy cows.Materials and Methods AnimalsTwenty lactating Holstein cows ranging from 2 to 11 years51of age, cycling and at least 40 days postpartum were selectedfor the study between May 1991 and January 1992 from theUniversity of British Columbia dairy herd. Cows received amixed ration of alfalfa cubes and a 16% protein dairy texturedgrain, according to milk production. As well cows were providedwith good quality alfalfa hay. Cows were milked twice dailybetween 0230 and 0500 h and 1430 and 1700 h. Cows were checkedfor signs of estrus at both milkings and for approximately 1/2hduring the evening.TreatmentCows were allocated at random to two treatment groups,control (n=10) and hCG-treated (n=10). Cows assigned to the hCGgroup recieved 1000i.u. hCG (A.P.L., Ayerst Laboratories,Montreal) on Day 7 of the cycle (Day 0=day of standing estrus),after confirming the presence of the dominant follicle of thefirst follicular wave with ultrasonographic examination. Bothcontrol and hCG-treated cows were superovulated on Days 9 to 13of the cycle by injecting intramuscularly (i.m.) 400mgFolltropin-V (NIH-FSH-P1, Vetrepharm Inc., Canada) at adecreasing dose of 60; 50; 40; 30 and 20mg b.i.d. at 0830 and2030 daily. A total of 25mg and 10mg PGF2a (Lutalyse, UpjohnCo., Canada) were administered with the seventh and eighth FSHinjections, respectively.Estrus detection, breeding and embryo recoveryCows were observed three times daily after PGF 2a for signsof estrus and standing estrus with the help of Kamar heat mount52detectors (Kamar Inc, Steamboat Springs, Colorado). Cows wereinseminated twice, about 12 and 24 h after standing estrus.Thawed semen was used for inseminations. Cows were flushed 7days after breeding by a non-surgical procedure describedearlier (Rajamahendran et al., 1987). The flushings werecollected into a sterilized collecting bottle, siphoned andfiltered to remove excess media. Embryos were located using adissecting microscope and quality was evaluated using a systemdescribed by Reynard and Heyman, 1979.Ultrasound examinationA real-time ultrasound scanning device (Tokyo Keiki LS300,Tokyo Keiki Co., Japan) equipped with a 5MHz rectal probe wasused to examine the ovaries as described previously (Taylor andRajamahendran, 1991). The ovaries of all cows were examined onDay 7 of the cycle and thereafter every other day until embryorecovery to monitor ovarian status at the initiation of FSHtreatment, follicular growth following FSH, corpus luteumregression following PGF2a and formation of multiple corporalutea following ovulation of the developed follicles. Desiredimages of the ovaries could be frozen on the screen,measurements taken using built-in calipers and hardcopies madeusing a video processing unit (Mitsubishi Electronics Co.,Japan).Blood sample collection and progesterone radioimmunoassayBlood was collected from a coccygeal blood vessel intoheparinized tubes from all cows on Day 7, the first day of FSH53treatment (Day 9), the day of PGF 2a (Day 12), at standing estrusfollowing PGF2, and the day of embryo collection. Plasma wasseparated and stored at -20C until progesterone analysis.Progesterone was measured using a commercially availablesolid-phase radioimmunoassay kit, Coat-A-Count (DiagnosticsCorp., Los Angeles, CA). Plasma or reference standard (0.1mL)was added to tubes coated with specific antibody. Standardscontained 0-40ng/mL progesterone. Buffered 131-labelledprogesterone (1.0mL) was added to all tubes; tubes were vortexedand incubated for 3h at room temperature. The tubes were thendecanted and cleaned with a cotton swab above the lmL mark toremove excess tracer. The tubes were counted for one minute ina gamma counter (Packard Auto-Gamma 500. Packard instruments,Downer's Grove, IL). The limit of detection of the assay is0.05ng/mL. Intra- and inter-assay coefficents of variation were7.9% and 10.3%. Counts were converted to values in ng/mL usingSAS (SAS, 1987). This procedure has been validated formeasuring progesterone in bovine plasma (Rajamahendran andTaylor, 1990).Statistical analysesMean numbers of follicles > 10mm at standing estrus,number of ovulations, total and transferrable embryos recoveredwere analyzed using the GLM proc and mean procedures from SAS(SAS, 1987) to determine differences between treatment groups.Least square means analysis was done using Duncan's test.Covariate analysis among treatment and progesteroneconcentrations and superovulatory parameters were performed54using SAS, 1987 using the following models:Yu=g + T i + D9 1 + D12 11 + En^Model 1Yn=p + T i + D9 1 + D12 n + Fn + En^Model 2Yn=11 + T i + D9 1 + D12 n + F 1 + CLn + E 1 ^Model 3Yn=p + T i + D9 11 + D12 n + Fn + CL 1 + coll 1 + E 1 ^Model 4T i is the effect of treatment, of which there were two, controland hCG. The covariates were D9 n and D12 n , the progesteronevalues of the ith treatment and the jth cow duringsuperovulatory treatment. F 1 , CLn and coll n were respectively,the number of follicles, corpora lutea and progesterone level atthe time of embryo collection in the ith treatment and jth cow.The value Yu = superovulatory parameter such as number ofcorpora lutea, progesterone level at embryo collection, total ortransferrable embryos, or % transferrable embryos.Results Plates 2.2.1 and 2.2.2 demonstrate the response tosuperovulation, measured by ultrasound, in a control and hCG-treated cow, respectively. Plate 2.2.1a, demonstrates the ovaryat the time of initiation of FSH treatment; the corpus luteum inthe left (L) ovary is indicated by a large arrow and a dominantfollicle is indicated by a smaller arrow in the right (R) ovary.A follicle stimulated by FSH is shown by an arrow in Plate2.2.1b. Ovarian responses to FSH treatment at embryocollection, in terms of a corpus luteum (large arrow) in theleft ovary and an unovulated follicle (small arrow) in the rightovary, are shown in a control cow in Plate 2.2.1c. Plate 2.2.2ademonstrates the presence of a spontaneous corpus luteum (large55L RL.EFT OVARY^ IGHT OVARV29X27 MM CL 2 MM FOL^23 MM FOLBLEFT .0\"--OUARY3 FOLLICLESEFT OVARY^ I6HT OVARY1^,HIAPlate 2.2.1^Superovulatory responses in a control cowsuperovulated in the mid-luteal phase of theestrous cycle.56BOTH LEFT OUPRY $OTN RIGHMM FOLSMALL FOLAB0TH RIGHT \u00B0WIRYPlate 2.2.2. Superovulatory responses in a cow treated withhCG on Day 7 of the estrous cycle to ovulate thedominant follicle prior to superovulation.57arrow) at Day 7 in the left ovary of an hCG-treated cow. Adominant follicle was located on the right ovary (small arrow).Plate 2.2.2b demonstrates the spontaneous corpus luteum (largearrow) in the left ovary, an hCG-induced corpus luteum in theright ovary (small arrow) and FSH-stimulated follicles(triangles) in both ovaries. Corpora lutea (large arrows) atthe time of embryo collection in an hCG-treated cow are shown inPlate 2.2.2c.All cows on Day 7 possessed a dominant follicle (12 to 20mmin diameter) and nine of ten hCG-treated cows ovulatedthe dominant follicle and formed an accessory corpus luteum. Inthe cow which failed to ovulate the dominant follicle followinghCG, the dominant follicle persisted until embryo recovery.Following superovulatory treatment with FSH, development ofmultiple follicles were observed in both control and hCG-treatedcows. The mean interval to standing estrus following PGF 2a wasnot different (p > 0.05) between control and hCG-treated cows(47 vs. 53 h). The mean number of follicles > 10mm at estrus;mean numbers of ovulations, total and transferrable embryosrecovered at embryo collection were not different (p > 0.05)between control and hCG-treated cows (Table 2.2.1). However,there was a tendency towards improved superovulatory responsesin hCG-treated cows.Mean (\u00B1 s.e.) plasma progesterone levels for control andhCG-treated cows are presented in Table 2.2.2. Plasmaprogesterone levels were not significantly different58Table 2.2.1 Mean (\u00B1 s.e.) numbers of follicles, corporalutea, total ova and transferrable embryosrecovered in control and hCG-treated cowsfollowing FSH/PGF2a treatment.TreatmentGroupFollicles( > 10mm)CorporaLuteaTotalOvaTransfer.EmbryosControl(n=10)10.1 \u00B1 1.9 a 8.7 \u00B1 2.0 a 5.0 \u00B1 2.1 a 3.1 \u00B1 1.4 ahCG(n=10)12.4 \u00B1 1.9 a 9.5 \u00B1 1.8 a 7.8 \u00B1^2.3 a 4.8 \u00B1^1.5aValues in columns with e same superscripts do not i er(p > 0.05).Table 2.2.2 Mean (\u00B1 s.e.) plasma progesterone concentration(ng/mL) in control and hCG-treated cows atdifferent times during superovulatory treatment.TreatmentGroupDay 7 Day 9Day 1 FSHDay 12At PGF2-Estrus EmbryoRecoveryControl(n=10) 2.6 \u00B1 0.2a 3.5 \u00B1 0.3b 4.0 \u00B1 0.4d 0.2 \u00B1 0.1 a 13.5 \u00B1 4.0ahCG(n=10) 2.6 \u00B1 0.3a 5.5 \u00B1 0.5c 6.1 \u00B1 0.5e 0.4 \u00B1 0.1 a 12.3 \u00B1 2.9aValues in columns with i eren superscripts differ.b,c p < 0.01d,e p < 0.00559(p > 0.05) in control and hCG-treated cows on Day 7, day ofestrus or at embryo collection.^However, significantdifferences in^progesterone concentrations were observedbetween hCG-treated cows and control cows on the first day ofFSH treatment and on the day of PGF 2a injection. Plasmaprogesterone levels (mean \u00B1s.e.) for control and hCG-treatedcows respectively, at the time of initiation of FSH treatmentwere 3.50 \u00B1 0.33 vs. 5.54 \u00B1 0.55 (p < 0.01) and at PGF 2a were4.04 \u00B1 0.45 vs. 6.13 \u00B1 0.46 (p < 0.005).DiscussionIt has been reported previously that the dominant folliclearising the a pool of follicles suppresses the growth of itscohort and possibily causes atresia (Ireland and Roche, 1987; Koet al., 1991). Presence of a dominant follicle at the time ofinitiation of superovulation treatment has beenreported to decrease superovulatory responses (Guilbault et al.,1991; Huhtinen et al., 1992) or to have no effect onsuperovulation (Wilson et al., 1990). In the present study,ovulation of the dominant follicle with hCG prior tosuperovulation treatment did not significantly improvefollicular development, ovulations or numbers of embryosrecovered. This implies that intrinsic factors other than thepresence of a dominant follicle may be responsible for thevariation in superovulatory response. However, possible effectsof hCG on small antral follicles should not be ignored, as thesemay have interfered with the beneficial effect of ovulating thedominant follicle prior to superovulation. If hCG does have60inhibitory effects on the small antral follicle populations inthe ovary, then elimination of the dominant follicle bycauterization or ultrasound-guided aspiration beforesuperovulatory treatment may improve the superovulatoryresponse.In this study, nine of ten cows treated with hCG on Day 7ovulated the dominant follicle and formed an accessory corpusluteum. This is in agreement with previous findings(Rajamahendran and Sianangama, 1992). Significant increases inplasma progesterone following hCG treatment observed in thisexperiment are in agreement with previous results (Eduvin andSequin, 1982; Breuel et al., 1990; Rajamahendran and Sianangama,1992). The increased progesterone levels could have resultedfrom hypertrophy of luteal cells in the spontaneous corpusluteum (Veenhuizen et al., 1972; Helmer and Britt, 1986) andfrom the accessory corpus luteum formation.In this experiment, in nine of ten cows treated with hCG toremove the dominant follicle prior to superovulation, both thespontaneous and the induced corpus luteum regressed followingadministration of a luteolytic dose of PGF 2, and the time fromPGF2, to standing estrus was not different between control andhCG-treated cows. This suggests that the hCG-induced corpusluteum is sensitive to PGF 2a as early as four days after it isformed. This finding is in contrast to Rusbridge and Webb(1991) where the interval from PGF2, to estrus was extended inheifers with hCG-induced corpora lutea compared to controls.61Howard and Britt (1990) examined responses of CL to PGF a 2, 4,or 6 days after estrus (spontaneous CL) and 2, 4, or 6 daysafter hCG given Day 10 of the estrous cycle (hCG-induced CL).Progesterone declined immediately after PGFa treatment on day 6for spontaneous CL or day 2-6 after induction of hCG-inducedovulation at mid-cycle. However, the time from PGF a injectionto estrus was extended in hCG-treated heifers.There are conflicting reports in the literature about therelationship between plasma progesterone at the initiation ofFSH treatment and superovulatory responses. Several studies,(Saumande et al., 1975; Sreenan and Gosling, 1977; Tamboura etal., 1985; Walton and Stubbings, 1986) failed to find anyrelationship between concentration of progesterone on the firstday of FSH treatment and superovulatory response. Others (Yadavet al., 1986; Goto et al., 1987) have reported that progesteronelevel on the first day of FSH treatment is related to embryoquality. Goto et al. (1988) found significant differences (p <0.01) in CL numbers, total and transferrable embryos betweencows which had under 3 ng/mL and over 3 ng/mL progesterone inplasma on the first day of superovulation treatment. In thisexperiment, progesterone concentrations in plasma were increasedon the first day of FSH treatment (p < 0.01) and on the day ofPGFa (p < 0.005) in hCG-treated cows compared to controls.Covariate analysis of the data showed no significantrelationships among treatment or progesterone values on thefirst day of FSH or at PGFa and the number of folliclesstimulated by superovulation, the number of ovulations, total or62transferrable embryos recovered and the percentage of embryosthat were transferrable (p > 0.05). It appears that the levelsuggested by Goto et al. (1987), 3 ng/ml progesterone in plasma,may be a permissive level for superovulation. Animals withunder 3 ng/ml tended to have poorer superovulatory responses.The number of follicles > 10mm at estrus, detected withultrasound, had a significant effect on the number of ovulations(p < 0.0001), progesterone level at embryo collection (p <0.005), and the numbers of total (p < 0.0001) and transferrableembryos recovered (p < 0.05). These high relationships areexpected because most follicles over 10mm in size will ovulateand release ova capable of being fertilized following the LHsurge. The number of follicles stimulated by FSH treatmentexplained most of the variation in superovulatory response. Forexample, the first model compared treatment and progesteroneconcentrations on the first day of FSH and day of PGF 2a andexplained only a small amount of variation in CL numbersr2=0.091718. The inclusion of follicle number in second modelexplained much more of the variation in response, r2=0.781896.Similarly, the second model increased the amount of variabilityexplained for progesterone values at collection, and total andtransferrable embryos recovered. However, the number offollicles generated by superovulation has little effect on theproportion of transferrable embryos (p > 0.05, r 2=0.128063). Asfollicle numbers increase, CL numbers and total andtransferrable embryos usually increase. The number of folliclesdeveloped limits the number of transferrable embryos obtainable63from a donor.Inclusion of CL numbers, as detected with ultrasound at thetime of embryo collection, into the third model explainedadditional variation in superovulatory responses. CL numbershad significant relationship with total embryos (p < 0.05) andprogesterone levels at embryo collection (p < 0.001). CLnumbers tended to have a relationship with transferrable embryosrecovered (p < 0.10). Numbers of CL also had a significantrelationship with the proportion of embryos recovered which weretransferrable (p < 0.05).Inclusion of the plasma progesterone values at embryocollection into the fourth model also helped to explain somevariability in reponse. Plasma progesterone concentration atthe time of embryo collection had a significant relationshipwith transferrable embryos (p < 0.05). However, plasmaprogesterone at embryo collection was not significantly relatedwith the proportion of transferrable embryos(p > 0.05).In conclusion, the results of this study demonstrated thatelimination of the dominant follicle with hCG prior tosuperovulation did not improve superovulatory responses ordecrease response variability. Progesterone levels wereincreased during FSH treatment in hCG-treated cows, however thisdid not have significant effects on superovulatory parameters.The number of follicles generated during superovulation64treatment was the best predictor of the subsequent ovulatory andcollection responses, however the number of follicles generatedduring treatment did not affect the quality of embryosrecovered. Follicles, CL numbers and progesterone levels atembryo collection can help predict the numbers of embryosrecoverable from a donor, and can influence decisions aboutwhether it is worthwhile to breed or attempt embryo collectionfrom a donor. However, it would be beneficial to have a markerto predict or increase response before superovulation isactually carried out. It has been postulated that bettersuperovulatory responses are obtained when superovulation isinitiated at mid-cycle when a dominant follicle may not bepresent (Goulding et al., 1990). Because estrous cycles mayhave two or three follicular waves (Roche and Boland, 1991), andthe timing of the window between periods of follicular dominancemay not be easily identifiable without the use of ultrasound; itwould be useful to investigate other methods to initiatesuperovulation in the absence of a dominant follicle. Althoughremoval of the dominant follicle with hCG prior tosuperovulation tended to increase the response; the experimentmay have been confounded by actions of hCG on small follicles inthe ovary.65Chapter 3 Experiment OneIMMUNIZATION OF FEMALE MICE AGAINST MALE SPLENIC CELLS ANDDETERMINATION OF ANTIBODY TITRE AGAINST H-Y ANTIGENSummaryC57BL\6 female mice were immunized against H-Y antigenthrough injections of thirty million male mouse splenic cellsand Freund's adjuvant weekly for six weeks. Blood was collectedfrom the retro-orbital sinus or tail vein and serum wascollected to determine H-Y antiserum titre. Two ELISAs weredeveloped to measure H-Y antisera titre (male spleen cell-basedand Daudi cell culture supernatant-based). However, largeamounts of non-specific antibody cross-reactivity were noted,such that it was not possible to accurately determine specificH-Y antibody titre.IntroductionBoth sperm (Goldberg et al., 1971) and male embryos (Krcoand Goldberg, 1976) are known to possess H-Y antigen. Manyexperiments have been designed in the past to attempt to changethe sex ratio of offspring to be born. H-Y antiserum, raised inmale spleen-immunized C57BL/6 female mice, has been used in manyexperiments and can be used to the kill male pre-implantationembryos in cytolytic assays (White et al., 1983). As well, H-Yantisera has been used to affect the sex ratio after artificialinsemination (Bennett and Boyse, 1973; Zavos, 1983) after invitro fertilization (Hoppe and Koo, 1984) and after breeding ofH-Y-immunized females (McLaren, 1962; Lappe and Schalk, 1971;Shalev et al., 1980; Rao et al., 1981); with inconsistent66effects on the sex ratio of litters.H-Y antibody titre has previously been assayed incytotoxicity assays (Goldberg et al., 1971) and in enzyme-linkedimmunosorbent assays (ELISA) (Brunner et al., 1984, 1988; Iyeret al., 1989).An experiment was designed to investigate immunization ofC57BL/6 female mice against male splenic cells to raise H-Yantisera and to develop an ELISA system to measure H-Y antiserumtitre.Materials and Methods Immunization Procedure and Sera CollectionVirgin C57BL/6 (B6) male and female mice were originallyobtained from Charles River. Thereafter, a colony of B6 micewas established. Six to eight week old females were immunizedat weekly intervals for six weeks against male B6 spleens.Males were euthanized with CO2 gas, abdomens opened using steriledissecting instruments and spleens removed aseptically andplaced into a 35mm petri dish in Dulbecco's phosphate bufferedsaline (PBS). The PBS was injected into several sites in eachspleen using a 1mL syringe and 26g needle, until the spleenturned pale and the PBS became quite red. Thus a single cellsuspension of splenocytes was made. Dislodged splenocytes werepooled and centrifuged at 1000g for 5 minutes. The cell pelletwas resuspended in PBS and an aliquot was counted on ahemocytometer. The suspension was then made up to contain 30067million splenocytes/mL in PBS.^Emulsions were made withcomplete Freund's adjuvant (CFA) in the first week ofimmunization and incomplete Freund's adjuvant (IFA) thereafter.A 1:1 concentration of splenocytes in PBS and adjuvant was madeand emulsification was accomplished using a sterile glasssyringe in a sterile glass beaker. Male-immunized females wereinjected with splenocyte/adjuvant mixture containingapproximately 30 million cells, subcutaneously in the abdomen infour sites, for a total volume of 0.2mL. Control immunizedfemales received 0.2mL 1:1 PBS and CFA in first week and PBS andIFA thereafter.Blood collection^Blood was^collected^from anaesthetized^(sodiumpentobarbitol i.p. or halothane inhalation) mice from theretro-orbital plexus or by cutting off a small piece of tailwith a scalpel blade and bleeding into heparinizedmicrocapillary tubes. Blood was placed into labelled 1.5mLmicrocentrifuge tubes and centrifuged at 1000g for 5 minutes.Sera was collected and placed into fresh microcentrifuge tubes.Serum samples were heat-inactivated at 56C in a water bath for30 minutes and then stored at -20C until assayed for anti-H-Ytitre. In addition, some control females were bled from thetail vein prior to adjuvant immunization and some controlfemales and males were euthanized by CO 2 gas and blood wascollected directly from the opened chest cavity.68Titre determinationDaudi Burkitt lymphoma is a cancer cell which lacks 13-microglobulin and thus can not bind H-Y antigen. Daudi cellculture contains a high concentration of free H-Y antigen(Beutler et al., 1978). Daudi Burkitt lymphoma (CCL 213) wasobtained from American Type Culture Collection, Maryland. Daudicells were cultured in RPMI containing 20% fetal calf serum in5% CO2 at 37C in tissue culture flasks under aseptic conditionsin Dr. Lee's Andrology laboratory, U.B.C. Hospital. Every otherday, culture supernatant was placed into sterile 50mL Falcontubes and centrifuged for 10 minutes. The supernatant wasdiscarded, media replaced and cell density checked. When largenumbers of cells were present, cells were washed and resuspendedin serum-free RPMI and cultured overnight in 5% CO 2 at 37C in24-well Nunc dishes. The supernatant was pooled and stored at-20C until used for an assay. An H-Y monoclonal antibodyhybridoma culture (HY3-11.27, HB8116) was also obtained fromATCC for use as a positive H-Y antibody control for ELISAs. Itwas grown in RPMI and 10% fetal calf serum under 5% CO2 in airat 37C, media was replaced every other day and supernatantcollected and stored at -20C until use.Development of ELISAs to measure anti-H-Y antigen titreSeveral preliminary experiments were carried out todetermine the proper concentrations of Daudi supernatant (H-Yantigen), monoclonal antibody and sera for use in developing anELISA. Daudi supernatant was diluted in carbonate- bicarbonatebuffer, pH 9.6 (1.59 g Na 2CO3 and 2.93g NaHCO3 in 1L distilled69water) at 1-1/20 dilutions, which was expected to produceapproximate concentrations of 0.5-1011g/mL protein per well. Onehundred microlitres diluted Daudi was added to Falcon 96-wellround-bottom plates and held at 4C overnight. Plates were thenblocked with 200pL blocking solution containing 0.1Mtris(hyroxymethyl)amino-methane [0.1M Tris-HC1], 0.15M NaC1, 2%sucrose, 0.1% thimerosal and 0.5% bovine serum albumin (BSA) for1h at 37C. Plates were then washed three times with PBS and0.05% tween [polyoxyethylene sorbitan monolaureate] and threetimes with distilled water, which constituted the standard platewashing regime. Monoclonal antibody was diluted in PBS with0.5% BSA and 0.1% thimerosal (PBS-BSA) and tested at 1-1/20,000dilutions. Positive and negative sera were initially tested at1/50-1/200 dilutions. One hundred microlitres of the mouse seraor monoclonal antibody was added to blocked plates and incubatedinitially at 37C for 1h, followed by the standard wash. Next100gL of the secondary antibody, goat anti-mouse F'c-alkalinephosphatase conjugate (Helix Biotech, Richmond, B.C.) diluted1/1000 in PBS-BSA was added and incubated for 1h at 37C,followed by the standard wash. Then 100pL of the alkalinephosphatase substrate containing 2.6mg/mL p-nitrophenylphosphate in 1.OM diethanolamine buffer pH 10 was added andincubated in the dark at 37C until adequate colour development.Optical density (O.D.) of the plates were read at various timesduring colour development at 405nm using an ELISA plate reader.The H-Y antibody hybridoma culture never showed evidence ofcolour development at any of the dilutions tested. Even after10x concentration with (NH 4 ) 25304 and assay with an antibody typing70kit, little or no IgM anti-H-Y activity was noted, andthereafter the supernatant was not used as a positive control.The assay was then designed to directly compare O.D. from seraobtained from male-immunized and adjuvant-only immunizedfemales.Based on preliminary results, Daudi supernatant was used at1/2 dilution in carbonate-bicarbonate pH 9.6 buffer and left tocoat plates overnight at 4C. Plates were blocked and washed asabove. One hundred microlitres H-Y immunized and control mousesera were tested in duplicate at 1/200 and 1/800 dilutions inPBS-BSA, and was incubated for 3h at 37h. Male mouse sera wassimilarly diluted and served as a background for antibodybinding. Another negative control using PBS alone in place ofthe mouse sera was done in order to measure the amount ofnon-specific secondary antibody binding to Daudi-coated wells.Following the standard wash, 100p1 goat anti-mouse F'c-alkalinephosphatase diluted 1/1000 in PBS-BSA was added and incubatedfor lh at 37C. Plates were washed and 100pL substrate was addedand plates were incubated in the dark. Plates were read at 15,30 and 45 minutes. All of the early samples were assayed in oneday. O.D. results were analyzed using SAS, 1987 (covariateanalysis).Sera from the final bleeding of H-Y-immunized andadjuvant-immunized females of the third replication, malecontrols, parous female and control females before and afteradjuvant immunization were assayed using two methods. The first71method was as above using Daudi cells. The second method was bya procedure similar to Iyer et al. (1989). One B6 male waseuthanized using CO 2 gas, and the spleen aseptically removed.The spleen was then dissociated by pushing it through a wiremesh in PBS using a 5mL syringe. The concentration ofsplenocytes was counted using a hemocytometer and cell countsadjusted to 10 million cells/mL using PBS. Fifty thousand malespleen cells in 50pL were added to each well of 96-well cellculture plates and left to dry at 37C overnight. The splenocytecells were fixed to the wells by the addition of 100pL 0.5%glutaraldehyde at room temperature for 10 minutes. Plates werewashed using the standard wash. Sera was initially tested at1/100 and 1/400 in PBS-BSA for 3h at 37C and secondary antibodyat 1/1000 in PBS-BSA for 1h at 37C, but little effect ofdilution was noted. Thereafter, 1/200 and 1/800 dilutions ofsera and 1/2000 of the secondary antibody were used. Onehundred microlitres of 2.6mg/mL p-nitrophenyl phosphate in 1.0Mdiethanolamine buffer pH 10 was added and incubated in the dark.Optical density of the wells was recorded at 405nm on the ELISAplate reader after 45 minutes incubation with the enzymesubstrate.Results Overall the results were variable and there were severalunexpected findings. However, all dilutions of serum followedexpected patterns, i.e. serum diluted at 1:200 always had ahigher reaction than serum at 1:800. Male serum was used as anegative control in this assay, because males were not expected72to have antibodies to H-Y antigen. Despite this, some maleserum samples exhibited higher reactivity in the ELISA systemthan some female serum samples. As well, any female exposure toadjuvant tended to increase reaction in the ELISA. One femalewhich had littered several times, yet had not been immunized orexposed to adjuvant, had a relatively high reaction in the assaysystem. Control females which had been immunized againstadjuvant alone (female controls) and had also been bred severaltimes displayed, at various times, H-Y antibody titres (asmeasured by both Daudi and spleen cell based ELISA systems)higher than H-Y immunized females. The two assay systems testedproduced similar results.DiscussionSerum titres of immunized mice could not be accuratelydetermined in this experiment. The ELISAs developed did measuresome antibody reactions, however these seemed to be nonspecific.Male serum was used as a negative control, because males werenot expected to have antibodies to H-Y (a self) antigen.Despite this, some male serum samples exhibited higherreactivity in the ELISA system than some female serum samples.As well, any female exposure to adjuvant tended to increasereaction in the ELISA. Overall, this indicates that the ELISAsystem may not have measured H-Y antibody titres, although alldilutions of serum followed expected patterns and were alwayshigher than PBS alone. The fact that two different assaysystems were tested and produced similar results, may indicatethat there is a high level of non-specific antibody cross-73reactivity in these assay systems. This has also been reportedby Savikurki et al. (1983).One female which had littered, but had not been immunizedor exposed to adjuvant, had a relatively high reaction in theassay system. Control females, immunized against adjuvantalone, had also been bred several times. They exhibited H-Yantibody titres higher than H-Y immunized females. Theseresults may be explained by the fact that females withsuccessive pregnancies (repeated exposure to H-Y antigen onsperm and/or male embryos) may generate H-Y antibodies (Krupen-Brown and Wachtel, 1979).Male spleens have been used in many previous experiments togenerate H-Y antisera (Wachtel et al., 1975; White et al, 1984),yet it is known that male spleen is not a pure source of H-Yantigen. Doubtless, some antibodies react in the ELISA systems,yet it is not known how much binding was due to: non-specificcross-reactive antibodies; adjuvant-stimulated non-specificcross-reactive antibodies; H-Y antigen specific reactivitygenerated by breeding and pregnancy; and H-Y specific reactivitystimulated by immunization to male spleens.Overall it appears difficult to obtain specific high titreH-Y antibody in mice (Savikurki et al., 1983). This may be dueto several factors: a) H-Y antigen is a weak antigenic stimulusb) only a few strains of mice respond to H-Y antigen c) theaccuracy of tests measuring anti-H-Y titre is confounded by the74presence of non-specific antibodies.75Chapter 3 Experiment TwoEFFECT OF MATERNAL IMMUNIZATION AGAINST H-Y ANTIGEN ON LITTERSIZE AND SEX RATIOSummaryBecause H-Y antigen is known to be present on spermatozoa(Goldberg et al., 1971) and male embryos (Krco and Goldberg,1976); an experiment was designed to test the effect of maternalH-Y antibody production on litter sizes and sex ratios ofimmunized females. Overall, no effects of treatment, breedingnumber, sire of litter, replication or treatment by breedinginteractions were found by covariate analysis. However, leastsquare means analysis showed a significant difference in theproportion of males born in the first litters of control and H-Yimmunized females (p < 0.05). H-Y immunized females had moremales in the first litter. This may be related to a beneficialmaternal reaction to the foreign H-Y antigen carried by malefetuses, such that male fetuses were advantaged at the time ofimplantation.Materials and Methods BreedingAfter the first bleeding following the sixth week ofimmunizations, described earlier, females were paired singlywith CD-1 or B6 males to allow mating. CD-1 males were used tostudy whether males which differed from the females in antigensother than H-Y antigen, would produce altered litter sizes orsex ratios. Females remained in the male's cage for two weeks.Females were then placed into individual cages to litter.76Litters were examined within 48 hours of birth and wereinitially sexed at this time. Final sexing was accomplished atweaning at 21 days. Control and male-immunized females were notimmunized during breeding, pregnancy or lactation periods.After the first litter was weaned, females were immunizedapproximately every two weeks. Sera was collected as above atvarious times during the experiment.Litter size and proportions of males born in a litter wereexamined through covariate analysis using SAS (SAS, 1987) anduse of Duncan's test to study least square mean separation. Themodel used for proportion of males was:Model 1 Y ijkl= p + T i + Rj + Bk + M I + ( TB) ik + OD + E ijuWhere T. is the ith (1-2, control or male-immunized)treatment, Rj is the jth replication (1-3), Bk the kth breeding(1-4), M t the lth male (1-2, CD-1 or C57BL/6) used for breeding.TB is the interaction betweeen treatment and breeding, and OD isthe ELISA optical density reading. ; At = the number of malesborn in a litter. A similar model was used for litter size,with Y ijk = litter size:Model 2 Y ijk= p + T i + R j + Bk + (TB)ik + ElkOptical density and breed of male were excluded from this modelbecause they had insignificant effects on litter size.Results No significant effects of treatment, replication, breeding,sire or optical density were noted on the arcsine-transformedproportion of males in the litters. Proportions of males in77litters appeared normally distributed and further analyses weredone using untransformed male proportions per litter. Overall,model 1 explained very little of the variation in the numbers ofmales born per litter, r 2=0.117722. Main effects of treatment,replication, breeding, sire and the interaction of breeding andtreatment were not significant. However, the overall proportionof males in first litters born to immunized females was 0.61while it was 0.49 in control females. Least square meansanalysis demonstrated that the proportion of males in firstlitters of control and immunized females were significantlydifferent (p < 0.05). Table 3.2.1 demonstrates litter size andsex ratio of the first litter of control and H-Y immunizedfemales in the first replication. The third replication alsohad an excess of males in the first litter, but litter sex ratiowas 1:1 in the second replication.Litter size was not affected by (anti-H-Y) ELISA O.D.value, sire or immunization treatment. The breeding numberaffected litter size (p < 0.05). Litter size in the fourthlitter was low, however, few females produced four litters.DiscussionPrevious studies using antibodies against H-Y antigen toaffect the sex ratio have given differing effects. H-Y antigenis known to be present on spermatozoa (Goldberg et al., 1971).This lead to development of experiments to test the effects ofanti-H-Y antisera on fertility. Bennett and Boyse (1973)combined mouse spermatozoa with H-Y antisera and complement78Figure 3.2.1 The number of males and females in thefirst litter of control and H-Y immunizedfemales in replication 1.CONTROL^ IMMUNIZEDSire M F Total Sire M F TotalCD-1 2 4 6 CD-1 2 3 5CD-1 3 5 8 CD-1 8 0 8CD-1 4 6 10 CD-1 6 1 7CD-1 4 3 7 CD-1 5 1 6CD-1 6 1 7 CD-1 C* C CCD-1 6 3 9 B6 0 0 0B6 0 0 0 B6 C C CB6 5 3 8 B6 0 0 0B6 3 4 7 B6 2 1 3B6 1 3 4 B6 7 0 7B6 2 3 5 B6 2 4 6B6 2 5 7B6 7 1 8Total 45 41 86 Total 32 10 42Mean 3.8 3.4 7.2 Mean 4.6 1.4 6.0S.D. 1.9 1.5 1.6 S.D. 2.6 1.5 1.6C cannibalized litter79in a cytolytic assay prior to use for artificialinsemination and saw an increase in the numbers of femalesborn.^Similarly, Zavos (1983) instilled H-Y antiseraintravaginally in rabbits prior to artificial insemination andalso showed an increase in the proportion of females borncompared to controls. Hoppe and Koo (1984) reacted mouse spermwith monoclonal H-Y antibodies in a sedimentation assay withprotein A-sheep red blood cells.^Unsedimented (less H-Yantigen) sperm were then used for in vitro fertilization ofmouse eggs.^Fertilized eggs were then transferred topseudopregnant recipients and no effect on sex ratio was seen.Since female mouse response to H-Y antigen is known to vary withstrain (Eichwald and Silmser, 1955); different strains of micefor use in antisera production, egg and sperm donors, andrecipients may have affected the numbers of males born in theprevious experiments. Krackow and Gruber (1990) have shown thatsex ratio of the litter depends on the strain of mice and modeof conception (matings postpartum or after lactationalanoestrus). Postpartum matings in some strains, resulted inlarger numbers of females born.Goldberg et al. (1971) saw no reason to suppose thatY-chromosome bearing sperm should carry more H-Y antigen. Theamount of H-Y antigen present is believed to depend on thematurational state of the sperm. H-Y antigen is found ondiploid spermatozoa precursors in the testis, but theconcentration of H-Y antigen bound to sperm decreases as spermmove through the epididymis (Hoppe and Koo, 1984). Therefore,80it appears that H-Y antigen expression is highest on immaturesperm and is not related to whether the sperm is X- or Y-chromosome bearing.H-Y antigen is found on sperm (Goldberg et al., 1971) andmale embryos after the eight-cell stage (Krco and Goldberg,1976). It was believed at the initiation of this project, thatmaternal response to H-Y antigen would affect the number ofviable sperm or would harm male embryos around the implantationstage. This would be reflected in decreased litter sizes orfewer males born. The results demonstrate that immunizationagainst H-Y antigen does not affect litter size and may provideevidence that sperm are normally inaccessible to attack in thefemale tract. Alternately, due to large numbers of spermavailable, enough sperm may survive immunological attack toprovide normal rates of fertilization. The lack of change inlitter size in immunized females agrees with previous reports(McLaren, 1962; Rao et al., 1981).Covariate analysis of breedings of male spleen-immunizedfemales and control adjuvant-immunized females in thisexperiment indicate that there were increased numbers of malesborn in the first litter of male-immunized females (p < 0.05).Previously, reported effects of H-Y immunization on sex ratiosof litters of immunized females has been controversial.Evidence exists that maternal H-Y immunization prior to breedingcaused female-biased litters (Pechan, 1985; Singh and Verma,1987), no change in the sex ratio (McLaren 1962, Rao et al.,811981) or male-biased litters in splenectomized and H-Y immunizedfemales (Lappe and Schalk, 1971; Shalev et al., 1980; Hings andBillingham, 1984). The increase in the number of males in thefirst litter of intact H-Y immunized females in this experimentwas an unexpected result.The increase of males born in the first litter to immunizedfemales, may have been due to a beneficial reaction in the H-Yimmunized mother that increased the number of males developing,while maintaining litter size. Previous studies have shown thatallogeneic pregnancies are routinely accepted by the mother,despite the presence of foreign antigens on the fetuses.Pregnancy has been shown to increase the number of cells inmaternal lymph nodes which drain the uterus, and this effect ismore marked in allogeneic pregnancies (Beer et al., 1975). Pre-sensitization to foreign paternal antigens improved, notdecreased, reproductive performance (Beer et al., 1975). Lymphnode cells may be part of an important maternal reaction whichincreases angiogenesis at implantation sites. Athanasskis etal. (1987) found that maternal T-cell lymphokines stimulateplacental growth.Lappe and Schalk (1971) found 30% more eggs are ovulatedthan embryos which implant in mice. Therefore, if 10 eggsovulate, only 7 embryos will implant. At fertilization, a 1:1sex ratio would result in 5 male and 5 female embryos. However,if males were favoured at implantation, then 5 males may implantbut then only 2 of the female embryos could implant, due to82space and other constraints in the uterus. Females immunized toH-Y antigen in this study may have reacted to the H-Y antigencarried on male embryos and thus improved male implantationrate, thereby increasing the sex ratio.The overall effect of immunizing females to H-Y antigen isweak and the effect is only noted in the first litter. It hasbeen claimed that the response to H-Y antigen depends on thestrength of the antisera (Piedrahita and Anderson, 1985) andthat it is relatively easy to induce tolerance to the H-Yantigen (McLaren, 1962; Piedrahita and Anderson, 1985).Therefore, it may well be that an immunized female's response tomale embryos carrying the H-Y antigen depends on the strength ofmaternal immunization. This is in line with the theories of(Krupen-Brown and Wachtel, 1979; Bell and Billington, 1980) thatindicate that successive pregnancies may induce tolerance to H-Yantigen and production of non-complement fixing H-Y antibodies.Prehn (1960) noted that C57BL/6 females which had borne severallitters were tolerant to male skin grafts. Either tolerance toH-Y antigen or production of non-complement fixing H-Yantibodies may indicate that the females are no longer able toreact to H-Y antigen. Thus any advantage that male fetusesgained in the first pregnancy due to maternal immunization maybe lost in successive pregnancies. Further evidence shows thatsplenectomized-immunized females, which normally had an excessof male progeny, had normal sex ratios if they were re-immunized(Shalev et al., 1980).83Although tests have been developed which can sex embryoswith 80% accuracy in vitro (White et al., 1983, 1987); resultsfrom experiments carried out partially or wholly in vivo produceinconsistent shifts in sex ratio. This is the first experimentwhich reported increases in males born to H-Y immunized females,however the effect was not seen in later pregnancies possiblydue to induction of tolerance to H-Y antigen through repeatedpregnancies (Krupen-Brown and Wachtel, 1979) or through repeatedimmunizations (Shalev et al., 1980). In conclusion, it does notappear that maternal immunization to H-Y antigen will be able toaffect large departures from the 1:1 sex ratio.84Chapter 3 Experiment ThreeSEXING OF BOVINE PRE IMPLANTATION EMBRYOS BY KARYOTYPE ANALYSISSummaryDue to the fact that sex selection of semen is unreliableand detection of fetal sex at mid-gestation is inefficient, mostof the emphasis for obtaining offspring of the desired sex hasfocussed on sex determination of pre-implantation embryos priorto embryo transfer. Sexing bovine embryos by karyotype analysisis relatively easy due to the ease in identifying the X- and Y-chromosomes. Embryos may be processed and sexed within about6h, which allows embryos to be transferred fresh or frozen.Although accuracy of sexing embryos is quite high, sexing rateis only 60% due to embryonic and technical difficulties.IntroductionThere are two main methods of sex selection: non-invasivetechniques which generally do not affect embryo viability, andinvasive techniques which although more accurate may have somedetrimental effects on embryo viability. Because phenotypic sexusually agrees with genotypic sex, embryos may be sexed bydirect identification of sex chromosomes. Melander reported in1959 that the chromosomal complement of cattle was 2n=60 with 58acrocentric autosomes and a large metacentric X-chromosome andsmall metacentric Y-chromosome. Because X- and Y-chromosomesare easily identifiable, it is possible to determine embryonicsex by karyotype analysis.Early experiments used large pieces of hatched embryos for85karyotype analysis (McFeely in 1960, Hare et al., 1976). Hareet al. (1976) prepared chromosomes from relatively large piecesof trophoblast from day 14 bovine embryos, and was able to sex20/34 embryos. After transfer of the biopsied embryos a 37.5%pregnancy rate resulted.Due to the ease of collection and ability to freezeembryos, most work with embryo transfer procedures is done withDay 6-8 embryos. Correspondingly, most karyotype analysis isalso done on the morula/blastocyst stage bovine embryo.Tarkowsky (1966) first used the air-dry method to preparechromosomal spreads from zona-enclosed mouse embryos. Wholemouse embryos were exposed to colcemid for 1-2h, 1% citrate for5-15 minutes at room temperature and then were fixed with 3:1ethanol-acetic acid while blowing air onto the specimen. Kinget al. (1979) used 1:1 methanol-acetic acid, followed byfixation in 3:1 methanol-acetic acid and was able to analyze18/20 bovine embryos. Some workers have bisected embryos toform demi-embryos and cultured these for four to ten hoursbefore processing for karyotype analysis to find that 60% weresexable (Picard et al., 1984; Rall and Leibo, 1987). Transferof the demi-embryos gave 50-60% pregnancy rates (Picard et al.,1984; Rall and Leibo, 1987). However, Murray et al. (1985) wereonly able to sex 40% of processed embryos after a ten hourculture. Hare et al. (1980) found that the limitations onability to karyoype embryos were mainly due to the small numberof cells available.86This experiment was carried out to learn the techniques ofembryo karyotyping as a method of preimplantation sexdetermination. Analysis of the bovine karyotype can be used asa primary method for sex determination or for confirming the sexof embryos sexed using other methods.Materials and Methods First culture of bovine lymphocytes was done in order tohave a large numbers of cells to process and gain familiaritywith the bovine karyotype. Blood was collected from coccygealbleeding from cows or male calves into heparinized test tubes.Blood was cultured to produce lymphocyte chromosomal spreadsaccording to the method of Eldridge (1985). Four hundredmicrolitres of whole blood was placed into 4mL Ham's F-10 mediawith 0.5mL fetal calf serum and 1 pg/mL phytohemagglutinin-M.This was cultured for 2-3 days at 37C in 5% CO 2 . Then theculture was poured into a graduated test tube and 0.067Mpotassium chloride was added to make 8mL. This stayed at roomtemperature for 30 minutes. Then lmL of 3:1 mixture ofmethanol/glacial acetic acid was added and the tubes centrifugedat 1000g for 5 minutes. The supernatant was removed and thepellet resuspended in 5mL 3:1 methanol-acetic acid andcentrifuged at 1000g for 5 minutes. This process was repeatedwith three washes with 3mL methanol-acetic acid. Then thepellet was resuspended in lmL methanol-acetic acid. A pasteurpipette was used to drop a few drops of the fixed cells onto aclean slide from a height of about 20cm. The slide was allowedto dry and was stained for 5 minutes with 4% Giemsa.87After enough experience had been gained with lymphocytes,good quality bovine embryos obtained from the two experiments onsuperovulation were karyotyped. Embryos were cultured in 35mmpetri dishes containing 2mL Ham's F-10 with 10% serum (fetalcalf or estrous cow) for 4-5h at 37C in 5% CO 2 in air,essentially according to King (1984). Colchicine (0.5pg/mL) wasused instead of 0.05pg/mL colcemid. After culture, embryos wereplaced into a 35mm petri dish containing 0.9-1.0% tri-sodiumcitrate for 5-20 minutes. Embryos were then placed onto a cleangrease-free slide in a small amount of the hypotonic solution.A few drops of 4:3:1 (methanol-distilled water-acetic acid) wereadded to soften and digest the zona pellucida. Then a few dropsof 1:1 methanol-acetic acid were placed on the embryos and theslides dried at room temperature. The fixation process wasobserved under a dissection microscope at 50x. Slides werefixed overnight in a Copplin jar with 3:1 methanol-acetic acid.Slides were dried before staining with 4% Giemsa.Photomicrographs of embryos were taken using oil immersion at1000x.Results Plate 3.3.1 and 3.3.2 show chromosomal spreads preparedfrom female and male lymphocytes respectively. The largemetacentric X-chromosomes are indicated by arrows in Plate3.3.1. In the male karyotype on Plate 3.3.2, the large X-chromosome (large arrow) and the small metacentric Y-chromosome(small arrow) are indicated. Plate 3.3.3 is a photomicrographof chromosomes prepared from a bovine embryo. One X-chromosome88Plate 3.3.2 Photomicrograph (1000x) of lymphocytesdemonstrating the male bovine karyotype.Plate 3.3.3. Photomicrograph of chromosomes prepared from abovine embryo (1000x).can be easily identified (arrow).^There are no other X-chromosomes visible, but due to chromosomal morphology it isdifficult to identify the Y-chromosome, although it is likely tobe one of the smaller chromosomes. This embryo is tentativelysexed as male.Of approximately 50 morula/blastocyst bovine embryosprocessed for karyotyping, only about 15 had good chromosomalspreads. Embryos had from 0-6 metaphases. After staining manyof the chromosomes appeared to have indistinct morphology.DiscussionKaryotype analysis of bovine embryos has been used as areliable method for embryo sexing and will allow the selectionof the sex of the calf before embryo transfer. The technique ishighly accurate due to the ease in identification of the sexchromosomes in cattle.In this experiment using whole embryos, only about 25% ofthe embryos processed had good metaphase chromosomal spreads.Some difficulties in obtaining good quality metaphases forsexing have been reported. Some of the difficulties experiencedin this experiment were: 1) limited number of cells availablefor sexing 2) lack of metaphases 3) poor spreading due toincomplete digestion of the zona pellucida before fixing 4) poormorphology of the chromosomes 5) incomplete chromosomal spreadsand 6) lost embryos. The major difficulty in the course of thisexperiment was in achieving complete removal of the zona92pellucida. When the zona pellucida was not completely removedbefore fixation, the embryo would not spread on the slide andeven if metaphases were visible, the chromosomes could not beseen clearly due to overlapping in the zona-constrained space.Most authors use micromanipulators to cut the embryo intodemi-embryos or to take small biopsies of embryos for sexing,thus avoiding the problem of trying to digest an intact zonapellucida. The other major difficulty in trying to karyotypeembryos was due to a lack of metaphases. Hare et al. (1980)noted that the ability to sex embryos is related to the numberof cells available for sexing. Metaphase spreads appeared to bein better condition before staining under phase contrast thanafter Giemsa staining. It may be that too long an exposure tostain (1h) and change in pH of stain after long storage affectedembryonic chromosomal morphology.Even in experienced hands, only about 60% of embryos can besexed and can be transferred fresh with about 60% pregnancy rate(Picard et al., 1984; Rall and Leibo, 1987). This causes aproblem for dealing with the 40% unsexable embryos. If 60/100embryos are sexable, and only the 30 of the desired sex aretransferred then 30 recipients will be required, and only 18calves of the desired sex are born from 100 embryos. If the 30embryos of the desired sex and the 40 unsexed embryos aretransferred, then 70 recipients will be required and 30 (18 fromsexed embryos and 12 from unsexed embryos) calves of the desiredsex and 12 of the undesired sex will result. If embryos werenot sexed then at a 60% pregnancy rate 100 recipients will be93required and 30 of the desired sex and 30 of the undesired sexof calf will be born. Usefulness of the karyotyping procedurewill depend on the efficiency and cost of the sexing procedure,the value of the desired and undesired sexes, and the cost tomaintain recipients. The karyotyping technique would increasein usefulness if procedures can be developed which increasesexability rate.94Chapter 4CONCLUDING DISCUSSIONInitiation of superovulatory treatment in the presence ofa dominant follicle has been shown to decrease superovulatoryresponses (Grasso et al., 1989; Guilbault et al., 1991; Huhtinenet al., 1992) although other authors have reported no effect ofa dominant follicle on superovulatory responses (Wilson et al.,1990). Two experiments were carried out to investigate thesuccess of superovulation in the absence of a dominant follicle.Initiating superovulation treatment in the early estrous cycleto avoid the influence of a dominant follicleIn the first experiment, follicular growth, ovulation andembryo recovery rates were compared between cows which weresuperovulated at Day 2 of the estrous cycle, before a dominantfollicle is present, and control cows superovulated at mid-cyclewhen a dominant follicle may have been present. Previousresearchers have noted that there were poorer superovulatoryresponses when superovulation was initiated at the beginning ofthe estrous cycle (Philippo and Rowson, 1975; Sreenan andGosling, 1977; Lindsell et al., 1985). Recently, Goulding etal. (1990) reported that fewer corpora lutea and embryos werefound after slaughter in cows superovulated at Day 2 of thecycle compared to Day 10. However, follicular developmentduring superovulation treatment was not measured in any of thesestudies.In this experiment, follicular development and ovulation95rate were monitored after superovulation induction in cowsbeginning superovulatory treatment at Day 2 or at mid-cycle Days9-13. All cows responded to FSH with adequate folliculardevelopment. However, few cows which were superovulated on Day2 demonstrated estrus and most failed to ovulate. Regressingfollicles were noted at the expected time of embryo collection.Very few embryos were recovered from cows which weresuperovulated early in the cycle. This experiment demonstratedthat although follicular development was adequate in response tosuperovulation induction early in the cycle, ovulation rateswere low. Lack of ovulation of the FSH-stimulated folliclesafter superovulation on Day 2, may have been due to severalfactors. Most of the Day 2 cows failed to demonstrate estrus.Using ultrasound, it was evident that in many cows, the corpusluteum failed to undergo complete luteolysis. Lindsell et al.(1985) noted that few animals superovulated on Day 3 of theestrous cycle demonstrated estrus. Prostaglandin F24 was givenon Day 7 of the estrous cycle in Day 2 cows, at a time when theCL is normally responsive (King et al., 1982; Momont et al.,1984). Therefore, it is likely that administration ofgonadotrophins early in the estrous cycle may have affected CLresponse to PGF 2a . Also it was likely that many of the folliclesstimulated by Day 2 FSH treatment were atretic because they wereunable to ovulate following hCG.Removal of the dominant follicle at Day 7 of the estrous cyclewith hCG prior to superovulatory treatmentIn the second experiment, superovulation parameters were96compared between animals which began treatment at mid-cycle,when a dominant follicle may have been present; and animals inwhich the dominant follicle was removed with hCG on Day 7 of theestrous cycle before initiation of superovulation. Humanchorionic gonadotrophin previously has been used to ovulate thedominant follicle present on Day 7 of the cycle and increaseprogesterone levels (Rajamahendran and Sianangama, 1992).Progesterone values during superovulation treatment havepreviously been shown to affect superovulatory responses by someworkers (Yadav et al., 1986; Goto et al., 1987) but not byothers (Sreenan and Gosling, 1977; Saumande et al., 1985;Tamboura et al., 1985). In this experiment, progesterone valuesduring superovulation treatment did not significantly affectsuperovulation responses.Cows which had the dominant follicle removed by hCGtreatment prior to superovulation induction tended to havebetter follicular development, ovulation rate and embryosrecovered, however these values were not significantly differentbetween control cows and hCG-treated cows. Bettersuperovulatory responses have been noted at Days 8-12 (Philippoand Rowson, 1975; Lindsell et al., 1985) than at other days ofthe cycle. Goulding et al. (1990) have postulated that thisresponse \"window\" is related to the time in the estrous cyclewhen a dominant follicle may not be present. Timing of the\"window\" may vary depending on whether the estrous cycle willhave two or three waves of follicular development. Optimalresponses may have occurred in both the hCG-treated and control97groups in this experiment because all cows were superovulatedbeginning on Days 8-11. In addition, the beneficial effect ofinitiating superovulation in the absence of a dominant folliclemay have been confounded by the effect of hCG on small antralfollicles. Investigation of the effect of initiatingsuperovulation in the absence of a dominant follicle mightbetter be resolved by removal of the dominant follicle throughother means such as laparotomy and electrocautery or throughultrasound-guided follicular aspiration.It would be useful if a method to allow superovulation inthe absence of a dominant follicle could be selected withoutreference to ultrasound monitoring. Because a dominant follicleis usually found on Day 7 of the estrous cycle in either two orthree wave cycles, it may be possible to use an agent such ashCG to remove the dominant follicle to create a more approriateenvironment for initiation of superovulation.Conclusions from superovulation experimentsBoth superovulation experiments were conducted at a time inthe estrous cycle when a dominant follicle would not be present.Although administration of gonadotrophins early in the estrouscycle caused follicular development, the majority of these FSH-stimulated follicles were unable to ovulate. However, in thesecond experiment, removal of the Day 7 dominant follicle withhCG did not significantly increase the numbers of follicles,corpora lutea or embryos recovered from superovulated donors.It may be that the dominant follicle does not have a large98effect on superovulatory success, hCG itself affectssuperovulatory success, or other intrinsic factors affectsuperovulatory parameters. Investigations on other methods ofremoving the dominant follicle may help resolve this issue.Sexing preimplantation embryos H-Y antisera raised in mice have been used previously inseveral experiments to sex preimplantation embryos (White etal., 1983, 1987) with 80% accuracy. Attempts were made toimmunize C57BL/6 female mice against male splenic cells to raiseH-Y antisera. The titre of the antisera was then measured intwo ELISA systems. Both ELISAs demonstrated that there was highnon-specific cross-reactivity in sera from control and immunizedmice. H-Y immunized females were bred to assess the effect ofmaternal H-Y antibodies in vivo on litter size and sex ratio.The litter size was unaffected by treatment. There wereincreased numbers of males in the first litter of immunizedfemales. This may have been due to beneficial immunologicalreaction of the mother to H-Y antigen on male fetuses.Tolerance may have been induced to H-Y antigen due to repeatedexposures of immunized females, and this may be the reason fornormal sex ratios in later litters.Another method used to sex embryos before embryo transferis karyotype analysis. A few cells can be removed from bovinepreimplantation embryos to perform karyotype analysis. Incattle, the X- and Y-chromosome are easily identified in goodchromosomal preparations. Previously, using karyotyping99techniques to assess embryonic sex, about 60% embryos could besuccessfully sexed (Picard et al., 1984; Rall and Leibo, 1987).In this experiment, whole embryos were used for karyotypeanalysis. There were some difficulties in preparing goodchromosomal spreads. Sexing embryos by karyotype analysis islimited by the small number of cells available. 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"10.14288/1.0086653"@en . "eng"@en . "Animal Science"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Studies on superovulation and embryo sexing in dairy cattle"@en . "Text"@en . "http://hdl.handle.net/2429/3256"@en .